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###
### Running command:
###
###   /home/biocbuild/bbs-3.22-bioc/R/bin/R CMD check --install=check:FLAMES.install-out.txt --library=/home/biocbuild/bbs-3.22-bioc/R/site-library --timings FLAMES_2.3.4.tar.gz
###
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* using log directory ‘/home/biocbuild/bbs-3.22-bioc/meat/FLAMES.Rcheck’
* using R version 4.5.1 (2025-06-13)
* using platform: x86_64-pc-linux-gnu
* R was compiled by
    gcc (Ubuntu 13.3.0-6ubuntu2~24.04) 13.3.0
    GNU Fortran (Ubuntu 13.3.0-6ubuntu2~24.04) 13.3.0
* running under: Ubuntu 24.04.2 LTS
* using session charset: UTF-8
* checking for file ‘FLAMES/DESCRIPTION’ ... OK
* this is package ‘FLAMES’ version ‘2.3.4’
* package encoding: UTF-8
* checking package namespace information ... OK
* checking package dependencies ... INFO
Imports includes 50 non-default packages.
Importing from so many packages makes the package vulnerable to any of
them becoming unavailable.  Move as many as possible to Suggests and
use conditionally.
* checking if this is a source package ... OK
* checking if there is a namespace ... OK
* checking for hidden files and directories ... OK
* checking for portable file names ... OK
* checking for sufficient/correct file permissions ... OK
* checking whether package ‘FLAMES’ can be installed ... NOTE
Found the following notes/warnings:
  Non-staged installation was used
See ‘/home/biocbuild/bbs-3.22-bioc/meat/FLAMES.Rcheck/00install.out’ for details.
* used C++ compiler: ‘g++ (Ubuntu 13.3.0-6ubuntu2~24.04) 13.3.0’
* checking C++ specification ... OK
* checking installed package size ... INFO
  installed size is  5.9Mb
  sub-directories of 1Mb or more:
    bin    1.1Mb
    data   1.8Mb
    libs   1.5Mb
* checking package directory ... OK
* checking ‘build’ directory ... OK
* checking DESCRIPTION meta-information ... OK
* checking top-level files ... OK
* checking for left-over files ... OK
* checking index information ... OK
* checking package subdirectories ... OK
* checking code files for non-ASCII characters ... OK
* checking R files for syntax errors ... OK
* checking whether the package can be loaded ... OK
* checking whether the package can be loaded with stated dependencies ... OK
* checking whether the package can be unloaded cleanly ... OK
* checking whether the namespace can be loaded with stated dependencies ... OK
* checking whether the namespace can be unloaded cleanly ... OK
* checking loading without being on the library search path ... OK
* checking dependencies in R code ... NOTE
There are ::: calls to the package's namespace in its code. A package
  almost never needs to use ::: for its own objects:
  'blaze' 'find_barcode' 'gene_quantification' 'isoform_identification'
  'minimap2_align' 'transcript_quantification'
* checking S3 generic/method consistency ... OK
* checking replacement functions ... OK
* checking foreign function calls ... OK
* checking R code for possible problems ... NOTE
BulkPipeline: no visible global function definition for ‘new’
MultiSampleSCPipeline: no visible global function definition for ‘new’
SingleCellPipeline: no visible global function definition for ‘new’
addRowRanges: no visible global function definition for ‘head’
addRowRanges: no visible global function definition for ‘as’
cache_dir: no visible global function definition for ‘packageVersion’
chisq_test_by_gene: no visible global function definition for
  ‘chisq.test’
create_spe: no visible binding for global variable ‘barcode’
create_spe: no visible binding for global variable ‘in_tissue’
download_oarfish: no visible global function definition for
  ‘download.file’
download_oarfish: no visible global function definition for ‘unzip’
filter_coverage: no visible global function definition for
  ‘starts_with’
filter_coverage: no visible binding for global variable ‘filter_res’
find_barcode: no visible binding for global variable ‘Sample’
find_barcode: no visible binding for global variable ‘Outfile’
find_variants_grange: no visible binding for global variable
  ‘which_label’
find_variants_grange: no visible binding for global variable
  ‘nucleotide’
find_variants_grange: no visible binding for global variable ‘pos’
find_variants_grange: no visible binding for global variable ‘count’
find_variants_grange: no visible binding for global variable
  ‘counts_no_ins’
find_variants_grange: no visible binding for global variable ‘ref’
generate_sc_sce: no visible binding for global variable ‘FSM_match’
get_coverage: no visible binding for global variable ‘Freq’
homopolymer_pct : <anonymous>: no visible binding for global variable
  ‘Freq’
homopolymer_pct : <anonymous>: no visible binding for global variable
  ‘pct’
plot_coverage: no visible binding for global variable ‘tr_length’
plot_coverage: no visible binding for global variable ‘read_counts’
plot_coverage: no visible binding for global variable ‘total_counts’
plot_coverage: no visible binding for global variable ‘cumpct’
plot_coverage: no visible binding for global variable ‘length_bin’
plot_coverage: no visible binding for global variable ‘min_length’
plot_coverage: no visible binding for global variable ‘max_length’
plot_coverage: no visible global function definition for ‘head’
plot_coverage: no visible binding for global variable ‘transcript’
plot_demultiplex_raw: no visible binding for global variable ‘Sample’
plot_demultiplex_raw: no visible binding for global variable
  ‘CellBarcode’
plot_demultiplex_raw: no visible binding for global variable ‘UMI’
plot_demultiplex_raw: no visible binding for global variable
  ‘UMI_count’
plot_demultiplex_raw: no visible binding for global variable
  ‘barcode_rank’
plot_demultiplex_raw: no visible binding for global variable
  ‘FlankEditDist’
plot_demultiplex_raw: no visible binding for global variable ‘n_reads’
plot_demultiplex_raw: no visible binding for global variable
  ‘BarcodeEditDist’
plot_demultiplex_raw: no visible binding for global variable ‘total
  reads’
plot_demultiplex_raw: no visible binding for global variable
  ‘demultiplexed reads’
plot_demultiplex_raw: no visible binding for global variable ‘single
  match reads’
plot_demultiplex_raw: no visible binding for global variable
  ‘undemultiplexted reads’
plot_demultiplex_raw: no visible binding for global variable
  ‘multi-matching reads’
plot_demultiplex_raw: no visible binding for global variable ‘Type’
plot_demultiplex_raw: no visible binding for global variable ‘Reads’
plot_demultiplex_raw: no visible binding for global variable ‘input’
plot_demultiplex_raw: no visible binding for global variable ‘output’
plot_demultiplex_raw: no visible binding for global variable
  ‘read1_with_adapter’
plot_demultiplex_raw: no visible binding for global variable ‘Count’
plot_flagstat: no visible global function definition for ‘everything’
plot_flagstat: no visible binding for global variable ‘name’
plot_flagstat: no visible binding for global variable ‘value’
plot_isoform_reduced_dim: no visible binding for global variable ‘x’
plot_isoform_reduced_dim: no visible binding for global variable ‘y’
plot_isoform_reduced_dim: no visible binding for global variable ‘expr’
plot_spatial: no visible binding for global variable ‘imageX’
plot_spatial: no visible binding for global variable ‘imageY’
plot_spatial_feature: no visible binding for global variable ‘imageX’
plot_spatial_feature: no visible binding for global variable ‘imageY’
plot_spatial_feature: no visible binding for global variable ‘x’
plot_spatial_feature: no visible binding for global variable ‘y’
plot_spatial_feature: no visible global function definition for
  ‘scale_alpha_continuous’
plot_spatial_feature: no visible global function definition for
  ‘scale_colour_gradient’
plot_spatial_isoform: no visible global function definition for ‘head’
plot_spatial_pie: no visible global function definition for ‘head’
plot_spatial_pie: no visible binding for global variable ‘imageX’
plot_spatial_pie: no visible binding for global variable ‘imageY’
sc_mutations: no visible binding for global variable ‘mutation_index’
sc_mutations: no visible binding for global variable ‘bam_index’
sc_transcript_usage_chisq: no visible global function definition for
  ‘as’
sc_transcript_usage_chisq: no visible binding for global variable
  ‘p.value’
sc_transcript_usage_chisq: no visible binding for global variable
  ‘adj.p.value’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘total’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘test’
sc_transcript_usage_permutation: no visible global function definition
  for ‘as’
sc_transcript_usage_permutation : <anonymous>: no visible global
  function definition for ‘as’
sc_transcript_usage_permutation : <anonymous> : <anonymous>: no visible
  global function definition for ‘na.omit’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘transcript’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘p.value’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘adj.p.value’
variant_count_tb: no visible binding for global variable ‘barcode’
variant_count_tb: no visible binding for global variable ‘allele_count’
variant_count_tb: no visible binding for global variable
  ‘cell_total_reads’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘expect_cell_number’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘fastq’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘outdir’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘demultiplexed_fastq’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘barcodes_file’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘expect_cell_number’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘fastq’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘outdir’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘demultiplexed_fastq’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘barcodes_file’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘j’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘genome_bam’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘minimap2’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘samtools’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘threads’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘outdir’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘j’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘transcriptome_assembly’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘transcriptome_bam’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘minimap2’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘samtools’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘outdir’
resume_FLAMES,FLAMES.Pipeline : <anonymous>: no visible global function
  definition for ‘capture.output’
run_FLAMES,FLAMES.Pipeline : <anonymous>: no visible global function
  definition for ‘capture.output’
Undefined global functions or variables:
  BarcodeEditDist CellBarcode Count FSM_match FlankEditDist Freq
  Outfile Reads Sample Type UMI UMI_count adj.p.value allele_count as
  bam_index barcode barcode_rank barcodes_file capture.output
  cell_total_reads chisq.test count counts_no_ins cumpct demultiplexed
  reads demultiplexed_fastq download.file everything expect_cell_number
  expr fastq filter_res genome_bam head imageX imageY in_tissue input j
  length_bin max_length min_length minimap2 multi-matching reads
  mutation_index n_reads na.omit name new nucleotide outdir output
  p.value packageVersion pct pos read1_with_adapter read_counts ref
  samtools scale_alpha_continuous scale_colour_gradient single match
  reads starts_with test threads total total reads total_counts
  tr_length transcript transcriptome_assembly transcriptome_bam
  undemultiplexted reads unzip value which_label x y
Consider adding
  importFrom("base", "match", "single")
  importFrom("methods", "as", "new")
  importFrom("stats", "chisq.test", "na.omit")
  importFrom("utils", "capture.output", "download.file", "head",
             "packageVersion", "unzip")
to your NAMESPACE file (and ensure that your DESCRIPTION Imports field
contains 'methods').
* checking Rd files ... OK
* checking Rd metadata ... OK
* checking Rd cross-references ... OK
* checking for missing documentation entries ... OK
* checking for code/documentation mismatches ... OK
* checking Rd \usage sections ... OK
* checking Rd contents ... OK
* checking for unstated dependencies in examples ... OK
* checking contents of ‘data’ directory ... OK
* checking data for non-ASCII characters ... OK
* checking data for ASCII and uncompressed saves ... OK
* checking line endings in shell scripts ... OK
* checking line endings in C/C++/Fortran sources/headers ... OK
* checking line endings in Makefiles ... OK
* checking compilation flags in Makevars ... OK
* checking for GNU extensions in Makefiles ... INFO
GNU make is a SystemRequirements.
* checking for portable use of $(BLAS_LIBS) and $(LAPACK_LIBS) ... OK
* checking use of PKG_*FLAGS in Makefiles ... OK
* checking include directives in Makefiles ... OK
* checking compiled code ... NOTE
Note: information on .o files is not available
File ‘/home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/libs/FLAMES.so’:
  Found ‘abort’, possibly from ‘abort’ (C)
  Found ‘exit’, possibly from ‘exit’ (C)
  Found ‘stderr’, possibly from ‘stderr’ (C)
  Found ‘stdout’, possibly from ‘stdout’ (C)

Compiled code should not call entry points which might terminate R nor
write to stdout/stderr instead of to the console, nor use Fortran I/O
nor system RNGs nor [v]sprintf. The detected symbols are linked into
the code but might come from libraries and not actually be called.

See ‘Writing portable packages’ in the ‘Writing R Extensions’ manual.
* checking files in ‘vignettes’ ... OK
* checking examples ... OK
Examples with CPU (user + system) or elapsed time > 5s
                               user system elapsed
blaze                         5.359 21.490  14.927
plot_isoform_reduced_dim     22.927  0.488  23.416
find_variants                19.683  0.398  19.397
sc_long_multisample_pipeline 13.080  5.911  12.791
bulk_long_pipeline            2.868 14.334   3.066
MultiSampleSCPipeline        14.629  0.948  15.817
sc_DTU_analysis              10.051  2.142   9.888
create_sce_from_dir           6.004  3.211   7.059
plot_isoform_heatmap          6.962  0.210   7.172
SingleCellPipeline            5.068  1.678   4.465
sc_long_pipeline              4.738  1.625   4.169
* checking for unstated dependencies in ‘tests’ ... OK
* checking tests ...
  Running ‘testthat.R’
 OK
* checking for unstated dependencies in vignettes ... OK
* checking package vignettes ... OK
* checking re-building of vignette outputs ... OK
* checking PDF version of manual ... OK
* DONE

Status: 4 NOTEs
See
  ‘/home/biocbuild/bbs-3.22-bioc/meat/FLAMES.Rcheck/00check.log’
for details.

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###
### Running command:
###
###   /home/biocbuild/bbs-3.22-bioc/R/bin/R CMD INSTALL FLAMES
###
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* installing to library ‘/home/biocbuild/bbs-3.22-bioc/R/site-library’
* installing *source* package ‘FLAMES’ ...
** this is package ‘FLAMES’ version ‘2.3.4’
** using non-staged installation via StagedInstall field
** libs
using C++ compiler: ‘g++ (Ubuntu 13.3.0-6ubuntu2~24.04) 13.3.0’
using C++17
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c RcppExports.cpp -o RcppExports.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c RcppFunctions.cpp -o RcppFunctions.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c classes/BamRecord.cpp -o classes/BamRecord.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c classes/GFFRecord.cpp -o classes/GFFRecord.o
In file included from classes/GFFRecord.cpp:8:
classes/../utility/utility.h: In function ‘std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > parseSpace(const std::string&)’:
classes/../utility/utility.h:255:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  255 |         for (int i = 0; i < s.size(); i++) {
      |                         ~~^~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c classes/GeneAnnotationParser.cpp -o classes/GeneAnnotationParser.o
In file included from classes/GeneAnnotationParser.cpp:15:
classes/../utility/utility.h: In function ‘std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > parseSpace(const std::string&)’:
classes/../utility/utility.h:255:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  255 |         for (int i = 0; i < s.size(); i++) {
      |                         ~~^~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c classes/Isoforms.cpp -o classes/Isoforms.o
In file included from classes/Isoforms.cpp:16:
classes/../utility/utility.h: In function ‘std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > parseSpace(const std::string&)’:
classes/../utility/utility.h:255:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  255 |         for (int i = 0; i < s.size(); i++) {
      |                         ~~^~~~~~~~~~
classes/Isoforms.cpp: In member function ‘void Isoforms::update_all_splice()’:
classes/Isoforms.cpp:233:35: warning: comparison of integer expressions of different signedness: ‘std::vector<StartEndPair>::size_type’ {aka ‘long unsigned int’} and ‘int’ [-Wsign-compare]
  233 |                 if (blocks.size() >= (int)this->Min_sup_cnt) {
      |                     ~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~~~~~~
classes/Isoforms.cpp: In member function ‘void Isoforms::filter_TSS_TES(std::ofstream&, DoubleJunctions, float)’:
classes/Isoforms.cpp:368:33: warning: comparison of integer expressions of different signedness: ‘std::unordered_map<int, int>::size_type’ {aka ‘long unsigned int’} and ‘int’ [-Wsign-compare]
  368 |         if ((left_counts.size() < (int)this->Min_sup_cnt) ||
      |              ~~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~~~~~
classes/Isoforms.cpp:369:38: warning: comparison of integer expressions of different signedness: ‘std::unordered_map<int, int>::size_type’ {aka ‘long unsigned int’} and ‘int’ [-Wsign-compare]
  369 |                 (right_counts.size() < (int)this->Min_sup_cnt)) {
      |                  ~~~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~~~~~
classes/Isoforms.cpp: In member function ‘void Isoforms::match_known_annotation(const std::unordered_map<std::__cxx11::basic_string<char>, Junctions>&, const std::unordered_map<std::__cxx11::basic_string<char>, Pos>&, const std::unordered_map<std::__cxx11::basic_string<char>, std::vector<StartEndPair> >&, GeneBlocks, std::unordered_map<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> >)’:
classes/Isoforms.cpp:662:51: warning: comparison of integer expressions of different signedness: ‘int’ and ‘const long unsigned int’ [-Wsign-compare]
  662 |                                 for (int j = 0; j < std::min(raw_iso_key.size() - 1, junction.junctions.size()); j++) {
      |                                                 ~~^~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
classes/Isoforms.cpp:713:43: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<int>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  713 |                         for (int i = 0; i < new_exons.size(); ++i) {
      |                                         ~~^~~~~~~~~~~~~~~~~~
classes/Isoforms.cpp:725:46: warning: comparisons like ‘X<=Y<=Z’ do not have their mathematical meaning [-Wparentheses]
  725 |                                 } else if (0 < i < raw_iso_key.size() - 1) { // a site from the middle somewhere
      |                                            ~~^~~
classes/Isoforms.cpp: In member function ‘std::string Isoforms::isoform_to_gff3(float)’:
classes/Isoforms.cpp:1140:43: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<int>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
 1140 |                         for (int i = 0; i < exons.size(); i+=2) {
      |                                         ~~^~~~~~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c classes/junctions.cpp -o classes/junctions.o
In file included from classes/junctions.cpp:12:
classes/../utility/utility.h: In function ‘std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > parseSpace(const std::string&)’:
classes/../utility/utility.h:255:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  255 |         for (int i = 0; i < s.size(); i++) {
      |                         ~~^~~~~~~~~~
classes/junctions.cpp: In function ‘std::unordered_map<std::__cxx11::basic_string<char>, std::vector<StartEndPair> > get_gene_flat(const std::unordered_map<std::__cxx11::basic_string<char>, std::vector<std::__cxx11::basic_string<char> > >&, const std::unordered_map<std::__cxx11::basic_string<char>, std::vector<StartEndPair> >&)’:
classes/junctions.cpp:166:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<StartEndPair>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  166 |         for (int i = 1; i < exons.size(); i++) {
      |                         ~~^~~~~~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c main-functions/find_isoform.cpp -o main-functions/find_isoform.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c main-functions/flexiplex.cpp -o main-functions/flexiplex.o
main-functions/flexiplex.cpp: In function ‘unsigned int edit_distance(const std::string&, const std::string&, unsigned int&, int)’:
main-functions/flexiplex.cpp:122:21: warning: comparison of integer expressions of different signedness: ‘unsigned int’ and ‘int’ [-Wsign-compare]
  122 |       if (min_value <= max_editd)
      |           ~~~~~~~~~~^~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘std::string get_umi(const std::string&, const std::vector<std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > >&, const std::vector<int>&, int, int, bool, int, int)’:
main-functions/flexiplex.cpp:163:22: warning: comparison of integer expressions of different signedness: ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} and ‘int’ [-Wsign-compare]
  163 |     if (seq.length() < umi_start + umi_length) {
      |         ~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:187:36: warning: comparison of integer expressions of different signedness: ‘std::vector<int>::size_type’ {aka ‘long unsigned int’} and ‘int’ [-Wsign-compare]
  187 |     if (read_to_subpatterns.size() > umi_index + 1) {
      |         ~~~~~~~~~~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘Barcode get_barcode(const std::string&, const std::unordered_set<std::__cxx11::basic_string<char> >&, int, int, const std::vector<std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > >&)’:
main-functions/flexiplex.cpp:295:19: warning: comparison of integer expressions of different signedness: ‘int’ and ‘__gnu_cxx::__alloc_traits<std::allocator<long unsigned int>, long unsigned int>::value_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  295 |     if (i_pattern >= subpattern_ends[i_subpattern]) {
main-functions/flexiplex.cpp:354:22: warning: comparison of integer expressions of different signedness: ‘unsigned int’ and ‘int’ [-Wsign-compare]
  354 |     if (editDistance == barcode.editd) {
      |         ~~~~~~~~~~~~~^~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:356:29: warning: comparison of integer expressions of different signedness: ‘unsigned int’ and ‘int’ [-Wsign-compare]
  356 |     } else if (editDistance < barcode.editd &&
      |                ~~~~~~~~~~~~~^~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:357:29: warning: comparison of integer expressions of different signedness: ‘unsigned int’ and ‘int’ [-Wsign-compare]
  357 |                editDistance <= barcode_max_editd) { // if best so far, update
      |                ~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘std::vector<Barcode> big_barcode_search(const std::string&, const std::unordered_set<std::__cxx11::basic_string<char> >&, int, int, const std::vector<std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > >&)’:
main-functions/flexiplex.cpp:396:29: warning: comparison of integer expressions of different signedness: ‘const int’ and ‘const std::__cxx11::basic_string<char>::size_type’ {aka ‘const long unsigned int’} [-Wsign-compare]
  396 |       if (barcode.flank_end == std::string::npos) {
      |           ~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘void print_stats(const std::string&, const std::vector<Barcode>&, std::ostream&)’:
main-functions/flexiplex.cpp:421:38: warning: comparison of integer expressions of different signedness: ‘const int’ and ‘const std::__cxx11::basic_string<char>::size_type’ {aka ‘const long unsigned int’} [-Wsign-compare]
  421 |                << (barcode.flank_end == std::string::npos ? "True" : "False")
      |                    ~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘void print_read(const std::string&, const std::string&, const std::string&, const std::vector<Barcode>&, gzFile, std::unordered_set<std::__cxx11::basic_string<char> >&, bool, bool, bool)’:
main-functions/flexiplex.cpp:462:21: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<Barcode>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  462 |   for (int b = 0; b < vec_bc.size(); b++) {
      |                   ~~^~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:477:32: warning: comparison of integer expressions of different signedness: ‘const int’ and ‘const std::__cxx11::basic_string<char>::size_type’ {aka ‘const long unsigned int’} [-Wsign-compare]
  477 |     if (vec_bc.at(b).flank_end == std::string::npos) {
      |         ~~~~~~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:482:23: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<Barcode>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  482 |     for (int f = 0; f < vec_bc.size(); f++) {
      |                     ~~^~~~~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘Rcpp::IntegerVector flexiplex_cpp(Rcpp::StringVector, Rcpp::String, bool, int, int, Rcpp::StringVector, Rcpp::String, Rcpp::String, Rcpp::String, bool, int)’:
main-functions/flexiplex.cpp:740:25: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<std::vector<SearchResult> >::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  740 |       for (int t = 0; t < sr_v.size();
      |                       ~~^~~~~~~~~~~~~
main-functions/flexiplex.cpp:745:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<SearchResult>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  745 |         for (int r = 0; r < sr_v[t].size(); r++) { // loop over the reads
      |                         ~~^~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:747:29: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<Barcode>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  747 |           for (int b = 0; b < sr_v[t][r].vec_bc_for.size(); b++)
      |                           ~~^~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:749:29: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<Barcode>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  749 |           for (int b = 0; b < sr_v[t][r].vec_bc_rev.size(); b++)
      |                           ~~^~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c main-functions/get_transcript_seq.cpp -o main-functions/get_transcript_seq.o
main-functions/get_transcript_seq.cpp: In function ‘void write_fa(const std::string&, const std::string&, const std::string&, int)’:
main-functions/get_transcript_seq.cpp:87:14: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
   87 |     while (i < seq.size()) {
      |            ~~^~~~~~~~~~~~
main-functions/get_transcript_seq.cpp:88:26: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
   88 |         if (i + wrap_len > seq.size()) {
      |             ~~~~~~~~~~~~~^~~~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c main-functions/group_bam2isoform.cpp -o main-functions/group_bam2isoform.o
In file included from main-functions/group_bam2isoform.cpp:18:
main-functions/../utility/utility.h: In function ‘std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > parseSpace(const std::string&)’:
main-functions/../utility/utility.h:255:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  255 |         for (int i = 0; i < s.size(); i++) {
      |                         ~~^~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c main-functions/pileup_readid.cpp -o main-functions/pileup_readid.o
main-functions/pileup_readid.cpp: In function ‘Rcpp::NumericMatrix variant_count_matrix_cpp(Rcpp::String, Rcpp::String, int, bool, bool)’:
main-functions/pileup_readid.cpp:268:21: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  268 |   for (int i = 0; i < BASES.size(); i++) {
      |                   ~~^~~~~~~~~~~~~~
main-functions/pileup_readid.cpp: In instantiation of ‘std::unordered_map<std::__cxx11::basic_string<char>, std::vector<std::vector<std::__cxx11::basic_string<char> > > > group_umis(const TableType&, int) [with TableType = std::unordered_map<std::__cxx11::basic_string<char>, std::unordered_map<std::__cxx11::basic_string<char>, std::unordered_map<std::__cxx11::basic_string<char>, unsigned int> > >]’:
main-functions/pileup_readid.cpp:342:30:   required from here
main-functions/pileup_readid.cpp:86:16: warning: unused variable ‘end’ [-Wunused-variable]
   86 |   unsigned int end;
      |                ^~~
main-functions/pileup_readid.cpp: In instantiation of ‘std::unordered_map<std::__cxx11::basic_string<char>, std::vector<std::vector<std::__cxx11::basic_string<char> > > > group_umis(const TableType&, int) [with TableType = std::unordered_map<std::__cxx11::basic_string<char>, std::unordered_map<std::__cxx11::basic_string<char>, std::array<unsigned int, 5> > >]’:
main-functions/pileup_readid.cpp:344:30:   required from here
main-functions/pileup_readid.cpp:86:16: warning: unused variable ‘end’ [-Wunused-variable]
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c tests/test-junctions.cpp -o tests/test-junctions.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c tests/test-parsing.cpp -o tests/test-parsing.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c utility/cigars.cpp -o utility/cigars.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c utility/edlib-1.2.7/edlib.cpp -o utility/edlib-1.2.7/edlib.o
gcc -std=gnu2x -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security -c utility/bam.c -o utility/bam.o
g++ -std=gnu++17 -shared -L/home/biocbuild/bbs-3.22-bioc/R/lib -L/usr/local/lib -o FLAMES.so RcppExports.o RcppFunctions.o classes/BamRecord.o classes/GFFRecord.o classes/GeneAnnotationParser.o classes/Isoforms.o classes/junctions.o main-functions/find_isoform.o main-functions/flexiplex.o main-functions/get_transcript_seq.o main-functions/group_bam2isoform.o main-functions/pileup_readid.o tests/test-junctions.o tests/test-parsing.o utility/cigars.o utility/edlib-1.2.7/edlib.o utility/bam.o -pthread -lz /home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/usrlib/libhts.a -lcurl -lbz2 -llzma -lz -L/home/biocbuild/bbs-3.22-bioc/R/lib -lR
if test -e "/usr/bin/strip" & test -e "/bin/uname" & [[ `uname` == "Linux" ]] ; then /usr/bin/strip --strip-debug FLAMES.so; fi
Building for x86_64
(cd submodule/minimap2 && make -f Makefile CFLAGS="-g -O2  -Wall -Werror=format-security -Wno-unused-result" minimap2)
make[1]: Entering directory '/home/biocbuild/bbs-3.22-bioc/meat/FLAMES/src/submodule/minimap2'
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  main.c -o main.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  kthread.c -o kthread.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  kalloc.c -o kalloc.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  misc.c -o misc.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  bseq.c -o bseq.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  sketch.c -o sketch.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  sdust.c -o sdust.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  options.c -o options.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  index.c -o index.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  lchain.c -o lchain.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  align.c -o align.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  hit.c -o hit.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  seed.c -o seed.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  jump.c -o jump.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  map.c -o map.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  format.c -o format.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  pe.c -o pe.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  esterr.c -o esterr.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  splitidx.c -o splitidx.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse2 -DHAVE_KALLOC  ksw2_ll_sse.c -o ksw2_ll_sse.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH  ksw2_extz2_sse.c -o ksw2_extz2_sse41.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH  ksw2_extd2_sse.c -o ksw2_extd2_sse41.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH  ksw2_exts2_sse.c -o ksw2_exts2_sse41.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse2 -mno-sse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH -DKSW_SSE2_ONLY  ksw2_extz2_sse.c -o ksw2_extz2_sse2.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse2 -mno-sse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH -DKSW_SSE2_ONLY  ksw2_extd2_sse.c -o ksw2_extd2_sse2.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse2 -mno-sse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH -DKSW_SSE2_ONLY  ksw2_exts2_sse.c -o ksw2_exts2_sse2.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH  ksw2_dispatch.c -o ksw2_dispatch.o
ar -csru libminimap2.a kthread.o kalloc.o misc.o bseq.o sketch.o sdust.o options.o index.o lchain.o align.o hit.o seed.o jump.o map.o format.o pe.o esterr.o splitidx.o ksw2_ll_sse.o ksw2_extz2_sse41.o ksw2_extd2_sse41.o ksw2_exts2_sse41.o ksw2_extz2_sse2.o ksw2_extd2_sse2.o ksw2_exts2_sse2.o ksw2_dispatch.o
ar: `u' modifier ignored since `D' is the default (see `U')
cc -g -O2  -Wall -Werror=format-security -Wno-unused-result main.o -o minimap2 -L. -lminimap2 -lm -lz -lpthread
make[1]: Leaving directory '/home/biocbuild/bbs-3.22-bioc/meat/FLAMES/src/submodule/minimap2'
echo "Installing binary to /home/biocbuild/bbs-3.22-bioc/meat/FLAMES/src/../inst/bin"
Installing binary to /home/biocbuild/bbs-3.22-bioc/meat/FLAMES/src/../inst/bin
mkdir -p ../inst/bin
cp submodule/minimap2/minimap2 ../inst/bin/
Rust version too old for edition 2024, skipping oarfish installation.
installing to /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/libs
** R
** data
** inst
** byte-compile and prepare package for lazy loading
** help
*** installing help indices
*** copying figures
** building package indices
** installing vignettes
** testing if installed package can be loaded
* DONE (FLAMES)

R version 4.5.1 (2025-06-13) -- "Great Square Root"
Copyright (C) 2025 The R Foundation for Statistical Computing
Platform: x86_64-pc-linux-gnu

R is free software and comes with ABSOLUTELY NO WARRANTY.
You are welcome to redistribute it under certain conditions.
Type 'license()' or 'licence()' for distribution details.

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Type 'contributors()' for more information and
'citation()' on how to cite R or R packages in publications.

Type 'demo()' for some demos, 'help()' for on-line help, or
'help.start()' for an HTML browser interface to help.
Type 'q()' to quit R.

> # This file is part of the standard setup for testthat.
> # It is recommended that you do not modify it.
> #
> # Where should you do additional test configuration?
> # Learn more about the roles of various files in:
> # * https://r-pkgs.org/tests.html
> # * https://testthat.r-lib.org/reference/test_package.html#special-files
> 
> library(testthat)
> library(FLAMES)
> 
> test_check("FLAMES")
Writing configuration parameters to:  /tmp/RtmpwBZQAu/file9c8885f054bcc/config_file_641160.json 
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpwBZQAu/file9c888497833f1/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Skipping TSO trimming...
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpwBZQAu/file9c88811631665/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Skipping TSO trimming...
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpwBZQAu/file9c88811631665/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpwBZQAu/file9c888465018be/musc_rps24_1.fastq
Searching for barcodes...
Processing file: /tmp/RtmpwBZQAu/file9c888465018be/musc_rps24_2.fastq
Searching for barcodes...
Processing file: /tmp/RtmpwBZQAu/file9c888465018be/musc_rps24_3.fastq
Searching for barcodes...
Processing file: /tmp/RtmpwBZQAu/file9c888465018be/musc_rps24_4.fastq
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Skipping TSO trimming...
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpwBZQAu/file9c8885fd593dc/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Skipping TSO trimming...
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpwBZQAu/file9c8885fd593dc/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpwBZQAu/file9c8885a46f36d/musc_rps24_1.fastq
Searching for barcodes...
Processing file: /tmp/RtmpwBZQAu/file9c8885a46f36d/musc_rps24_2.fastq
Searching for barcodes...
Processing file: /tmp/RtmpwBZQAu/file9c8885a46f36d/musc_rps24_3.fastq
Searching for barcodes...
Processing file: /tmp/RtmpwBZQAu/file9c8885a46f36d/musc_rps24_4.fastq
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpwBZQAu/file9c8885fd593dc/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Skipping TSO trimming...
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpwBZQAu/file9c888768f7535/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Skipping TSO trimming...
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpwBZQAu/file9c88878be47ce/config_file_641160.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Fri Aug  8 22:36:01 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpwBZQAu/file9c88878be47ce/sample1_align2genome.bam
[M::mm_idx_gen::0.001*1.66] collected minimizers
[M::mm_idx_gen::0.002*1.46] sorted minimizers
[M::main::0.002*1.45] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*1.42] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*1.40] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.171*1.01] mapped 100 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/RtmpwBZQAu/file9c88878be47ce/reference.bed --junc-bonus 1 /tmp/RtmpwBZQAu/file9c88878be47ce/rps24.fa /tmp/RtmpwBZQAu/file9c88878be47ce/fastq/sample1.fq.gz
[M::main] Real time: 0.171 sec; CPU: 0.172 sec; Peak RSS: 0.006 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpwBZQAu/file9c88878be47ce/sample2_align2genome.bam
[M::mm_idx_gen::0.001*1.01] collected minimizers
[M::mm_idx_gen::0.002*1.01] sorted minimizers
[M::main::0.002*1.01] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*1.01] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*1.01] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.171*1.00] mapped 100 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/RtmpwBZQAu/file9c88878be47ce/reference.bed --junc-bonus 1 /tmp/RtmpwBZQAu/file9c88878be47ce/rps24.fa /tmp/RtmpwBZQAu/file9c88878be47ce/fastq/sample2.fq.gz
[M::main] Real time: 0.172 sec; CPU: 0.172 sec; Peak RSS: 0.004 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpwBZQAu/file9c88878be47ce/sample3_align2genome.bam
[M::mm_idx_gen::0.001*1.57] collected minimizers
[M::mm_idx_gen::0.002*1.37] sorted minimizers
[M::main::0.002*1.37] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*1.34] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*1.33] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.332*1.00] mapped 193 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/RtmpwBZQAu/file9c88878be47ce/reference.bed --junc-bonus 1 /tmp/RtmpwBZQAu/file9c88878be47ce/rps24.fa /tmp/RtmpwBZQAu/file9c88878be47ce/fastq/sample3.fq.gz
[M::main] Real time: 0.333 sec; CPU: 0.333 sec; Peak RSS: 0.006 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Fri Aug  8 22:36:04 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

  |                                                                            
  |                                                                      |   0%[22:36:11] WARNING: src/learner.cc:553: 
  If you are loading a serialized model (like pickle in Python, RDS in R) generated by
  older XGBoost, please export the model by calling `Booster.save_model` from that version
  first, then load it back in current version. See:

    https://xgboost.readthedocs.io/en/latest/tutorials/saving_model.html

  for more details about differences between saving model and serializing.

[22:36:11] WARNING: src/learner.cc:553: 
  If you are loading a serialized model (like pickle in Python, RDS in R) generated by
  older XGBoost, please export the model by calling `Booster.save_model` from that version
  first, then load it back in current version. See:

    https://xgboost.readthedocs.io/en/latest/tutorials/saving_model.html

  for more details about differences between saving model and serializing.

[22:36:11] WARNING: src/learner.cc:553: 
  If you are loading a serialized model (like pickle in Python, RDS in R) generated by
  older XGBoost, please export the model by calling `Booster.save_model` from that version
  first, then load it back in current version. See:

    https://xgboost.readthedocs.io/en/latest/tutorials/saving_model.html

  for more details about differences between saving model and serializing.

[22:36:11] WARNING: src/learner.cc:553: 
  If you are loading a serialized model (like pickle in Python, RDS in R) generated by
  older XGBoost, please export the model by calling `Booster.save_model` from that version
  first, then load it back in current version. See:

    https://xgboost.readthedocs.io/en/latest/tutorials/saving_model.html

  for more details about differences between saving model and serializing.

[22:36:13] WARNING: src/learner.cc:553: 
  If you are loading a serialized model (like pickle in Python, RDS in R) generated by
  older XGBoost, please export the model by calling `Booster.save_model` from that version
  first, then load it back in current version. See:

    https://xgboost.readthedocs.io/en/latest/tutorials/saving_model.html

  for more details about differences between saving model and serializing.

[22:36:13] WARNING: src/learner.cc:553: 
  If you are loading a serialized model (like pickle in Python, RDS in R) generated by
  older XGBoost, please export the model by calling `Booster.save_model` from that version
  first, then load it back in current version. See:

    https://xgboost.readthedocs.io/en/latest/tutorials/saving_model.html

  for more details about differences between saving model and serializing.


  |                                                                            
  |=======================                                               |  33%
  |                                                                            
  |===============================================                       |  67%
  |                                                                            
  |======================================================================| 100%

Detected 4 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
-- Running step: read_realignment @ Fri Aug  8 22:36:31 2025 -------------------
Realigning sample sample1 -> /tmp/RtmpwBZQAu/file9c88878be47ce/sample1_realign2transcript.bam
[M::mm_idx_gen::0.001*1.00] collected minimizers
[M::mm_idx_gen::0.001*1.00] sorted minimizers
[M::main::0.001*1.00] loaded/built the index for 5 target sequence(s)
[M::mm_mapopt_update::0.001*1.00] mid_occ = 10
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 5
[M::mm_idx_stat::0.001*1.00] distinct minimizers: 198 (33.33% are singletons); average occurrences: 2.884; average spacing: 5.196; total length: 2967
[M::worker_pipeline::0.082*1.00] mapped 100 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 --eqx -N 100 -ax map-ont -t 1 --seed 2022 /tmp/RtmpwBZQAu/file9c88878be47ce/transcript_assembly.fa /tmp/RtmpwBZQAu/file9c88878be47ce/fastq/sample1.fq.gz
[M::main] Real time: 0.082 sec; CPU: 0.082 sec; Peak RSS: 0.004 GB
Skipped sorting BAM files.

Realigning sample sample2 -> /tmp/RtmpwBZQAu/file9c88878be47ce/sample2_realign2transcript.bam
[M::mm_idx_gen::0.001*2.02] collected minimizers
[M::mm_idx_gen::0.001*1.80] sorted minimizers
[M::main::0.001*1.79] loaded/built the index for 5 target sequence(s)
[M::mm_mapopt_update::0.001*1.76] mid_occ = 10
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 5
[M::mm_idx_stat::0.001*1.72] distinct minimizers: 198 (33.33% are singletons); average occurrences: 2.884; average spacing: 5.196; total length: 2967
[M::worker_pipeline::0.087*1.01] mapped 100 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 --eqx -N 100 -ax map-ont -t 1 --seed 2022 /tmp/RtmpwBZQAu/file9c88878be47ce/transcript_assembly.fa /tmp/RtmpwBZQAu/file9c88878be47ce/fastq/sample2.fq.gz
[M::main] Real time: 0.088 sec; CPU: 0.089 sec; Peak RSS: 0.004 GB
Skipped sorting BAM files.

Realigning sample sample3 -> /tmp/RtmpwBZQAu/file9c88878be47ce/sample3_realign2transcript.bam
[M::mm_idx_gen::0.001*1.22] collected minimizers
[M::mm_idx_gen::0.001*1.16] sorted minimizers
[M::main::0.001*1.16] loaded/built the index for 5 target sequence(s)
[M::mm_mapopt_update::0.001*1.15] mid_occ = 10
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 5
[M::mm_idx_stat::0.001*1.15] distinct minimizers: 198 (33.33% are singletons); average occurrences: 2.884; average spacing: 5.196; total length: 2967
[M::worker_pipeline::0.164*1.00] mapped 193 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 --eqx -N 100 -ax map-ont -t 1 --seed 2022 /tmp/RtmpwBZQAu/file9c88878be47ce/transcript_assembly.fa /tmp/RtmpwBZQAu/file9c88878be47ce/fastq/sample3.fq.gz
[M::main] Real time: 0.164 sec; CPU: 0.164 sec; Peak RSS: 0.004 GB
Skipped sorting BAM files.

-- Running step: transcript_quantification @ Fri Aug  8 22:36:31 2025 ----------
2025-08-09T02:36:31.888239Z  INFO oarfish: setting user-provided filter parameters.
2025-08-09T02:36:31.888698Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpwBZQAu/file9c88878be47ce/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-08-09T02:36:31.888707Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-09T02:36:31.888710Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-09T02:36:31.888771Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-09T02:36:31.888777Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-08-09T02:36:31.890308Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-08-09T02:36:31.890436Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 253   │
│ aligned fraction too low        │ 4     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 1     │
│ reads with valid best alignment │ 96    │
╰─────────────────────────────────┴───────╯

2025-08-09T02:36:31.890461Z  INFO oarfish::bulk: Total number of alignment records : 116
2025-08-09T02:36:31.890463Z  INFO oarfish::bulk: number of aligned reads : 96
2025-08-09T02:36:31.890465Z  INFO oarfish::bulk: number of unique alignments : 86
2025-08-09T02:36:31.891053Z  INFO oarfish: oarfish completed successfully.
2025-08-09T02:36:31.898055Z  INFO oarfish: setting user-provided filter parameters.
2025-08-09T02:36:31.898516Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpwBZQAu/file9c88878be47ce/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-08-09T02:36:31.898527Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-09T02:36:31.898531Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-09T02:36:31.898589Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-09T02:36:31.898594Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-08-09T02:36:31.900159Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-08-09T02:36:31.900275Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 251   │
│ aligned fraction too low        │ 5     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 2     │
│ reads with valid best alignment │ 95    │
╰─────────────────────────────────┴───────╯

2025-08-09T02:36:31.900294Z  INFO oarfish::bulk: Total number of alignment records : 114
2025-08-09T02:36:31.900297Z  INFO oarfish::bulk: number of aligned reads : 95
2025-08-09T02:36:31.900299Z  INFO oarfish::bulk: number of unique alignments : 82
2025-08-09T02:36:31.900871Z  INFO oarfish: oarfish completed successfully.
2025-08-09T02:36:31.907536Z  INFO oarfish: setting user-provided filter parameters.
2025-08-09T02:36:31.907949Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpwBZQAu/file9c88878be47ce/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-08-09T02:36:31.907958Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-09T02:36:31.907961Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-09T02:36:31.908008Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-09T02:36:31.908013Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-08-09T02:36:31.910692Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2025-08-09T02:36:31.910836Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 398   │
│ aligned fraction too low        │ 12    │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 5     │
│ reads with valid best alignment │ 179   │
╰─────────────────────────────────┴───────╯

2025-08-09T02:36:31.910862Z  INFO oarfish::bulk: Total number of alignment records : 239
2025-08-09T02:36:31.910865Z  INFO oarfish::bulk: number of aligned reads : 179
2025-08-09T02:36:31.910867Z  INFO oarfish::bulk: number of unique alignments : 143
2025-08-09T02:36:31.911547Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpwBZQAu/file9c88859760b6a/config_file_641160.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Fri Aug  8 22:36:32 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpwBZQAu/file9c88859760b6a/sample1_align2genome.bam
sample2 ->/tmp/RtmpwBZQAu/file9c88859760b6a/sample2_align2genome.bam
sample3 ->/tmp/RtmpwBZQAu/file9c88859760b6a/sample3_align2genome.bam
-- Running step: isoform_identification @ Fri Aug  8 22:36:55 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

  |                                                                            
  |                                                                      |   0%
  |                                                                            
  |=======================                                               |  33%
  |                                                                            
  |===============================================                       |  67%
  |                                                                            
  |======================================================================| 100%

Detected 4 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
-- Running step: read_realignment @ Fri Aug  8 22:37:14 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpwBZQAu/file9c88859760b6a/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpwBZQAu/file9c88859760b6a/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpwBZQAu/file9c88859760b6a/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Fri Aug  8 22:37:36 2025 ----------
2025-08-09T02:37:36.354409Z  INFO oarfish: setting user-provided filter parameters.
2025-08-09T02:37:36.354764Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpwBZQAu/file9c88859760b6a/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-08-09T02:37:36.354774Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-09T02:37:36.354778Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-09T02:37:36.354834Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-09T02:37:36.354840Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-08-09T02:37:36.356310Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-08-09T02:37:36.356428Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 253   │
│ aligned fraction too low        │ 4     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 1     │
│ reads with valid best alignment │ 96    │
╰─────────────────────────────────┴───────╯

2025-08-09T02:37:36.356457Z  INFO oarfish::bulk: Total number of alignment records : 116
2025-08-09T02:37:36.356460Z  INFO oarfish::bulk: number of aligned reads : 96
2025-08-09T02:37:36.356462Z  INFO oarfish::bulk: number of unique alignments : 86
2025-08-09T02:37:36.357030Z  INFO oarfish: oarfish completed successfully.
2025-08-09T02:37:36.363955Z  INFO oarfish: setting user-provided filter parameters.
2025-08-09T02:37:36.364320Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpwBZQAu/file9c88859760b6a/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-08-09T02:37:36.364331Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-09T02:37:36.364334Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-09T02:37:36.364392Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-09T02:37:36.364397Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-08-09T02:37:36.365911Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-08-09T02:37:36.366026Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 251   │
│ aligned fraction too low        │ 5     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 2     │
│ reads with valid best alignment │ 95    │
╰─────────────────────────────────┴───────╯

2025-08-09T02:37:36.366050Z  INFO oarfish::bulk: Total number of alignment records : 114
2025-08-09T02:37:36.366052Z  INFO oarfish::bulk: number of aligned reads : 95
2025-08-09T02:37:36.366054Z  INFO oarfish::bulk: number of unique alignments : 82
2025-08-09T02:37:36.366620Z  INFO oarfish: oarfish completed successfully.
2025-08-09T02:37:36.373516Z  INFO oarfish: setting user-provided filter parameters.
2025-08-09T02:37:36.373845Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpwBZQAu/file9c88859760b6a/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-08-09T02:37:36.373853Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-09T02:37:36.373866Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-09T02:37:36.373916Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-09T02:37:36.373921Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-08-09T02:37:36.376484Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2025-08-09T02:37:36.376628Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 398   │
│ aligned fraction too low        │ 12    │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 5     │
│ reads with valid best alignment │ 179   │
╰─────────────────────────────────┴───────╯

2025-08-09T02:37:36.376654Z  INFO oarfish::bulk: Total number of alignment records : 239
2025-08-09T02:37:36.376656Z  INFO oarfish::bulk: number of aligned reads : 179
2025-08-09T02:37:36.376659Z  INFO oarfish::bulk: number of unique alignments : 143
2025-08-09T02:37:36.377311Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpwBZQAu/file9c88859fa41f1/config_file_641160.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Fri Aug  8 22:37:36 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpwBZQAu/file9c88859fa41f1/sample1_align2genome.bam
[M::mm_idx_gen::0.001*1.10] collected minimizers
[M::mm_idx_gen::0.002*1.07] sorted minimizers
[M::main::0.002*1.07] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*1.06] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*1.06] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.169*1.00] mapped 100 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/RtmpwBZQAu/file9c88859fa41f1/reference.bed --junc-bonus 1 /tmp/RtmpwBZQAu/file9c88859fa41f1/rps24.fa /tmp/RtmpwBZQAu/file9c88859fa41f1/fastq/sample1.fq.gz
[M::main] Real time: 0.170 sec; CPU: 0.170 sec; Peak RSS: 0.006 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpwBZQAu/file9c88859fa41f1/sample2_align2genome.bam
[M::mm_idx_gen::0.003*1.22] collected minimizers
[M::mm_idx_gen::0.003*1.18] sorted minimizers
[M::main::0.003*1.17] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.004*1.17] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.004*1.16] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.173*1.00] mapped 100 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/RtmpwBZQAu/file9c88859fa41f1/reference.bed --junc-bonus 1 /tmp/RtmpwBZQAu/file9c88859fa41f1/rps24.fa /tmp/RtmpwBZQAu/file9c88859fa41f1/fastq/sample2.fq.gz
[M::main] Real time: 0.174 sec; CPU: 0.175 sec; Peak RSS: 0.004 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpwBZQAu/file9c88859fa41f1/sample3_align2genome.bam
[M::mm_idx_gen::0.001*0.91] collected minimizers
[M::mm_idx_gen::0.002*0.94] sorted minimizers
[M::main::0.002*0.94] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*0.94] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*0.94] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.331*1.00] mapped 193 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/RtmpwBZQAu/file9c88859fa41f1/reference.bed --junc-bonus 1 /tmp/RtmpwBZQAu/file9c88859fa41f1/rps24.fa /tmp/RtmpwBZQAu/file9c88859fa41f1/fastq/sample3.fq.gz
[M::main] Real time: 0.332 sec; CPU: 0.332 sec; Peak RSS: 0.006 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Fri Aug  8 22:37:37 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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Detected 4 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
-- Running step: read_realignment @ Fri Aug  8 22:37:56 2025 -------------------
Realigning sample sample1 -> /tmp/RtmpwBZQAu/file9c88859fa41f1/sample1_realign2transcript.bam
[M::mm_idx_gen::0.002*1.51] collected minimizers
[M::mm_idx_gen::0.002*1.45] sorted minimizers
[M::main::0.002*1.45] loaded/built the index for 5 target sequence(s)
[M::mm_mapopt_update::0.002*1.44] mid_occ = 10
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 5
[M::mm_idx_stat::0.002*1.43] distinct minimizers: 198 (33.33% are singletons); average occurrences: 2.884; average spacing: 5.196; total length: 2967
[M::worker_pipeline::0.071*1.01] mapped 100 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax map-ont -p 0.9 --end-bonus 10 -N 3 -t 1 --seed 2022 /tmp/RtmpwBZQAu/file9c88859fa41f1/transcript_assembly.fa /tmp/RtmpwBZQAu/file9c88859fa41f1/fastq/sample1.fq.gz
[M::main] Real time: 0.071 sec; CPU: 0.072 sec; Peak RSS: 0.003 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample2 -> /tmp/RtmpwBZQAu/file9c88859fa41f1/sample2_realign2transcript.bam
[M::mm_idx_gen::0.001*1.61] collected minimizers
[M::mm_idx_gen::0.001*1.48] sorted minimizers
[M::main::0.001*1.47] loaded/built the index for 5 target sequence(s)
[M::mm_mapopt_update::0.001*1.45] mid_occ = 10
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 5
[M::mm_idx_stat::0.001*1.43] distinct minimizers: 198 (33.33% are singletons); average occurrences: 2.884; average spacing: 5.196; total length: 2967
[M::worker_pipeline::0.070*1.01] mapped 100 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax map-ont -p 0.9 --end-bonus 10 -N 3 -t 1 --seed 2022 /tmp/RtmpwBZQAu/file9c88859fa41f1/transcript_assembly.fa /tmp/RtmpwBZQAu/file9c88859fa41f1/fastq/sample2.fq.gz
[M::main] Real time: 0.070 sec; CPU: 0.071 sec; Peak RSS: 0.003 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample3 -> /tmp/RtmpwBZQAu/file9c88859fa41f1/sample3_realign2transcript.bam
[M::mm_idx_gen::0.001*0.96] collected minimizers
[M::mm_idx_gen::0.001*0.97] sorted minimizers
[M::main::0.001*0.97] loaded/built the index for 5 target sequence(s)
[M::mm_mapopt_update::0.001*0.97] mid_occ = 10
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 5
[M::mm_idx_stat::0.001*0.97] distinct minimizers: 198 (33.33% are singletons); average occurrences: 2.884; average spacing: 5.196; total length: 2967
[M::worker_pipeline::0.137*1.00] mapped 193 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax map-ont -p 0.9 --end-bonus 10 -N 3 -t 1 --seed 2022 /tmp/RtmpwBZQAu/file9c88859fa41f1/transcript_assembly.fa /tmp/RtmpwBZQAu/file9c88859fa41f1/fastq/sample3.fq.gz
[M::main] Real time: 0.137 sec; CPU: 0.137 sec; Peak RSS: 0.004 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Fri Aug  8 22:37:56 2025 ----------
22:37:56 Fri Aug 08 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpwBZQAu/file9c8883751e8ba/config_file_641160.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Fri Aug  8 22:37:57 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpwBZQAu/file9c8883751e8ba/sample1_align2genome.bam
sample2 ->/tmp/RtmpwBZQAu/file9c8883751e8ba/sample2_align2genome.bam
sample3 ->/tmp/RtmpwBZQAu/file9c8883751e8ba/sample3_align2genome.bam
-- Running step: isoform_identification @ Fri Aug  8 22:38:19 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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Detected 4 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
-- Running step: read_realignment @ Fri Aug  8 22:38:38 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpwBZQAu/file9c8883751e8ba/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpwBZQAu/file9c8883751e8ba/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpwBZQAu/file9c8883751e8ba/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Fri Aug  8 22:39:01 2025 ----------
22:39:01 Fri Aug 08 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Inputs:  ['/tmp/RtmpwBZQAu/file9c88859fa41f1/sample1_realign2transcript.bam', '/tmp/RtmpwBZQAu/file9c88859fa41f1/sample2_realign2transcript.bam', '/tmp/RtmpwBZQAu/file9c88859fa41f1/sample3_realign2transcript.bam'] /tmp/RtmpwBZQAu/file9c88859fa41f1/transcript_assembly.fa.fai 5 0.4 0.4
	Counter({'counted_reads': 375, 'not_enough_coverage': 16, 'unmapped': 2})
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpwBZQAu/file9c88831d01e92/config_file_641160.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Fri Aug  8 22:39:01 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpwBZQAu/file9c88831d01e92/sample1_align2genome.bam
[M::mm_idx_gen::0.001*1.44] collected minimizers
[M::mm_idx_gen::0.002*1.30] sorted minimizers
[M::main::0.002*1.30] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*1.28] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*1.26] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.169*1.00] mapped 100 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/RtmpwBZQAu/file9c88831d01e92/reference.bed --junc-bonus 1 /tmp/RtmpwBZQAu/file9c88831d01e92/rps24.fa /tmp/RtmpwBZQAu/file9c88831d01e92/fastq/sample1.fq.gz
[M::main] Real time: 0.169 sec; CPU: 0.170 sec; Peak RSS: 0.005 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpwBZQAu/file9c88831d01e92/sample2_align2genome.bam
[M::mm_idx_gen::0.001*1.26] collected minimizers
[M::mm_idx_gen::0.002*1.17] sorted minimizers
[M::main::0.002*1.17] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*1.16] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*1.15] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.170*1.00] mapped 100 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/RtmpwBZQAu/file9c88831d01e92/reference.bed --junc-bonus 1 /tmp/RtmpwBZQAu/file9c88831d01e92/rps24.fa /tmp/RtmpwBZQAu/file9c88831d01e92/fastq/sample2.fq.gz
[M::main] Real time: 0.170 sec; CPU: 0.170 sec; Peak RSS: 0.004 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpwBZQAu/file9c88831d01e92/sample3_align2genome.bam
[M::mm_idx_gen::0.002*1.28] collected minimizers
[M::mm_idx_gen::0.003*1.22] sorted minimizers
[M::main::0.003*1.22] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.003*1.21] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.003*1.20] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.333*1.00] mapped 193 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/RtmpwBZQAu/file9c88831d01e92/reference.bed --junc-bonus 1 /tmp/RtmpwBZQAu/file9c88831d01e92/rps24.fa /tmp/RtmpwBZQAu/file9c88831d01e92/fastq/sample3.fq.gz
[M::main] Real time: 0.334 sec; CPU: 0.335 sec; Peak RSS: 0.007 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Fri Aug  8 22:39:02 2025 -------------
Inputs:  ['/tmp/RtmpwBZQAu/file9c8883751e8ba/sample1_realign2transcript.bam', '/tmp/RtmpwBZQAu/file9c8883751e8ba/sample2_realign2transcript.bam', '/tmp/RtmpwBZQAu/file9c8883751e8ba/sample3_realign2transcript.bam'] /tmp/RtmpwBZQAu/file9c8883751e8ba/transcript_assembly.fa.fai 5 0.4 0.4
	Counter({'counted_reads': 375, 'not_enough_coverage': 16, 'unmapped': 2})
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
-- Running step: read_realignment @ Fri Aug  8 22:39:03 2025 -------------------
Realigning sample sample1 -> /tmp/RtmpwBZQAu/file9c88831d01e92/sample1_realign2transcript.bam
[M::mm_idx_gen::0.003*1.26] collected minimizers
[M::mm_idx_gen::0.003*1.23] sorted minimizers
[M::main::0.003*1.23] loaded/built the index for 10 target sequence(s)
[M::mm_mapopt_update::0.003*1.22] mid_occ = 11
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 10
[M::mm_idx_stat::0.003*1.21] distinct minimizers: 611 (72.01% are singletons); average occurrences: 2.597; average spacing: 5.171; total length: 8207
[M::worker_pipeline::0.175*1.00] mapped 100 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 --eqx -N 100 -ax map-ont -t 1 --seed 2022 /tmp/RtmpwBZQAu/file9c88831d01e92/transcript_assembly.fa /tmp/RtmpwBZQAu/file9c88831d01e92/fastq/sample1.fq.gz
[M::main] Real time: 0.175 sec; CPU: 0.176 sec; Peak RSS: 0.004 GB
Skipped sorting BAM files.

Realigning sample sample2 -> /tmp/RtmpwBZQAu/file9c88831d01e92/sample2_realign2transcript.bam
[M::mm_idx_gen::0.001*1.20] collected minimizers
[M::mm_idx_gen::0.002*1.15] sorted minimizers
[M::main::0.002*1.15] loaded/built the index for 10 target sequence(s)
[M::mm_mapopt_update::0.002*1.15] mid_occ = 11
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 10
[M::mm_idx_stat::0.002*1.14] distinct minimizers: 611 (72.01% are singletons); average occurrences: 2.597; average spacing: 5.171; total length: 8207
[M::worker_pipeline::0.172*1.00] mapped 100 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 --eqx -N 100 -ax map-ont -t 1 --seed 2022 /tmp/RtmpwBZQAu/file9c88831d01e92/transcript_assembly.fa /tmp/RtmpwBZQAu/file9c88831d01e92/fastq/sample2.fq.gz
[M::main] Real time: 0.173 sec; CPU: 0.173 sec; Peak RSS: 0.003 GB
Skipped sorting BAM files.

Realigning sample sample3 -> /tmp/RtmpwBZQAu/file9c88831d01e92/sample3_realign2transcript.bam
[M::mm_idx_gen::0.001*1.05] collected minimizers
[M::mm_idx_gen::0.002*1.04] sorted minimizers
[M::main::0.002*1.04] loaded/built the index for 10 target sequence(s)
[M::mm_mapopt_update::0.002*1.04] mid_occ = 11
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 10
[M::mm_idx_stat::0.002*1.04] distinct minimizers: 611 (72.01% are singletons); average occurrences: 2.597; average spacing: 5.171; total length: 8207
[M::worker_pipeline::0.305*1.00] mapped 193 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 --eqx -N 100 -ax map-ont -t 1 --seed 2022 /tmp/RtmpwBZQAu/file9c88831d01e92/transcript_assembly.fa /tmp/RtmpwBZQAu/file9c88831d01e92/fastq/sample3.fq.gz
[M::main] Real time: 0.306 sec; CPU: 0.306 sec; Peak RSS: 0.006 GB
Skipped sorting BAM files.

-- Running step: transcript_quantification @ Fri Aug  8 22:39:04 2025 ----------
2025-08-09T02:39:04.517634Z  INFO oarfish: setting user-provided filter parameters.
2025-08-09T02:39:04.518150Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpwBZQAu/file9c88831d01e92/sample1_realign2transcript.bam, contains 10 reference sequences.
2025-08-09T02:39:04.518162Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-09T02:39:04.518165Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-09T02:39:04.518232Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-09T02:39:04.518238Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-08-09T02:39:04.520729Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-08-09T02:39:04.520871Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 707   │
│ aligned fraction too low        │ 2     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 98    │
╰─────────────────────────────────┴───────╯

2025-08-09T02:39:04.520895Z  INFO oarfish::bulk: Total number of alignment records : 125
2025-08-09T02:39:04.520898Z  INFO oarfish::bulk: number of aligned reads : 98
2025-08-09T02:39:04.520901Z  INFO oarfish::bulk: number of unique alignments : 86
2025-08-09T02:39:04.521511Z  INFO oarfish: oarfish completed successfully.
2025-08-09T02:39:04.528956Z  INFO oarfish: setting user-provided filter parameters.
2025-08-09T02:39:04.529323Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpwBZQAu/file9c88831d01e92/sample2_realign2transcript.bam, contains 10 reference sequences.
2025-08-09T02:39:04.529333Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-09T02:39:04.529336Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-09T02:39:04.529410Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-09T02:39:04.529416Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-08-09T02:39:04.531954Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-08-09T02:39:04.532095Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 701   │
│ aligned fraction too low        │ 3     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 97    │
╰─────────────────────────────────┴───────╯

2025-08-09T02:39:04.532123Z  INFO oarfish::bulk: Total number of alignment records : 136
2025-08-09T02:39:04.532126Z  INFO oarfish::bulk: number of aligned reads : 97
2025-08-09T02:39:04.532128Z  INFO oarfish::bulk: number of unique alignments : 79
2025-08-09T02:39:04.532707Z  INFO oarfish: oarfish completed successfully.
2025-08-09T02:39:04.540051Z  INFO oarfish: setting user-provided filter parameters.
2025-08-09T02:39:04.540411Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpwBZQAu/file9c88831d01e92/sample3_realign2transcript.bam, contains 10 reference sequences.
2025-08-09T02:39:04.540422Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-09T02:39:04.540425Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-09T02:39:04.540489Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-09T02:39:04.540497Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-08-09T02:39:04.544763Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-08-09T02:39:04.544920Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 1060  │
│ aligned fraction too low        │ 6     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 187   │
╰─────────────────────────────────┴───────╯

2025-08-09T02:39:04.544948Z  INFO oarfish::bulk: Total number of alignment records : 272
2025-08-09T02:39:04.544951Z  INFO oarfish::bulk: number of aligned reads : 187
2025-08-09T02:39:04.544953Z  INFO oarfish::bulk: number of unique alignments : 140
2025-08-09T02:39:04.545657Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpwBZQAu/file9c888dd4ff34/config_file_641160.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Fri Aug  8 22:39:04 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpwBZQAu/file9c888dd4ff34/sample1_align2genome.bam
sample2 ->/tmp/RtmpwBZQAu/file9c888dd4ff34/sample2_align2genome.bam
sample3 ->/tmp/RtmpwBZQAu/file9c888dd4ff34/sample3_align2genome.bam
-- Running step: isoform_identification @ Fri Aug  8 22:39:26 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
-- Running step: read_realignment @ Fri Aug  8 22:39:26 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpwBZQAu/file9c888dd4ff34/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpwBZQAu/file9c888dd4ff34/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpwBZQAu/file9c888dd4ff34/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Fri Aug  8 22:39:49 2025 ----------
2025-08-09T02:39:49.362660Z  INFO oarfish: setting user-provided filter parameters.
2025-08-09T02:39:49.363084Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpwBZQAu/file9c888dd4ff34/sample1_realign2transcript.bam, contains 10 reference sequences.
2025-08-09T02:39:49.363108Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-09T02:39:49.363111Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-09T02:39:49.363189Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-09T02:39:49.363196Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-08-09T02:39:49.365685Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-08-09T02:39:49.365822Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 707   │
│ aligned fraction too low        │ 2     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 98    │
╰─────────────────────────────────┴───────╯

2025-08-09T02:39:49.365850Z  INFO oarfish::bulk: Total number of alignment records : 125
2025-08-09T02:39:49.365853Z  INFO oarfish::bulk: number of aligned reads : 98
2025-08-09T02:39:49.365857Z  INFO oarfish::bulk: number of unique alignments : 86
2025-08-09T02:39:49.366475Z  INFO oarfish: oarfish completed successfully.
2025-08-09T02:39:49.374872Z  INFO oarfish: setting user-provided filter parameters.
2025-08-09T02:39:49.375376Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpwBZQAu/file9c888dd4ff34/sample2_realign2transcript.bam, contains 10 reference sequences.
2025-08-09T02:39:49.375389Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-09T02:39:49.375392Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-09T02:39:49.375467Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-09T02:39:49.375474Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-08-09T02:39:49.378075Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-08-09T02:39:49.378225Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 701   │
│ aligned fraction too low        │ 3     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 97    │
╰─────────────────────────────────┴───────╯

2025-08-09T02:39:49.378253Z  INFO oarfish::bulk: Total number of alignment records : 136
2025-08-09T02:39:49.378257Z  INFO oarfish::bulk: number of aligned reads : 97
2025-08-09T02:39:49.378259Z  INFO oarfish::bulk: number of unique alignments : 79
2025-08-09T02:39:49.378890Z  INFO oarfish: oarfish completed successfully.
2025-08-09T02:39:49.386520Z  INFO oarfish: setting user-provided filter parameters.
2025-08-09T02:39:49.386872Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpwBZQAu/file9c888dd4ff34/sample3_realign2transcript.bam, contains 10 reference sequences.
2025-08-09T02:39:49.386881Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-09T02:39:49.386883Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-09T02:39:49.386961Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-09T02:39:49.386968Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-08-09T02:39:49.391162Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-08-09T02:39:49.391329Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 1060  │
│ aligned fraction too low        │ 6     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 187   │
╰─────────────────────────────────┴───────╯

2025-08-09T02:39:49.391367Z  INFO oarfish::bulk: Total number of alignment records : 272
2025-08-09T02:39:49.391369Z  INFO oarfish::bulk: number of aligned reads : 187
2025-08-09T02:39:49.391371Z  INFO oarfish::bulk: number of unique alignments : 140
2025-08-09T02:39:49.392059Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpwBZQAu/file9c888f5a642c/config_file_641160.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Fri Aug  8 22:39:49 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpwBZQAu/file9c888f5a642c/sample1_align2genome.bam
[M::mm_idx_gen::0.002*1.69] collected minimizers
[M::mm_idx_gen::0.003*1.51] sorted minimizers
[M::main::0.003*1.50] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.003*1.47] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.003*1.45] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.171*1.01] mapped 100 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/RtmpwBZQAu/file9c888f5a642c/reference.bed --junc-bonus 1 /tmp/RtmpwBZQAu/file9c888f5a642c/rps24.fa /tmp/RtmpwBZQAu/file9c888f5a642c/fastq/sample1.fq.gz
[M::main] Real time: 0.172 sec; CPU: 0.173 sec; Peak RSS: 0.006 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpwBZQAu/file9c888f5a642c/sample2_align2genome.bam
[M::mm_idx_gen::0.001*1.83] collected minimizers
[M::mm_idx_gen::0.002*1.57] sorted minimizers
[M::main::0.002*1.56] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*1.52] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*1.50] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.171*1.01] mapped 100 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/RtmpwBZQAu/file9c888f5a642c/reference.bed --junc-bonus 1 /tmp/RtmpwBZQAu/file9c888f5a642c/rps24.fa /tmp/RtmpwBZQAu/file9c888f5a642c/fastq/sample2.fq.gz
[M::main] Real time: 0.172 sec; CPU: 0.174 sec; Peak RSS: 0.004 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpwBZQAu/file9c888f5a642c/sample3_align2genome.bam
[M::mm_idx_gen::0.001*1.56] collected minimizers
[M::mm_idx_gen::0.002*1.38] sorted minimizers
[M::main::0.002*1.38] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*1.35] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*1.34] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.332*1.00] mapped 193 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/RtmpwBZQAu/file9c888f5a642c/reference.bed --junc-bonus 1 /tmp/RtmpwBZQAu/file9c888f5a642c/rps24.fa /tmp/RtmpwBZQAu/file9c888f5a642c/fastq/sample3.fq.gz
[M::main] Real time: 0.333 sec; CPU: 0.334 sec; Peak RSS: 0.006 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Fri Aug  8 22:39:50 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
-- Running step: read_realignment @ Fri Aug  8 22:39:51 2025 -------------------
Realigning sample sample1 -> /tmp/RtmpwBZQAu/file9c888f5a642c/sample1_realign2transcript.bam
[M::mm_idx_gen::0.001*1.35] collected minimizers
[M::mm_idx_gen::0.002*1.26] sorted minimizers
[M::main::0.002*1.26] loaded/built the index for 10 target sequence(s)
[M::mm_mapopt_update::0.002*1.25] mid_occ = 11
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 10
[M::mm_idx_stat::0.002*1.24] distinct minimizers: 611 (72.01% are singletons); average occurrences: 2.597; average spacing: 5.171; total length: 8207
[M::worker_pipeline::0.093*1.00] mapped 100 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax map-ont -p 0.9 --end-bonus 10 -N 3 -t 1 --seed 2022 /tmp/RtmpwBZQAu/file9c888f5a642c/transcript_assembly.fa /tmp/RtmpwBZQAu/file9c888f5a642c/fastq/sample1.fq.gz
[M::main] Real time: 0.094 sec; CPU: 0.094 sec; Peak RSS: 0.004 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample2 -> /tmp/RtmpwBZQAu/file9c888f5a642c/sample2_realign2transcript.bam
[M::mm_idx_gen::0.001*0.83] collected minimizers
[M::mm_idx_gen::0.002*0.87] sorted minimizers
[M::main::0.002*0.87] loaded/built the index for 10 target sequence(s)
[M::mm_mapopt_update::0.002*0.88] mid_occ = 11
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 10
[M::mm_idx_stat::0.002*0.88] distinct minimizers: 611 (72.01% are singletons); average occurrences: 2.597; average spacing: 5.171; total length: 8207
[M::worker_pipeline::0.091*1.00] mapped 100 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax map-ont -p 0.9 --end-bonus 10 -N 3 -t 1 --seed 2022 /tmp/RtmpwBZQAu/file9c888f5a642c/transcript_assembly.fa /tmp/RtmpwBZQAu/file9c888f5a642c/fastq/sample2.fq.gz
[M::main] Real time: 0.092 sec; CPU: 0.092 sec; Peak RSS: 0.003 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample3 -> /tmp/RtmpwBZQAu/file9c888f5a642c/sample3_realign2transcript.bam
[M::mm_idx_gen::0.002*1.45] collected minimizers
[M::mm_idx_gen::0.002*1.38] sorted minimizers
[M::main::0.002*1.38] loaded/built the index for 10 target sequence(s)
[M::mm_mapopt_update::0.002*1.36] mid_occ = 11
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 10
[M::mm_idx_stat::0.002*1.35] distinct minimizers: 611 (72.01% are singletons); average occurrences: 2.597; average spacing: 5.171; total length: 8207
[M::worker_pipeline::0.175*1.00] mapped 193 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax map-ont -p 0.9 --end-bonus 10 -N 3 -t 1 --seed 2022 /tmp/RtmpwBZQAu/file9c888f5a642c/transcript_assembly.fa /tmp/RtmpwBZQAu/file9c888f5a642c/fastq/sample3.fq.gz
[M::main] Real time: 0.175 sec; CPU: 0.176 sec; Peak RSS: 0.004 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Fri Aug  8 22:39:51 2025 ----------
22:39:51 Fri Aug 08 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpwBZQAu/file9c8881213d884/config_file_641160.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Fri Aug  8 22:39:52 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpwBZQAu/file9c8881213d884/sample1_align2genome.bam
sample2 ->/tmp/RtmpwBZQAu/file9c8881213d884/sample2_align2genome.bam
sample3 ->/tmp/RtmpwBZQAu/file9c8881213d884/sample3_align2genome.bam
-- Running step: isoform_identification @ Fri Aug  8 22:40:14 2025 -------------
Inputs:  ['/tmp/RtmpwBZQAu/file9c888f5a642c/sample1_realign2transcript.bam', '/tmp/RtmpwBZQAu/file9c888f5a642c/sample2_realign2transcript.bam', '/tmp/RtmpwBZQAu/file9c888f5a642c/sample3_realign2transcript.bam'] /tmp/RtmpwBZQAu/file9c888f5a642c/transcript_assembly.fa.fai 5 0.4 0.4
	Counter({'counted_reads': 391, 'not_enough_coverage': 2})
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
-- Running step: read_realignment @ Fri Aug  8 22:40:15 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpwBZQAu/file9c8881213d884/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpwBZQAu/file9c8881213d884/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpwBZQAu/file9c8881213d884/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Fri Aug  8 22:40:38 2025 ----------
22:40:38 Fri Aug 08 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpwBZQAu/file9c8886ac5cac3/config_file_641160.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Fri Aug  8 22:40:38 2025 ----------------
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpwBZQAu/file9c8886ac5cac3/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
-- Running step: genome_alignment @ Fri Aug  8 22:40:39 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpwBZQAu/file9c8886ac5cac3/matched_reads.fastq.gz -> /tmp/RtmpwBZQAu/file9c8886ac5cac3/align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
[M::mm_idx_gen::0.001*1.70] collected minimizers
[M::mm_idx_gen::0.002*1.14] sorted minimizers
[M::main::0.002*1.14] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*1.12] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*1.12] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.094*7.22] mapped 372 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 8 --seed 2022 --junc-bed /tmp/RtmpwBZQAu/file9c8886ac5cac3/reference.bed --junc-bonus 1 /tmp/RtmpwBZQAu/file9c8886ac5cac3/rps24.fa /tmp/RtmpwBZQAu/file9c8886ac5cac3/matched_reads.fastq.gz
[M::main] Real time: 0.095 sec; CPU: 0.680 sec; Peak RSS: 0.020 GB
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: gene_quantification @ Fri Aug  8 22:40:39 2025 ----------------
22:40:39 Fri Aug 08 2025 quantify genes 
Using BAM(s): '/tmp/RtmpwBZQAu/file9c8886ac5cac3/align2genome.bam'
Inputs:  ['/tmp/RtmpwBZQAu/file9c8881213d884/sample1_realign2transcript.bam', '/tmp/RtmpwBZQAu/file9c8881213d884/sample2_realign2transcript.bam', '/tmp/RtmpwBZQAu/file9c8881213d884/sample3_realign2transcript.bam'] /tmp/RtmpwBZQAu/file9c8881213d884/transcript_assembly.fa.fai 5 0.4 0.4
	Counter({'counted_reads': 391, 'not_enough_coverage': 2})
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.47gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.46gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 39448.62Read/s]
-- Running step: isoform_identification @ Fri Aug  8 22:40:40 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---
Detected 2 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
-- Running step: read_realignment @ Fri Aug  8 22:40:50 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpwBZQAu/file9c8886ac5cac3/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpwBZQAu/file9c8886ac5cac3/matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpwBZQAu/file9c8886ac5cac3/matched_reads_dedup.fastq.gz -> /tmp/RtmpwBZQAu/file9c8886ac5cac3/realign2transcript.bam
[M::mm_idx_gen::0.001*1.86] collected minimizers
[M::mm_idx_gen::0.002*2.12] sorted minimizers
[M::main::0.002*2.11] loaded/built the index for 5 target sequence(s)
[M::mm_mapopt_update::0.002*2.07] mid_occ = 10
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 5
[M::mm_idx_stat::0.002*2.05] distinct minimizers: 198 (33.33% are singletons); average occurrences: 2.884; average spacing: 5.196; total length: 2967
[M::worker_pipeline::0.054*6.40] mapped 358 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 --eqx -N 100 -ax map-ont -t 8 --seed 2022 -y /tmp/RtmpwBZQAu/file9c8886ac5cac3/transcript_assembly.fa /tmp/RtmpwBZQAu/file9c8886ac5cac3/matched_reads_dedup.fastq.gz
[M::main] Real time: 0.055 sec; CPU: 0.347 sec; Peak RSS: 0.007 GB
Sorting BAM files by 8 with CB threads...

[bam_sort_core] merging from 0 files and 8 in-memory blocks...
-- Running step: transcript_quantification @ Fri Aug  8 22:40:50 2025 ----------
2025-08-09T02:40:50.946081Z  INFO oarfish: setting user-provided filter parameters.
2025-08-09T02:40:50.946512Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpwBZQAu/file9c8886ac5cac3/realign2transcript.bam, contains 5 reference sequences.
2025-08-09T02:40:50.946520Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-09T02:40:50.946523Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-09T02:40:50.946575Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-09T02:40:50.946580Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-08-09T02:40:50.952528Z  INFO oarfish::single_cell: Processed 100 cells.
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpwBZQAu/file9c88878fc34ac/config_file_641160.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Fri Aug  8 22:40:52 2025 ----------------
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpwBZQAu/file9c88878fc34ac/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
-- Running step: genome_alignment @ Fri Aug  8 22:40:52 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpwBZQAu/file9c88878fc34ac/matched_reads.fastq.gz ->/tmp/RtmpwBZQAu/file9c88878fc34ac/align2genome.bam
-- Running step: gene_quantification @ Fri Aug  8 22:41:13 2025 ----------------
22:41:13 Fri Aug 08 2025 quantify genes 
Using BAM(s): '/tmp/RtmpwBZQAu/file9c88878fc34ac/align2genome.bam'
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  1.84gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  1.83gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 38865.08Read/s]
-- Running step: isoform_identification @ Fri Aug  8 22:41:14 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---
Detected 2 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
-- Running step: read_realignment @ Fri Aug  8 22:41:24 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpwBZQAu/file9c88878fc34ac/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpwBZQAu/file9c88878fc34ac/matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpwBZQAu/file9c88878fc34ac/matched_reads_dedup.fastq.gz ->/tmp/RtmpwBZQAu/file9c88878fc34ac/realign2transcript.bam
-- Running step: transcript_quantification @ Fri Aug  8 22:41:44 2025 ----------
2025-08-09T02:41:44.363831Z  INFO oarfish: setting user-provided filter parameters.
2025-08-09T02:41:44.364257Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpwBZQAu/file9c88878fc34ac/realign2transcript.bam, contains 5 reference sequences.
2025-08-09T02:41:44.364270Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-09T02:41:44.364273Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-09T02:41:44.364341Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-09T02:41:44.364347Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-08-09T02:41:44.371692Z  INFO oarfish::single_cell: Processed 100 cells.
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpwBZQAu/file9c88858b4a6c3/config_file_641160.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Fri Aug  8 22:41:46 2025 ----------------
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpwBZQAu/file9c88858b4a6c3/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
-- Running step: genome_alignment @ Fri Aug  8 22:41:46 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpwBZQAu/file9c88858b4a6c3/matched_reads.fastq.gz -> /tmp/RtmpwBZQAu/file9c88858b4a6c3/align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
[M::mm_idx_gen::0.001*1.35] collected minimizers
[M::mm_idx_gen::0.002*1.92] sorted minimizers
[M::main::0.002*1.91] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*1.85] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.003*1.81] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.096*7.15] mapped 372 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 8 --seed 2022 --junc-bed /tmp/RtmpwBZQAu/file9c88858b4a6c3/reference.bed --junc-bonus 1 /tmp/RtmpwBZQAu/file9c88858b4a6c3/rps24.fa /tmp/RtmpwBZQAu/file9c88858b4a6c3/matched_reads.fastq.gz
[M::main] Real time: 0.097 sec; CPU: 0.689 sec; Peak RSS: 0.019 GB
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: gene_quantification @ Fri Aug  8 22:41:46 2025 ----------------
22:41:46 Fri Aug 08 2025 quantify genes 
Using BAM(s): '/tmp/RtmpwBZQAu/file9c88858b4a6c3/align2genome.bam'
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.13gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.13gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 38820.76Read/s]
-- Running step: isoform_identification @ Fri Aug  8 22:41:47 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---
Detected 2 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
-- Running step: read_realignment @ Fri Aug  8 22:41:57 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpwBZQAu/file9c88858b4a6c3/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpwBZQAu/file9c88858b4a6c3/matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpwBZQAu/file9c88858b4a6c3/matched_reads_dedup.fastq.gz -> /tmp/RtmpwBZQAu/file9c88858b4a6c3/realign2transcript.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
[M::mm_idx_gen::0.001*1.78] collected minimizers
[M::mm_idx_gen::0.002*3.53] sorted minimizers
[M::main::0.002*3.49] loaded/built the index for 5 target sequence(s)
[M::mm_mapopt_update::0.002*3.40] mid_occ = 10
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 5
[M::mm_idx_stat::0.002*3.32] distinct minimizers: 198 (33.33% are singletons); average occurrences: 2.884; average spacing: 5.196; total length: 2967
[M::worker_pipeline::0.039*6.24] mapped 358 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax map-ont -p 0.9 --end-bonus 10 -N 3 -t 8 --seed 2022 /tmp/RtmpwBZQAu/file9c88858b4a6c3/transcript_assembly.fa /tmp/RtmpwBZQAu/file9c88858b4a6c3/matched_reads_dedup.fastq.gz
[M::main] Real time: 0.039 sec; CPU: 0.241 sec; Peak RSS: 0.007 GB
Sorting BAM files by genome coordinates with 8 threads...

[bam_sort_core] merging from 0 files and 8 in-memory blocks...
Indexing bam files
-- Running step: transcript_quantification @ Fri Aug  8 22:41:57 2025 ----------
22:41:57 Fri Aug 08 2025 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpwBZQAu/file9c888506a6ef2/config_file_641160.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Fri Aug  8 22:41:58 2025 ----------------
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpwBZQAu/file9c888506a6ef2/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
-- Running step: genome_alignment @ Fri Aug  8 22:41:58 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpwBZQAu/file9c888506a6ef2/matched_reads.fastq.gz ->/tmp/RtmpwBZQAu/file9c888506a6ef2/align2genome.bam
-- Running step: gene_quantification @ Fri Aug  8 22:42:19 2025 ----------------
22:42:19 Fri Aug 08 2025 quantify genes 
Using BAM(s): '/tmp/RtmpwBZQAu/file9c888506a6ef2/align2genome.bam'
	Counter({'counted_reads': 347, 'not_enough_coverage': 9, 'unmapped': 2})
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.07gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.07gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 39614.20Read/s]
-- Running step: isoform_identification @ Fri Aug  8 22:42:20 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---
Detected 2 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
-- Running step: read_realignment @ Fri Aug  8 22:42:30 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpwBZQAu/file9c888506a6ef2/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpwBZQAu/file9c888506a6ef2/matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpwBZQAu/file9c888506a6ef2/matched_reads_dedup.fastq.gz ->/tmp/RtmpwBZQAu/file9c888506a6ef2/realign2transcript.bam
-- Running step: transcript_quantification @ Fri Aug  8 22:42:50 2025 ----------
22:42:50 Fri Aug 08 2025 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpwBZQAu/file9c888654a70e2/config_file_641160.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Fri Aug  8 22:42:51 2025 ----------------
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpwBZQAu/file9c888654a70e2/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
-- Running step: genome_alignment @ Fri Aug  8 22:42:51 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpwBZQAu/file9c888654a70e2/matched_reads.fastq.gz -> /tmp/RtmpwBZQAu/file9c888654a70e2/align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
[M::mm_idx_gen::0.001*1.70] collected minimizers
[M::mm_idx_gen::0.002*1.16] sorted minimizers
[M::main::0.002*1.16] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*1.15] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.003*1.14] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.086*7.10] mapped 372 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 8 --seed 2022 --junc-bed /tmp/RtmpwBZQAu/file9c888654a70e2/reference.bed --junc-bonus 1 /tmp/RtmpwBZQAu/file9c888654a70e2/rps24.fa /tmp/RtmpwBZQAu/file9c888654a70e2/matched_reads.fastq.gz
[M::main] Real time: 0.086 sec; CPU: 0.607 sec; Peak RSS: 0.020 GB
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: gene_quantification @ Fri Aug  8 22:42:51 2025 ----------------
22:42:51 Fri Aug 08 2025 quantify genes 
Using BAM(s): '/tmp/RtmpwBZQAu/file9c888654a70e2/align2genome.bam'
	Counter({'counted_reads': 347, 'not_enough_coverage': 9, 'unmapped': 2})
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.26gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.26gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 39242.67Read/s]
-- Running step: isoform_identification @ Fri Aug  8 22:42:52 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
-- Running step: read_realignment @ Fri Aug  8 22:42:53 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpwBZQAu/file9c888654a70e2/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpwBZQAu/file9c888654a70e2/matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpwBZQAu/file9c888654a70e2/matched_reads_dedup.fastq.gz -> /tmp/RtmpwBZQAu/file9c888654a70e2/realign2transcript.bam
[M::mm_idx_gen::0.002*1.51] collected minimizers
[M::mm_idx_gen::0.003*2.36] sorted minimizers
[M::main::0.003*2.35] loaded/built the index for 10 target sequence(s)
[M::mm_mapopt_update::0.003*2.31] mid_occ = 11
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 10
[M::mm_idx_stat::0.003*2.27] distinct minimizers: 611 (72.01% are singletons); average occurrences: 2.597; average spacing: 5.171; total length: 8207
[M::worker_pipeline::0.084*6.46] mapped 358 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 --eqx -N 100 -ax map-ont -t 8 --seed 2022 -y /tmp/RtmpwBZQAu/file9c888654a70e2/transcript_assembly.fa /tmp/RtmpwBZQAu/file9c888654a70e2/matched_reads_dedup.fastq.gz
[M::main] Real time: 0.084 sec; CPU: 0.543 sec; Peak RSS: 0.009 GB
Sorting BAM files by 8 with CB threads...

[bam_sort_core] merging from 0 files and 8 in-memory blocks...
-- Running step: transcript_quantification @ Fri Aug  8 22:42:53 2025 ----------
2025-08-09T02:42:53.707934Z  INFO oarfish: setting user-provided filter parameters.
2025-08-09T02:42:53.708611Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpwBZQAu/file9c888654a70e2/realign2transcript.bam, contains 10 reference sequences.
2025-08-09T02:42:53.708622Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-09T02:42:53.708625Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-09T02:42:53.708701Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-09T02:42:53.708708Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-08-09T02:42:53.718241Z  INFO oarfish::single_cell: Processed 100 cells.
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpwBZQAu/file9c888ce384e0/config_file_641160.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Fri Aug  8 22:42:55 2025 ----------------
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpwBZQAu/file9c888ce384e0/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
-- Running step: genome_alignment @ Fri Aug  8 22:42:55 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpwBZQAu/file9c888ce384e0/matched_reads.fastq.gz ->/tmp/RtmpwBZQAu/file9c888ce384e0/align2genome.bam
-- Running step: gene_quantification @ Fri Aug  8 22:43:16 2025 ----------------
22:43:16 Fri Aug 08 2025 quantify genes 
Using BAM(s): '/tmp/RtmpwBZQAu/file9c888ce384e0/align2genome.bam'
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  1.98gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  1.98gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 38817.89Read/s]
-- Running step: isoform_identification @ Fri Aug  8 22:43:17 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
-- Running step: read_realignment @ Fri Aug  8 22:43:18 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpwBZQAu/file9c888ce384e0/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpwBZQAu/file9c888ce384e0/matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpwBZQAu/file9c888ce384e0/matched_reads_dedup.fastq.gz ->/tmp/RtmpwBZQAu/file9c888ce384e0/realign2transcript.bam
-- Running step: transcript_quantification @ Fri Aug  8 22:43:39 2025 ----------
2025-08-09T02:43:39.292887Z  INFO oarfish: setting user-provided filter parameters.
2025-08-09T02:43:39.293400Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpwBZQAu/file9c888ce384e0/realign2transcript.bam, contains 10 reference sequences.
2025-08-09T02:43:39.293412Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-09T02:43:39.293416Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-09T02:43:39.293485Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-09T02:43:39.293492Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-08-09T02:43:39.304996Z  INFO oarfish::single_cell: Processed 100 cells.
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpwBZQAu/file9c888611250f5/config_file_641160.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Fri Aug  8 22:43:41 2025 ----------------
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpwBZQAu/file9c888611250f5/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
-- Running step: genome_alignment @ Fri Aug  8 22:43:41 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpwBZQAu/file9c888611250f5/matched_reads.fastq.gz -> /tmp/RtmpwBZQAu/file9c888611250f5/align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
[M::mm_idx_gen::0.001*1.81] collected minimizers
[M::mm_idx_gen::0.002*1.61] sorted minimizers
[M::main::0.002*1.61] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*1.57] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.003*1.54] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.097*7.12] mapped 372 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 8 --seed 2022 --junc-bed /tmp/RtmpwBZQAu/file9c888611250f5/reference.bed --junc-bonus 1 /tmp/RtmpwBZQAu/file9c888611250f5/rps24.fa /tmp/RtmpwBZQAu/file9c888611250f5/matched_reads.fastq.gz
[M::main] Real time: 0.098 sec; CPU: 0.693 sec; Peak RSS: 0.020 GB
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: gene_quantification @ Fri Aug  8 22:43:41 2025 ----------------
22:43:41 Fri Aug 08 2025 quantify genes 
Using BAM(s): '/tmp/RtmpwBZQAu/file9c888611250f5/align2genome.bam'
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.23gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.22gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 38777.12Read/s]
-- Running step: isoform_identification @ Fri Aug  8 22:43:42 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
-- Running step: read_realignment @ Fri Aug  8 22:43:43 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpwBZQAu/file9c888611250f5/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpwBZQAu/file9c888611250f5/matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpwBZQAu/file9c888611250f5/matched_reads_dedup.fastq.gz -> /tmp/RtmpwBZQAu/file9c888611250f5/realign2transcript.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
[M::mm_idx_gen::0.001*1.50] collected minimizers
[M::mm_idx_gen::0.002*3.06] sorted minimizers
[M::main::0.002*3.04] loaded/built the index for 10 target sequence(s)
[M::mm_mapopt_update::0.003*2.96] mid_occ = 11
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 10
[M::mm_idx_stat::0.003*2.90] distinct minimizers: 611 (72.01% are singletons); average occurrences: 2.597; average spacing: 5.171; total length: 8207
[M::worker_pipeline::0.060*6.17] mapped 358 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax map-ont -p 0.9 --end-bonus 10 -N 3 -t 8 --seed 2022 /tmp/RtmpwBZQAu/file9c888611250f5/transcript_assembly.fa /tmp/RtmpwBZQAu/file9c888611250f5/matched_reads_dedup.fastq.gz
[M::main] Real time: 0.061 sec; CPU: 0.371 sec; Peak RSS: 0.008 GB
Sorting BAM files by genome coordinates with 8 threads...

[bam_sort_core] merging from 0 files and 8 in-memory blocks...
Indexing bam files
-- Running step: transcript_quantification @ Fri Aug  8 22:43:43 2025 ----------
22:43:43 Fri Aug 08 2025 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpwBZQAu/file9c8883d8926a3/config_file_641160.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Fri Aug  8 22:43:44 2025 ----------------
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpwBZQAu/file9c8883d8926a3/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
-- Running step: genome_alignment @ Fri Aug  8 22:43:44 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpwBZQAu/file9c8883d8926a3/matched_reads.fastq.gz ->/tmp/RtmpwBZQAu/file9c8883d8926a3/align2genome.bam
-- Running step: gene_quantification @ Fri Aug  8 22:44:06 2025 ----------------
22:44:06 Fri Aug 08 2025 quantify genes 
Using BAM(s): '/tmp/RtmpwBZQAu/file9c8883d8926a3/align2genome.bam'
	Counter({'counted_reads': 358})
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.32gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.32gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 38629.27Read/s]
-- Running step: isoform_identification @ Fri Aug  8 22:44:06 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
-- Running step: read_realignment @ Fri Aug  8 22:44:07 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpwBZQAu/file9c8883d8926a3/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpwBZQAu/file9c8883d8926a3/matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpwBZQAu/file9c8883d8926a3/matched_reads_dedup.fastq.gz ->/tmp/RtmpwBZQAu/file9c8883d8926a3/realign2transcript.bam
-- Running step: transcript_quantification @ Fri Aug  8 22:44:29 2025 ----------
22:44:29 Fri Aug 08 2025 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpwBZQAu/file9c8882cffc64d/config_file_641160.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Fri Aug  8 22:44:30 2025 ----------------
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpwBZQAu/file9c8882cffc64d/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpwBZQAu/file9c8882cffc64d/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpwBZQAu/file9c8882cffc64d/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpwBZQAu/file9c8882cffc64d/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpwBZQAu/file9c8882cffc64d/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpwBZQAu/file9c8882cffc64d/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 92
Number of reads with exactly one barcode match: 91
Number of chimera reads: 1
All done!
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpwBZQAu/file9c8882cffc64d/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpwBZQAu/file9c8882cffc64d/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 95
Number of reads with exactly one barcode match: 94
Number of chimera reads: 0
All done!
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpwBZQAu/file9c8882cffc64d/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpwBZQAu/file9c8882cffc64d/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
-- Running step: genome_alignment @ Fri Aug  8 22:44:31 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpwBZQAu/file9c8882cffc64d/sampleA_matched_reads.fastq.gz -> /tmp/RtmpwBZQAu/file9c8882cffc64d/sampleA_align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
[M::mm_idx_gen::0.002*1.27] collected minimizers
[M::mm_idx_gen::0.003*1.21] sorted minimizers
[M::main::0.003*1.20] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.003*1.19] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.003*1.19] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.601*1.00] mapped 372 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/RtmpwBZQAu/file9c8882cffc64d/reference.bed --junc-bonus 1 /tmp/RtmpwBZQAu/file9c8882cffc64d/rps24.fa /tmp/RtmpwBZQAu/file9c8882cffc64d/sampleA_matched_reads.fastq.gz
[M::main] Real time: 0.602 sec; CPU: 0.603 sec; Peak RSS: 0.006 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpwBZQAu/file9c8882cffc64d/sample1_matched_reads.fastq.gz -> /tmp/RtmpwBZQAu/file9c8882cffc64d/sample1_align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
[M::mm_idx_gen::0.002*1.04] collected minimizers
[M::mm_idx_gen::0.003*1.03] sorted minimizers
[M::main::0.003*1.03] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.003*1.03] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.003*1.03] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.154*1.00] mapped 93 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/RtmpwBZQAu/file9c8882cffc64d/reference.bed --junc-bonus 1 /tmp/RtmpwBZQAu/file9c8882cffc64d/rps24.fa /tmp/RtmpwBZQAu/file9c8882cffc64d/sample1_matched_reads.fastq.gz
[M::main] Real time: 0.155 sec; CPU: 0.155 sec; Peak RSS: 0.006 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpwBZQAu/file9c8882cffc64d/sample2_matched_reads.fastq.gz -> /tmp/RtmpwBZQAu/file9c8882cffc64d/sample2_align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
[M::mm_idx_gen::0.001*1.04] collected minimizers
[M::mm_idx_gen::0.002*1.03] sorted minimizers
[M::main::0.002*1.03] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*1.02] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*1.02] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.157*1.00] mapped 96 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/RtmpwBZQAu/file9c8882cffc64d/reference.bed --junc-bonus 1 /tmp/RtmpwBZQAu/file9c8882cffc64d/rps24.fa /tmp/RtmpwBZQAu/file9c8882cffc64d/sample2_matched_reads.fastq.gz
[M::main] Real time: 0.157 sec; CPU: 0.157 sec; Peak RSS: 0.004 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpwBZQAu/file9c8882cffc64d/sample3_matched_reads.fastq.gz -> /tmp/RtmpwBZQAu/file9c8882cffc64d/sample3_align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
[M::mm_idx_gen::0.001*1.09] collected minimizers
[M::mm_idx_gen::0.002*1.06] sorted minimizers
[M::main::0.002*1.06] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*1.06] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*1.05] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.300*1.00] mapped 183 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/RtmpwBZQAu/file9c8882cffc64d/reference.bed --junc-bonus 1 /tmp/RtmpwBZQAu/file9c8882cffc64d/rps24.fa /tmp/RtmpwBZQAu/file9c8882cffc64d/sample3_matched_reads.fastq.gz
[M::main] Real time: 0.300 sec; CPU: 0.300 sec; Peak RSS: 0.006 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Fri Aug  8 22:44:32 2025 ----------------
22:44:32 Fri Aug 08 2025 quantify genes 
Using BAM(s): '/tmp/RtmpwBZQAu/file9c8882cffc64d/sampleA_align2genome.bam',
'/tmp/RtmpwBZQAu/file9c8882cffc64d/sample1_align2genome.bam',
'/tmp/RtmpwBZQAu/file9c8882cffc64d/sample2_align2genome.bam', and
'/tmp/RtmpwBZQAu/file9c8882cffc64d/sample3_align2genome.bam'
	Counter({'counted_reads': 358})
parsing /tmp/RtmpwBZQAu/file9c8882cffc64d/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 20.37gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 38738.44Read/s]
parsing /tmp/RtmpwBZQAu/file9c8882cffc64d/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 48.35gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 187386.25Read/s]
parsing /tmp/RtmpwBZQAu/file9c8882cffc64d/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 49.35gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 153642.01Read/s]
parsing /tmp/RtmpwBZQAu/file9c8882cffc64d/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 35.76gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 76803.56Read/s]
-- Running step: isoform_identification @ Fri Aug  8 22:44:33 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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  |                                                                            
  |==================                                                    |  25%
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Detected 6 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
-- Running step: read_realignment @ Fri Aug  8 22:44:57 2025 -------------------
Checking for fastq file(s) /tmp/RtmpwBZQAu/file9c8882cffc64d/fastq, /tmp/RtmpwBZQAu/file9c8882cffc64d/fastq/sample1.fq.gz, /tmp/RtmpwBZQAu/file9c8882cffc64d/fastq/sample2.fq.gz, /tmp/RtmpwBZQAu/file9c8882cffc64d/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpwBZQAu/file9c8882cffc64d/sampleA_matched_reads.fastq.gz, /tmp/RtmpwBZQAu/file9c8882cffc64d/sample1_matched_reads.fastq.gz, /tmp/RtmpwBZQAu/file9c8882cffc64d/sample2_matched_reads.fastq.gz, /tmp/RtmpwBZQAu/file9c8882cffc64d/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpwBZQAu/file9c8882cffc64d/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpwBZQAu/file9c8882cffc64d/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpwBZQAu/file9c8882cffc64d/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpwBZQAu/file9c8882cffc64d/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpwBZQAu/file9c8882cffc64d/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpwBZQAu/file9c8882cffc64d/sampleA_realign2transcript.bam
[M::mm_idx_gen::0.001*1.07] collected minimizers
[M::mm_idx_gen::0.001*1.05] sorted minimizers
[M::main::0.001*1.05] loaded/built the index for 5 target sequence(s)
[M::mm_mapopt_update::0.001*1.05] mid_occ = 10
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 5
[M::mm_idx_stat::0.001*1.05] distinct minimizers: 198 (33.33% are singletons); average occurrences: 2.884; average spacing: 5.196; total length: 2967
[M::worker_pipeline::0.271*1.00] mapped 358 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 --eqx -N 100 -ax map-ont -t 1 --seed 2022 -y /tmp/RtmpwBZQAu/file9c8882cffc64d/transcript_assembly.fa /tmp/RtmpwBZQAu/file9c8882cffc64d/sampleA_matched_reads_dedup.fastq.gz
[M::main] Real time: 0.271 sec; CPU: 0.271 sec; Peak RSS: 0.006 GB
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpwBZQAu/file9c8882cffc64d/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpwBZQAu/file9c8882cffc64d/sample1_realign2transcript.bam
[M::mm_idx_gen::0.001*1.86] collected minimizers
[M::mm_idx_gen::0.001*1.68] sorted minimizers
[M::main::0.001*1.68] loaded/built the index for 5 target sequence(s)
[M::mm_mapopt_update::0.001*1.65] mid_occ = 10
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 5
[M::mm_idx_stat::0.001*1.62] distinct minimizers: 198 (33.33% are singletons); average occurrences: 2.884; average spacing: 5.196; total length: 2967
[M::worker_pipeline::0.070*1.01] mapped 91 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 --eqx -N 100 -ax map-ont -t 1 --seed 2022 -y /tmp/RtmpwBZQAu/file9c8882cffc64d/transcript_assembly.fa /tmp/RtmpwBZQAu/file9c8882cffc64d/sample1_matched_reads_dedup.fastq.gz
[M::main] Real time: 0.070 sec; CPU: 0.071 sec; Peak RSS: 0.003 GB
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpwBZQAu/file9c8882cffc64d/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpwBZQAu/file9c8882cffc64d/sample2_realign2transcript.bam
[M::mm_idx_gen::0.001*1.04] collected minimizers
[M::mm_idx_gen::0.001*1.03] sorted minimizers
[M::main::0.001*1.03] loaded/built the index for 5 target sequence(s)
[M::mm_mapopt_update::0.001*1.03] mid_occ = 10
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 5
[M::mm_idx_stat::0.001*1.03] distinct minimizers: 198 (33.33% are singletons); average occurrences: 2.884; average spacing: 5.196; total length: 2967
[M::worker_pipeline::0.079*1.00] mapped 95 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 --eqx -N 100 -ax map-ont -t 1 --seed 2022 -y /tmp/RtmpwBZQAu/file9c8882cffc64d/transcript_assembly.fa /tmp/RtmpwBZQAu/file9c8882cffc64d/sample2_matched_reads_dedup.fastq.gz
[M::main] Real time: 0.079 sec; CPU: 0.079 sec; Peak RSS: 0.004 GB
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpwBZQAu/file9c8882cffc64d/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpwBZQAu/file9c8882cffc64d/sample3_realign2transcript.bam
[M::mm_idx_gen::0.002*1.69] collected minimizers
[M::mm_idx_gen::0.002*1.57] sorted minimizers
[M::main::0.002*1.56] loaded/built the index for 5 target sequence(s)
[M::mm_mapopt_update::0.002*1.55] mid_occ = 10
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 5
[M::mm_idx_stat::0.002*1.54] distinct minimizers: 198 (33.33% are singletons); average occurrences: 2.884; average spacing: 5.196; total length: 2967
[M::worker_pipeline::0.138*1.01] mapped 176 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 --eqx -N 100 -ax map-ont -t 1 --seed 2022 -y /tmp/RtmpwBZQAu/file9c8882cffc64d/transcript_assembly.fa /tmp/RtmpwBZQAu/file9c8882cffc64d/sample3_matched_reads_dedup.fastq.gz
[M::main] Real time: 0.138 sec; CPU: 0.139 sec; Peak RSS: 0.005 GB
Sorting BAM files by 1 with CB threads...

-- Running step: transcript_quantification @ Fri Aug  8 22:44:58 2025 ----------
2025-08-09T02:44:58.194282Z  INFO oarfish: setting user-provided filter parameters.
2025-08-09T02:44:58.194747Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpwBZQAu/file9c8882cffc64d/sampleA_realign2transcript.bam, contains 5 reference sequences.
2025-08-09T02:44:58.194754Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-09T02:44:58.194757Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-09T02:44:58.194810Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-09T02:44:58.194815Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-08-09T02:44:58.200418Z  INFO oarfish::single_cell: Processed 100 cells.
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
2025-08-09T02:44:59.578168Z  INFO oarfish: setting user-provided filter parameters.
2025-08-09T02:44:59.578555Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpwBZQAu/file9c8882cffc64d/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-08-09T02:44:59.578563Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-09T02:44:59.578567Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-09T02:44:59.578627Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-09T02:44:59.578633Z  INFO oarfish: parsed reference information for 5 transcripts.
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
2025-08-09T02:45:00.950000Z  INFO oarfish: setting user-provided filter parameters.
2025-08-09T02:45:00.950391Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpwBZQAu/file9c8882cffc64d/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-08-09T02:45:00.950402Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-09T02:45:00.950405Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-09T02:45:00.950459Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-09T02:45:00.950464Z  INFO oarfish: parsed reference information for 5 transcripts.
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
2025-08-09T02:45:02.208728Z  INFO oarfish: setting user-provided filter parameters.
2025-08-09T02:45:02.209096Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpwBZQAu/file9c8882cffc64d/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-08-09T02:45:02.209107Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-09T02:45:02.209110Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-09T02:45:02.209161Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-09T02:45:02.209176Z  INFO oarfish: parsed reference information for 5 transcripts.
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpwBZQAu/file9c88830785193/config_file_641160.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Fri Aug  8 22:45:03 2025 ----------------
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpwBZQAu/file9c88830785193/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpwBZQAu/file9c88830785193/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpwBZQAu/file9c88830785193/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpwBZQAu/file9c88830785193/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpwBZQAu/file9c88830785193/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpwBZQAu/file9c88830785193/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 92
Number of reads with exactly one barcode match: 91
Number of chimera reads: 1
All done!
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpwBZQAu/file9c88830785193/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpwBZQAu/file9c88830785193/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 95
Number of reads with exactly one barcode match: 94
Number of chimera reads: 0
All done!
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpwBZQAu/file9c88830785193/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpwBZQAu/file9c88830785193/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
-- Running step: genome_alignment @ Fri Aug  8 22:45:04 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpwBZQAu/file9c88830785193/sampleA_matched_reads.fastq.gz ->/tmp/RtmpwBZQAu/file9c88830785193/sampleA_align2genome.bam
/tmp/RtmpwBZQAu/file9c88830785193/sample1_matched_reads.fastq.gz ->/tmp/RtmpwBZQAu/file9c88830785193/sample1_align2genome.bam
/tmp/RtmpwBZQAu/file9c88830785193/sample2_matched_reads.fastq.gz ->/tmp/RtmpwBZQAu/file9c88830785193/sample2_align2genome.bam
/tmp/RtmpwBZQAu/file9c88830785193/sample3_matched_reads.fastq.gz ->/tmp/RtmpwBZQAu/file9c88830785193/sample3_align2genome.bam
-- Running step: gene_quantification @ Fri Aug  8 22:45:28 2025 ----------------
22:45:28 Fri Aug 08 2025 quantify genes 
Using BAM(s): '/tmp/RtmpwBZQAu/file9c88830785193/sampleA_align2genome.bam',
'/tmp/RtmpwBZQAu/file9c88830785193/sample1_align2genome.bam',
'/tmp/RtmpwBZQAu/file9c88830785193/sample2_align2genome.bam', and
'/tmp/RtmpwBZQAu/file9c88830785193/sample3_align2genome.bam'
parsing /tmp/RtmpwBZQAu/file9c88830785193/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 21.44gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 39165.57Read/s]
parsing /tmp/RtmpwBZQAu/file9c88830785193/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 53.68gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 187530.36Read/s]
parsing /tmp/RtmpwBZQAu/file9c88830785193/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 48.49gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 153982.70Read/s]
parsing /tmp/RtmpwBZQAu/file9c88830785193/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 36.30gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 76812.56Read/s]
-- Running step: isoform_identification @ Fri Aug  8 22:45:29 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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  |==================                                                    |  25%
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Detected 6 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
-- Running step: read_realignment @ Fri Aug  8 22:45:52 2025 -------------------
Checking for fastq file(s) /tmp/RtmpwBZQAu/file9c88830785193/fastq, /tmp/RtmpwBZQAu/file9c88830785193/fastq/sample1.fq.gz, /tmp/RtmpwBZQAu/file9c88830785193/fastq/sample2.fq.gz, /tmp/RtmpwBZQAu/file9c88830785193/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpwBZQAu/file9c88830785193/sampleA_matched_reads.fastq.gz, /tmp/RtmpwBZQAu/file9c88830785193/sample1_matched_reads.fastq.gz, /tmp/RtmpwBZQAu/file9c88830785193/sample2_matched_reads.fastq.gz, /tmp/RtmpwBZQAu/file9c88830785193/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpwBZQAu/file9c88830785193/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpwBZQAu/file9c88830785193/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpwBZQAu/file9c88830785193/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpwBZQAu/file9c88830785193/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpwBZQAu/file9c88830785193/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpwBZQAu/file9c88830785193/sampleA_realign2transcript.bam
/tmp/RtmpwBZQAu/file9c88830785193/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpwBZQAu/file9c88830785193/sample1_realign2transcript.bam
/tmp/RtmpwBZQAu/file9c88830785193/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpwBZQAu/file9c88830785193/sample2_realign2transcript.bam
/tmp/RtmpwBZQAu/file9c88830785193/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpwBZQAu/file9c88830785193/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Fri Aug  8 22:46:13 2025 ----------
2025-08-09T02:46:13.585987Z  INFO oarfish: setting user-provided filter parameters.
2025-08-09T02:46:13.586478Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpwBZQAu/file9c88830785193/sampleA_realign2transcript.bam, contains 5 reference sequences.
2025-08-09T02:46:13.586490Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-09T02:46:13.586494Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-09T02:46:13.586558Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-09T02:46:13.586565Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-08-09T02:46:13.592774Z  INFO oarfish::single_cell: Processed 100 cells.
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
2025-08-09T02:46:15.224205Z  INFO oarfish: setting user-provided filter parameters.
2025-08-09T02:46:15.224720Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpwBZQAu/file9c88830785193/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-08-09T02:46:15.224732Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-09T02:46:15.224735Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-09T02:46:15.224803Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-09T02:46:15.224810Z  INFO oarfish: parsed reference information for 5 transcripts.
Error in x$.self$finalize() : attempt to apply non-function
Error in x$.self$finalize() : attempt to apply non-function
In addition: Warning message:
call dbDisconnect() when finished working with a connection 
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
2025-08-09T02:46:16.723260Z  INFO oarfish: setting user-provided filter parameters.
2025-08-09T02:46:16.723650Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpwBZQAu/file9c88830785193/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-08-09T02:46:16.723659Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-09T02:46:16.723662Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-09T02:46:16.723718Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-09T02:46:16.723723Z  INFO oarfish: parsed reference information for 5 transcripts.
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
2025-08-09T02:46:18.085355Z  INFO oarfish: setting user-provided filter parameters.
2025-08-09T02:46:18.085847Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpwBZQAu/file9c88830785193/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-08-09T02:46:18.085856Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-09T02:46:18.085858Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-09T02:46:18.085917Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-09T02:46:18.085923Z  INFO oarfish: parsed reference information for 5 transcripts.
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpwBZQAu/file9c888639d133f/config_file_641160.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Fri Aug  8 22:46:19 2025 ----------------
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpwBZQAu/file9c888639d133f/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpwBZQAu/file9c888639d133f/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpwBZQAu/file9c888639d133f/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpwBZQAu/file9c888639d133f/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpwBZQAu/file9c888639d133f/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpwBZQAu/file9c888639d133f/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 92
Number of reads with exactly one barcode match: 91
Number of chimera reads: 1
All done!
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpwBZQAu/file9c888639d133f/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpwBZQAu/file9c888639d133f/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 95
Number of reads with exactly one barcode match: 94
Number of chimera reads: 0
All done!
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpwBZQAu/file9c888639d133f/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpwBZQAu/file9c888639d133f/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
-- Running step: genome_alignment @ Fri Aug  8 22:46:20 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpwBZQAu/file9c888639d133f/sampleA_matched_reads.fastq.gz -> /tmp/RtmpwBZQAu/file9c888639d133f/sampleA_align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
[M::mm_idx_gen::0.001*1.30] collected minimizers
[M::mm_idx_gen::0.002*1.20] sorted minimizers
[M::main::0.002*1.20] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*1.18] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*1.17] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.601*1.00] mapped 372 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/RtmpwBZQAu/file9c888639d133f/reference.bed --junc-bonus 1 /tmp/RtmpwBZQAu/file9c888639d133f/rps24.fa /tmp/RtmpwBZQAu/file9c888639d133f/sampleA_matched_reads.fastq.gz
[M::main] Real time: 0.602 sec; CPU: 0.602 sec; Peak RSS: 0.006 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpwBZQAu/file9c888639d133f/sample1_matched_reads.fastq.gz -> /tmp/RtmpwBZQAu/file9c888639d133f/sample1_align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
[M::mm_idx_gen::0.003*1.37] collected minimizers
[M::mm_idx_gen::0.003*1.30] sorted minimizers
[M::main::0.003*1.30] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.004*1.29] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.004*1.28] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.156*1.01] mapped 93 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/RtmpwBZQAu/file9c888639d133f/reference.bed --junc-bonus 1 /tmp/RtmpwBZQAu/file9c888639d133f/rps24.fa /tmp/RtmpwBZQAu/file9c888639d133f/sample1_matched_reads.fastq.gz
[M::main] Real time: 0.156 sec; CPU: 0.157 sec; Peak RSS: 0.005 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpwBZQAu/file9c888639d133f/sample2_matched_reads.fastq.gz -> /tmp/RtmpwBZQAu/file9c888639d133f/sample2_align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
[M::mm_idx_gen::0.001*0.85] collected minimizers
[M::mm_idx_gen::0.002*0.90] sorted minimizers
[M::main::0.002*0.90] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*0.90] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*0.91] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.156*1.00] mapped 96 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/RtmpwBZQAu/file9c888639d133f/reference.bed --junc-bonus 1 /tmp/RtmpwBZQAu/file9c888639d133f/rps24.fa /tmp/RtmpwBZQAu/file9c888639d133f/sample2_matched_reads.fastq.gz
[M::main] Real time: 0.157 sec; CPU: 0.157 sec; Peak RSS: 0.004 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpwBZQAu/file9c888639d133f/sample3_matched_reads.fastq.gz -> /tmp/RtmpwBZQAu/file9c888639d133f/sample3_align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
[M::mm_idx_gen::0.001*1.03] collected minimizers
[M::mm_idx_gen::0.002*1.02] sorted minimizers
[M::main::0.002*1.02] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*1.02] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*1.02] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.299*1.00] mapped 183 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/RtmpwBZQAu/file9c888639d133f/reference.bed --junc-bonus 1 /tmp/RtmpwBZQAu/file9c888639d133f/rps24.fa /tmp/RtmpwBZQAu/file9c888639d133f/sample3_matched_reads.fastq.gz
[M::main] Real time: 0.300 sec; CPU: 0.300 sec; Peak RSS: 0.006 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Fri Aug  8 22:46:22 2025 ----------------
22:46:22 Fri Aug 08 2025 quantify genes 
Using BAM(s): '/tmp/RtmpwBZQAu/file9c888639d133f/sampleA_align2genome.bam',
'/tmp/RtmpwBZQAu/file9c888639d133f/sample1_align2genome.bam',
'/tmp/RtmpwBZQAu/file9c888639d133f/sample2_align2genome.bam', and
'/tmp/RtmpwBZQAu/file9c888639d133f/sample3_align2genome.bam'
parsing /tmp/RtmpwBZQAu/file9c888639d133f/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 21.80gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 39214.50Read/s]
parsing /tmp/RtmpwBZQAu/file9c888639d133f/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 53.23gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 189993.84Read/s]
parsing /tmp/RtmpwBZQAu/file9c888639d133f/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 52.51gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 151690.54Read/s]
parsing /tmp/RtmpwBZQAu/file9c888639d133f/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 34.83gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 77314.93Read/s]
-- Running step: isoform_identification @ Fri Aug  8 22:46:22 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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  |======================================================================| 100%

Detected 6 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
-- Running step: read_realignment @ Fri Aug  8 22:46:47 2025 -------------------
Checking for fastq file(s) /tmp/RtmpwBZQAu/file9c888639d133f/fastq, /tmp/RtmpwBZQAu/file9c888639d133f/fastq/sample1.fq.gz, /tmp/RtmpwBZQAu/file9c888639d133f/fastq/sample2.fq.gz, /tmp/RtmpwBZQAu/file9c888639d133f/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpwBZQAu/file9c888639d133f/sampleA_matched_reads.fastq.gz, /tmp/RtmpwBZQAu/file9c888639d133f/sample1_matched_reads.fastq.gz, /tmp/RtmpwBZQAu/file9c888639d133f/sample2_matched_reads.fastq.gz, /tmp/RtmpwBZQAu/file9c888639d133f/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpwBZQAu/file9c888639d133f/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpwBZQAu/file9c888639d133f/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpwBZQAu/file9c888639d133f/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpwBZQAu/file9c888639d133f/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpwBZQAu/file9c888639d133f/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpwBZQAu/file9c888639d133f/sampleA_realign2transcript.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
[M::mm_idx_gen::0.001*1.35] collected minimizers
[M::mm_idx_gen::0.001*1.28] sorted minimizers
[M::main::0.001*1.28] loaded/built the index for 5 target sequence(s)
[M::mm_mapopt_update::0.001*1.27] mid_occ = 10
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 5
[M::mm_idx_stat::0.001*1.25] distinct minimizers: 198 (33.33% are singletons); average occurrences: 2.884; average spacing: 5.196; total length: 2967
[M::worker_pipeline::0.231*1.00] mapped 358 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax map-ont -p 0.9 --end-bonus 10 -N 3 -t 1 --seed 2022 /tmp/RtmpwBZQAu/file9c888639d133f/transcript_assembly.fa /tmp/RtmpwBZQAu/file9c888639d133f/sampleA_matched_reads_dedup.fastq.gz
[M::main] Real time: 0.231 sec; CPU: 0.232 sec; Peak RSS: 0.004 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample /tmp/RtmpwBZQAu/file9c888639d133f/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpwBZQAu/file9c888639d133f/sample1_realign2transcript.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
[M::mm_idx_gen::0.001*0.86] collected minimizers
[M::mm_idx_gen::0.001*0.89] sorted minimizers
[M::main::0.001*0.89] loaded/built the index for 5 target sequence(s)
[M::mm_mapopt_update::0.001*0.89] mid_occ = 10
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 5
[M::mm_idx_stat::0.001*0.90] distinct minimizers: 198 (33.33% are singletons); average occurrences: 2.884; average spacing: 5.196; total length: 2967
[M::worker_pipeline::0.060*1.00] mapped 91 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax map-ont -p 0.9 --end-bonus 10 -N 3 -t 1 --seed 2022 /tmp/RtmpwBZQAu/file9c888639d133f/transcript_assembly.fa /tmp/RtmpwBZQAu/file9c888639d133f/sample1_matched_reads_dedup.fastq.gz
[M::main] Real time: 0.060 sec; CPU: 0.060 sec; Peak RSS: 0.003 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample /tmp/RtmpwBZQAu/file9c888639d133f/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpwBZQAu/file9c888639d133f/sample2_realign2transcript.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
[M::mm_idx_gen::0.001*2.45] collected minimizers
[M::mm_idx_gen::0.002*1.92] sorted minimizers
[M::main::0.002*1.91] loaded/built the index for 5 target sequence(s)
[M::mm_mapopt_update::0.002*1.88] mid_occ = 10
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 5
[M::mm_idx_stat::0.002*1.86] distinct minimizers: 198 (33.33% are singletons); average occurrences: 2.884; average spacing: 5.196; total length: 2967
[M::worker_pipeline::0.064*1.02] mapped 95 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax map-ont -p 0.9 --end-bonus 10 -N 3 -t 1 --seed 2022 /tmp/RtmpwBZQAu/file9c888639d133f/transcript_assembly.fa /tmp/RtmpwBZQAu/file9c888639d133f/sample2_matched_reads_dedup.fastq.gz
[M::main] Real time: 0.064 sec; CPU: 0.065 sec; Peak RSS: 0.003 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample /tmp/RtmpwBZQAu/file9c888639d133f/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpwBZQAu/file9c888639d133f/sample3_realign2transcript.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
[M::mm_idx_gen::0.002*1.41] collected minimizers
[M::mm_idx_gen::0.002*1.36] sorted minimizers
[M::main::0.002*1.36] loaded/built the index for 5 target sequence(s)
[M::mm_mapopt_update::0.002*1.35] mid_occ = 10
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 5
[M::mm_idx_stat::0.002*1.34] distinct minimizers: 198 (33.33% are singletons); average occurrences: 2.884; average spacing: 5.196; total length: 2967
[M::worker_pipeline::0.119*1.01] mapped 176 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax map-ont -p 0.9 --end-bonus 10 -N 3 -t 1 --seed 2022 /tmp/RtmpwBZQAu/file9c888639d133f/transcript_assembly.fa /tmp/RtmpwBZQAu/file9c888639d133f/sample3_matched_reads_dedup.fastq.gz
[M::main] Real time: 0.119 sec; CPU: 0.120 sec; Peak RSS: 0.004 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Fri Aug  8 22:46:48 2025 ----------
22:46:48 Fri Aug 08 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpwBZQAu/file9c888639d133f/sampleA_realign2transcript.bam...
parsing /tmp/RtmpwBZQAu/file9c888639d133f/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpwBZQAu/file9c888639d133f/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpwBZQAu/file9c888639d133f/sample1_realign2transcript.bam...
parsing /tmp/RtmpwBZQAu/file9c888639d133f/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpwBZQAu/file9c888639d133f/sample1_realign2transcript.bamdone
parsing /tmp/RtmpwBZQAu/file9c888639d133f/sample2_realign2transcript.bam...
parsing /tmp/RtmpwBZQAu/file9c888639d133f/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpwBZQAu/file9c888639d133f/sample2_realign2transcript.bamdone
parsing /tmp/RtmpwBZQAu/file9c888639d133f/sample3_realign2transcript.bam...
parsing /tmp/RtmpwBZQAu/file9c888639d133f/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpwBZQAu/file9c888639d133f/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpwBZQAu/file9c88860139b26/config_file_641160.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Fri Aug  8 22:46:51 2025 ----------------
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpwBZQAu/file9c88860139b26/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpwBZQAu/file9c88860139b26/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpwBZQAu/file9c88860139b26/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpwBZQAu/file9c88860139b26/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpwBZQAu/file9c88860139b26/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpwBZQAu/file9c88860139b26/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 92
Number of reads with exactly one barcode match: 91
Number of chimera reads: 1
All done!
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpwBZQAu/file9c88860139b26/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpwBZQAu/file9c88860139b26/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 95
Number of reads with exactly one barcode match: 94
Number of chimera reads: 0
All done!
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpwBZQAu/file9c88860139b26/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpwBZQAu/file9c88860139b26/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
-- Running step: genome_alignment @ Fri Aug  8 22:46:52 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpwBZQAu/file9c88860139b26/sampleA_matched_reads.fastq.gz ->/tmp/RtmpwBZQAu/file9c88860139b26/sampleA_align2genome.bam
/tmp/RtmpwBZQAu/file9c88860139b26/sample1_matched_reads.fastq.gz ->/tmp/RtmpwBZQAu/file9c88860139b26/sample1_align2genome.bam
/tmp/RtmpwBZQAu/file9c88860139b26/sample2_matched_reads.fastq.gz ->/tmp/RtmpwBZQAu/file9c88860139b26/sample2_align2genome.bam
/tmp/RtmpwBZQAu/file9c88860139b26/sample3_matched_reads.fastq.gz ->/tmp/RtmpwBZQAu/file9c88860139b26/sample3_align2genome.bam
-- Running step: gene_quantification @ Fri Aug  8 22:47:20 2025 ----------------
22:47:20 Fri Aug 08 2025 quantify genes 
Using BAM(s): '/tmp/RtmpwBZQAu/file9c88860139b26/sampleA_align2genome.bam',
'/tmp/RtmpwBZQAu/file9c88860139b26/sample1_align2genome.bam',
'/tmp/RtmpwBZQAu/file9c88860139b26/sample2_align2genome.bam', and
'/tmp/RtmpwBZQAu/file9c88860139b26/sample3_align2genome.bam'
	Counter({'counted_reads': 347, 'not_enough_coverage': 9, 'unmapped': 2})
	Counter({'counted_reads': 89, 'not_enough_coverage': 2})
	Counter({'counted_reads': 92, 'not_enough_coverage': 3})
	Counter({'counted_reads': 168, 'not_enough_coverage': 6, 'unmapped': 2})
parsing /tmp/RtmpwBZQAu/file9c88860139b26/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 20.39gene_group/s]
/home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/python/count_gene.py:704: RuntimeWarning: More than 20 figures have been opened. Figures created through the pyplot interface (`matplotlib.pyplot.figure`) are retained until explicitly closed and may consume too much memory. (To control this warning, see the rcParam `figure.max_open_warning`). Consider using `matplotlib.pyplot.close()`.
  plt.figure(figsize=(8, 6))
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 37776.31Read/s]
parsing /tmp/RtmpwBZQAu/file9c88860139b26/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 51.28gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 191098.39Read/s]
parsing /tmp/RtmpwBZQAu/file9c88860139b26/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 47.98gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 154892.54Read/s]
parsing /tmp/RtmpwBZQAu/file9c88860139b26/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 36.56gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 77187.44Read/s]
-- Running step: isoform_identification @ Fri Aug  8 22:47:21 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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Detected 6 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
-- Running step: read_realignment @ Fri Aug  8 22:47:45 2025 -------------------
Checking for fastq file(s) /tmp/RtmpwBZQAu/file9c88860139b26/fastq, /tmp/RtmpwBZQAu/file9c88860139b26/fastq/sample1.fq.gz, /tmp/RtmpwBZQAu/file9c88860139b26/fastq/sample2.fq.gz, /tmp/RtmpwBZQAu/file9c88860139b26/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpwBZQAu/file9c88860139b26/sampleA_matched_reads.fastq.gz, /tmp/RtmpwBZQAu/file9c88860139b26/sample1_matched_reads.fastq.gz, /tmp/RtmpwBZQAu/file9c88860139b26/sample2_matched_reads.fastq.gz, /tmp/RtmpwBZQAu/file9c88860139b26/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpwBZQAu/file9c88860139b26/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpwBZQAu/file9c88860139b26/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpwBZQAu/file9c88860139b26/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpwBZQAu/file9c88860139b26/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpwBZQAu/file9c88860139b26/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpwBZQAu/file9c88860139b26/sampleA_realign2transcript.bam
/tmp/RtmpwBZQAu/file9c88860139b26/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpwBZQAu/file9c88860139b26/sample1_realign2transcript.bam
/tmp/RtmpwBZQAu/file9c88860139b26/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpwBZQAu/file9c88860139b26/sample2_realign2transcript.bam
/tmp/RtmpwBZQAu/file9c88860139b26/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpwBZQAu/file9c88860139b26/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Fri Aug  8 22:48:06 2025 ----------
22:48:06 Fri Aug 08 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpwBZQAu/file9c88860139b26/sampleA_realign2transcript.bam...
parsing /tmp/RtmpwBZQAu/file9c88860139b26/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpwBZQAu/file9c88860139b26/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpwBZQAu/file9c88860139b26/sample1_realign2transcript.bam...
parsing /tmp/RtmpwBZQAu/file9c88860139b26/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpwBZQAu/file9c88860139b26/sample1_realign2transcript.bamdone
parsing /tmp/RtmpwBZQAu/file9c88860139b26/sample2_realign2transcript.bam...
parsing /tmp/RtmpwBZQAu/file9c88860139b26/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpwBZQAu/file9c88860139b26/sample2_realign2transcript.bamdone
parsing /tmp/RtmpwBZQAu/file9c88860139b26/sample3_realign2transcript.bam...
parsing /tmp/RtmpwBZQAu/file9c88860139b26/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpwBZQAu/file9c88860139b26/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpwBZQAu/file9c8883d174bd6/config_file_641160.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Fri Aug  8 22:48:10 2025 ----------------
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpwBZQAu/file9c8883d174bd6/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpwBZQAu/file9c8883d174bd6/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpwBZQAu/file9c8883d174bd6/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpwBZQAu/file9c8883d174bd6/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpwBZQAu/file9c8883d174bd6/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpwBZQAu/file9c8883d174bd6/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 92
Number of reads with exactly one barcode match: 91
Number of chimera reads: 1
All done!
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpwBZQAu/file9c8883d174bd6/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpwBZQAu/file9c8883d174bd6/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 95
Number of reads with exactly one barcode match: 94
Number of chimera reads: 0
All done!
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpwBZQAu/file9c8883d174bd6/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpwBZQAu/file9c8883d174bd6/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
-- Running step: genome_alignment @ Fri Aug  8 22:48:11 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpwBZQAu/file9c8883d174bd6/sampleA_matched_reads.fastq.gz -> /tmp/RtmpwBZQAu/file9c8883d174bd6/sampleA_align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
[M::mm_idx_gen::0.001*0.88] collected minimizers
[M::mm_idx_gen::0.002*0.93] sorted minimizers
[M::main::0.002*0.93] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*0.93] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*0.94] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.598*1.00] mapped 372 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/RtmpwBZQAu/file9c8883d174bd6/reference.bed --junc-bonus 1 /tmp/RtmpwBZQAu/file9c8883d174bd6/rps24.fa /tmp/RtmpwBZQAu/file9c8883d174bd6/sampleA_matched_reads.fastq.gz
[M::main] Real time: 0.599 sec; CPU: 0.598 sec; Peak RSS: 0.007 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpwBZQAu/file9c8883d174bd6/sample1_matched_reads.fastq.gz -> /tmp/RtmpwBZQAu/file9c8883d174bd6/sample1_align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
[M::mm_idx_gen::0.001*1.31] collected minimizers
[M::mm_idx_gen::0.002*1.20] sorted minimizers
[M::main::0.002*1.20] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*1.19] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*1.18] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.155*1.00] mapped 93 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/RtmpwBZQAu/file9c8883d174bd6/reference.bed --junc-bonus 1 /tmp/RtmpwBZQAu/file9c8883d174bd6/rps24.fa /tmp/RtmpwBZQAu/file9c8883d174bd6/sample1_matched_reads.fastq.gz
[M::main] Real time: 0.155 sec; CPU: 0.155 sec; Peak RSS: 0.005 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpwBZQAu/file9c8883d174bd6/sample2_matched_reads.fastq.gz -> /tmp/RtmpwBZQAu/file9c8883d174bd6/sample2_align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
[M::mm_idx_gen::0.001*1.88] collected minimizers
[M::mm_idx_gen::0.002*1.59] sorted minimizers
[M::main::0.002*1.58] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*1.54] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*1.51] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.157*1.01] mapped 96 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/RtmpwBZQAu/file9c8883d174bd6/reference.bed --junc-bonus 1 /tmp/RtmpwBZQAu/file9c8883d174bd6/rps24.fa /tmp/RtmpwBZQAu/file9c8883d174bd6/sample2_matched_reads.fastq.gz
[M::main] Real time: 0.157 sec; CPU: 0.158 sec; Peak RSS: 0.004 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpwBZQAu/file9c8883d174bd6/sample3_matched_reads.fastq.gz -> /tmp/RtmpwBZQAu/file9c8883d174bd6/sample3_align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
[M::mm_idx_gen::0.001*1.53] collected minimizers
[M::mm_idx_gen::0.002*1.33] sorted minimizers
[M::main::0.002*1.33] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*1.31] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*1.29] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.300*1.00] mapped 183 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/RtmpwBZQAu/file9c8883d174bd6/reference.bed --junc-bonus 1 /tmp/RtmpwBZQAu/file9c8883d174bd6/rps24.fa /tmp/RtmpwBZQAu/file9c8883d174bd6/sample3_matched_reads.fastq.gz
[M::main] Real time: 0.301 sec; CPU: 0.301 sec; Peak RSS: 0.006 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Fri Aug  8 22:48:12 2025 ----------------
22:48:12 Fri Aug 08 2025 quantify genes 
Using BAM(s): '/tmp/RtmpwBZQAu/file9c8883d174bd6/sampleA_align2genome.bam',
'/tmp/RtmpwBZQAu/file9c8883d174bd6/sample1_align2genome.bam',
'/tmp/RtmpwBZQAu/file9c8883d174bd6/sample2_align2genome.bam', and
'/tmp/RtmpwBZQAu/file9c8883d174bd6/sample3_align2genome.bam'
	Counter({'counted_reads': 347, 'not_enough_coverage': 9, 'unmapped': 2})
	Counter({'counted_reads': 89, 'not_enough_coverage': 2})
	Counter({'counted_reads': 92, 'not_enough_coverage': 3})
	Counter({'counted_reads': 168, 'not_enough_coverage': 6, 'unmapped': 2})
parsing /tmp/RtmpwBZQAu/file9c8883d174bd6/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 22.33gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 39018.23Read/s]
parsing /tmp/RtmpwBZQAu/file9c8883d174bd6/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 53.72gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 185868.30Read/s]
parsing /tmp/RtmpwBZQAu/file9c8883d174bd6/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 51.94gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 152635.59Read/s]
parsing /tmp/RtmpwBZQAu/file9c8883d174bd6/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 36.52gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 76958.01Read/s]
-- Running step: isoform_identification @ Fri Aug  8 22:48:13 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
-- Running step: read_realignment @ Fri Aug  8 22:48:14 2025 -------------------
Checking for fastq file(s) /tmp/RtmpwBZQAu/file9c8883d174bd6/fastq, /tmp/RtmpwBZQAu/file9c8883d174bd6/fastq/sample1.fq.gz, /tmp/RtmpwBZQAu/file9c8883d174bd6/fastq/sample2.fq.gz, /tmp/RtmpwBZQAu/file9c8883d174bd6/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpwBZQAu/file9c8883d174bd6/sampleA_matched_reads.fastq.gz, /tmp/RtmpwBZQAu/file9c8883d174bd6/sample1_matched_reads.fastq.gz, /tmp/RtmpwBZQAu/file9c8883d174bd6/sample2_matched_reads.fastq.gz, /tmp/RtmpwBZQAu/file9c8883d174bd6/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpwBZQAu/file9c8883d174bd6/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpwBZQAu/file9c8883d174bd6/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpwBZQAu/file9c8883d174bd6/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpwBZQAu/file9c8883d174bd6/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpwBZQAu/file9c8883d174bd6/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpwBZQAu/file9c8883d174bd6/sampleA_realign2transcript.bam
[M::mm_idx_gen::0.001*1.67] collected minimizers
[M::mm_idx_gen::0.002*1.53] sorted minimizers
[M::main::0.002*1.52] loaded/built the index for 14 target sequence(s)
[M::mm_mapopt_update::0.002*1.50] mid_occ = 15
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 14
[M::mm_idx_stat::0.002*1.48] distinct minimizers: 632 (64.56% are singletons); average occurrences: 3.326; average spacing: 5.176; total length: 10880
[M::worker_pipeline::0.725*1.00] mapped 358 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 --eqx -N 100 -ax map-ont -t 1 --seed 2022 -y /tmp/RtmpwBZQAu/file9c8883d174bd6/transcript_assembly.fa /tmp/RtmpwBZQAu/file9c8883d174bd6/sampleA_matched_reads_dedup.fastq.gz
[M::main] Real time: 0.726 sec; CPU: 0.726 sec; Peak RSS: 0.007 GB
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpwBZQAu/file9c8883d174bd6/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpwBZQAu/file9c8883d174bd6/sample1_realign2transcript.bam
[M::mm_idx_gen::0.001*1.80] collected minimizers
[M::mm_idx_gen::0.002*1.63] sorted minimizers
[M::main::0.002*1.62] loaded/built the index for 14 target sequence(s)
[M::mm_mapopt_update::0.002*1.59] mid_occ = 15
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 14
[M::mm_idx_stat::0.002*1.57] distinct minimizers: 632 (64.56% are singletons); average occurrences: 3.326; average spacing: 5.176; total length: 10880
[M::worker_pipeline::0.197*1.01] mapped 91 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 --eqx -N 100 -ax map-ont -t 1 --seed 2022 -y /tmp/RtmpwBZQAu/file9c8883d174bd6/transcript_assembly.fa /tmp/RtmpwBZQAu/file9c8883d174bd6/sample1_matched_reads_dedup.fastq.gz
[M::main] Real time: 0.198 sec; CPU: 0.199 sec; Peak RSS: 0.004 GB
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpwBZQAu/file9c8883d174bd6/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpwBZQAu/file9c8883d174bd6/sample2_realign2transcript.bam
[M::mm_idx_gen::0.001*1.15] collected minimizers
[M::mm_idx_gen::0.002*1.12] sorted minimizers
[M::main::0.002*1.11] loaded/built the index for 14 target sequence(s)
[M::mm_mapopt_update::0.002*1.11] mid_occ = 15
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 14
[M::mm_idx_stat::0.002*1.10] distinct minimizers: 632 (64.56% are singletons); average occurrences: 3.326; average spacing: 5.176; total length: 10880
[M::worker_pipeline::0.204*1.00] mapped 95 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 --eqx -N 100 -ax map-ont -t 1 --seed 2022 -y /tmp/RtmpwBZQAu/file9c8883d174bd6/transcript_assembly.fa /tmp/RtmpwBZQAu/file9c8883d174bd6/sample2_matched_reads_dedup.fastq.gz
[M::main] Real time: 0.204 sec; CPU: 0.204 sec; Peak RSS: 0.003 GB
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpwBZQAu/file9c8883d174bd6/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpwBZQAu/file9c8883d174bd6/sample3_realign2transcript.bam
[M::mm_idx_gen::0.002*0.98] collected minimizers
[M::mm_idx_gen::0.003*0.98] sorted minimizers
[M::main::0.003*0.98] loaded/built the index for 14 target sequence(s)
[M::mm_mapopt_update::0.003*0.98] mid_occ = 15
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 14
[M::mm_idx_stat::0.003*0.98] distinct minimizers: 632 (64.56% are singletons); average occurrences: 3.326; average spacing: 5.176; total length: 10880
[M::worker_pipeline::0.344*1.00] mapped 176 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 --eqx -N 100 -ax map-ont -t 1 --seed 2022 -y /tmp/RtmpwBZQAu/file9c8883d174bd6/transcript_assembly.fa /tmp/RtmpwBZQAu/file9c8883d174bd6/sample3_matched_reads_dedup.fastq.gz
[M::main] Real time: 0.345 sec; CPU: 0.344 sec; Peak RSS: 0.006 GB
Sorting BAM files by 1 with CB threads...

-- Running step: transcript_quantification @ Fri Aug  8 22:48:16 2025 ----------
2025-08-09T02:48:16.511886Z  INFO oarfish: setting user-provided filter parameters.
2025-08-09T02:48:16.512348Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpwBZQAu/file9c8883d174bd6/sampleA_realign2transcript.bam, contains 14 reference sequences.
2025-08-09T02:48:16.512360Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-09T02:48:16.512363Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-09T02:48:16.512448Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-09T02:48:16.512455Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-08-09T02:48:16.524901Z  INFO oarfish::single_cell: Processed 100 cells.
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
2025-08-09T02:48:18.078841Z  INFO oarfish: setting user-provided filter parameters.
2025-08-09T02:48:18.079311Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpwBZQAu/file9c8883d174bd6/sample1_realign2transcript.bam, contains 14 reference sequences.
2025-08-09T02:48:18.079323Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-09T02:48:18.079326Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-09T02:48:18.079405Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-09T02:48:18.079412Z  INFO oarfish: parsed reference information for 14 transcripts.
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
2025-08-09T02:48:19.705249Z  INFO oarfish: setting user-provided filter parameters.
2025-08-09T02:48:19.705832Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpwBZQAu/file9c8883d174bd6/sample2_realign2transcript.bam, contains 14 reference sequences.
2025-08-09T02:48:19.705843Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-09T02:48:19.705847Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-09T02:48:19.705934Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-09T02:48:19.705954Z  INFO oarfish: parsed reference information for 14 transcripts.
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
2025-08-09T02:48:21.235219Z  INFO oarfish: setting user-provided filter parameters.
2025-08-09T02:48:21.235827Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpwBZQAu/file9c8883d174bd6/sample3_realign2transcript.bam, contains 14 reference sequences.
2025-08-09T02:48:21.235836Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-09T02:48:21.235839Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-09T02:48:21.235922Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-09T02:48:21.235929Z  INFO oarfish: parsed reference information for 14 transcripts.
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpwBZQAu/file9c88819c7f17d/config_file_641160.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Fri Aug  8 22:48:23 2025 ----------------
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpwBZQAu/file9c88819c7f17d/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpwBZQAu/file9c88819c7f17d/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpwBZQAu/file9c88819c7f17d/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpwBZQAu/file9c88819c7f17d/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpwBZQAu/file9c88819c7f17d/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpwBZQAu/file9c88819c7f17d/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 92
Number of reads with exactly one barcode match: 91
Number of chimera reads: 1
All done!
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpwBZQAu/file9c88819c7f17d/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpwBZQAu/file9c88819c7f17d/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 95
Number of reads with exactly one barcode match: 94
Number of chimera reads: 0
All done!
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpwBZQAu/file9c88819c7f17d/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpwBZQAu/file9c88819c7f17d/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
-- Running step: genome_alignment @ Fri Aug  8 22:48:23 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpwBZQAu/file9c88819c7f17d/sampleA_matched_reads.fastq.gz ->/tmp/RtmpwBZQAu/file9c88819c7f17d/sampleA_align2genome.bam
/tmp/RtmpwBZQAu/file9c88819c7f17d/sample1_matched_reads.fastq.gz ->/tmp/RtmpwBZQAu/file9c88819c7f17d/sample1_align2genome.bam
/tmp/RtmpwBZQAu/file9c88819c7f17d/sample2_matched_reads.fastq.gz ->/tmp/RtmpwBZQAu/file9c88819c7f17d/sample2_align2genome.bam
/tmp/RtmpwBZQAu/file9c88819c7f17d/sample3_matched_reads.fastq.gz ->/tmp/RtmpwBZQAu/file9c88819c7f17d/sample3_align2genome.bam
-- Running step: gene_quantification @ Fri Aug  8 22:48:47 2025 ----------------
22:48:47 Fri Aug 08 2025 quantify genes 
Using BAM(s): '/tmp/RtmpwBZQAu/file9c88819c7f17d/sampleA_align2genome.bam',
'/tmp/RtmpwBZQAu/file9c88819c7f17d/sample1_align2genome.bam',
'/tmp/RtmpwBZQAu/file9c88819c7f17d/sample2_align2genome.bam', and
'/tmp/RtmpwBZQAu/file9c88819c7f17d/sample3_align2genome.bam'
parsing /tmp/RtmpwBZQAu/file9c88819c7f17d/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 22.48gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 38704.84Read/s]
parsing /tmp/RtmpwBZQAu/file9c88819c7f17d/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 53.05gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 186605.92Read/s]
parsing /tmp/RtmpwBZQAu/file9c88819c7f17d/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 51.48gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 153489.08Read/s]
parsing /tmp/RtmpwBZQAu/file9c88819c7f17d/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 35.32gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 76647.49Read/s]
-- Running step: isoform_identification @ Fri Aug  8 22:48:48 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
-- Running step: read_realignment @ Fri Aug  8 22:48:49 2025 -------------------
Checking for fastq file(s) /tmp/RtmpwBZQAu/file9c88819c7f17d/fastq, /tmp/RtmpwBZQAu/file9c88819c7f17d/fastq/sample1.fq.gz, /tmp/RtmpwBZQAu/file9c88819c7f17d/fastq/sample2.fq.gz, /tmp/RtmpwBZQAu/file9c88819c7f17d/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpwBZQAu/file9c88819c7f17d/sampleA_matched_reads.fastq.gz, /tmp/RtmpwBZQAu/file9c88819c7f17d/sample1_matched_reads.fastq.gz, /tmp/RtmpwBZQAu/file9c88819c7f17d/sample2_matched_reads.fastq.gz, /tmp/RtmpwBZQAu/file9c88819c7f17d/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpwBZQAu/file9c88819c7f17d/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpwBZQAu/file9c88819c7f17d/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpwBZQAu/file9c88819c7f17d/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpwBZQAu/file9c88819c7f17d/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpwBZQAu/file9c88819c7f17d/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpwBZQAu/file9c88819c7f17d/sampleA_realign2transcript.bam
/tmp/RtmpwBZQAu/file9c88819c7f17d/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpwBZQAu/file9c88819c7f17d/sample1_realign2transcript.bam
/tmp/RtmpwBZQAu/file9c88819c7f17d/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpwBZQAu/file9c88819c7f17d/sample2_realign2transcript.bam
/tmp/RtmpwBZQAu/file9c88819c7f17d/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpwBZQAu/file9c88819c7f17d/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Fri Aug  8 22:49:11 2025 ----------
2025-08-09T02:49:11.567661Z  INFO oarfish: setting user-provided filter parameters.
2025-08-09T02:49:11.568029Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpwBZQAu/file9c88819c7f17d/sampleA_realign2transcript.bam, contains 14 reference sequences.
2025-08-09T02:49:11.568038Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-09T02:49:11.568042Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-09T02:49:11.568130Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-09T02:49:11.568140Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-08-09T02:49:11.579795Z  INFO oarfish::single_cell: Processed 100 cells.
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
2025-08-09T02:49:13.538652Z  INFO oarfish: setting user-provided filter parameters.
2025-08-09T02:49:13.538981Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpwBZQAu/file9c88819c7f17d/sample1_realign2transcript.bam, contains 14 reference sequences.
2025-08-09T02:49:13.538990Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-09T02:49:13.538994Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-09T02:49:13.539066Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-09T02:49:13.539074Z  INFO oarfish: parsed reference information for 14 transcripts.
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
2025-08-09T02:49:15.144948Z  INFO oarfish: setting user-provided filter parameters.
2025-08-09T02:49:15.145311Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpwBZQAu/file9c88819c7f17d/sample2_realign2transcript.bam, contains 14 reference sequences.
2025-08-09T02:49:15.145322Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-09T02:49:15.145333Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-09T02:49:15.145417Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-09T02:49:15.145425Z  INFO oarfish: parsed reference information for 14 transcripts.
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
2025-08-09T02:49:16.779880Z  INFO oarfish: setting user-provided filter parameters.
2025-08-09T02:49:16.780275Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpwBZQAu/file9c88819c7f17d/sample3_realign2transcript.bam, contains 14 reference sequences.
2025-08-09T02:49:16.780288Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-09T02:49:16.780292Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-09T02:49:16.780377Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-09T02:49:16.780385Z  INFO oarfish: parsed reference information for 14 transcripts.
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpwBZQAu/file9c8881c7afa51/config_file_641160.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Fri Aug  8 22:49:18 2025 ----------------
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpwBZQAu/file9c8881c7afa51/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpwBZQAu/file9c8881c7afa51/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpwBZQAu/file9c8881c7afa51/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpwBZQAu/file9c8881c7afa51/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpwBZQAu/file9c8881c7afa51/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpwBZQAu/file9c8881c7afa51/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 92
Number of reads with exactly one barcode match: 91
Number of chimera reads: 1
All done!
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpwBZQAu/file9c8881c7afa51/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpwBZQAu/file9c8881c7afa51/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 95
Number of reads with exactly one barcode match: 94
Number of chimera reads: 0
All done!
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpwBZQAu/file9c8881c7afa51/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpwBZQAu/file9c8881c7afa51/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
-- Running step: genome_alignment @ Fri Aug  8 22:49:19 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpwBZQAu/file9c8881c7afa51/sampleA_matched_reads.fastq.gz -> /tmp/RtmpwBZQAu/file9c8881c7afa51/sampleA_align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
[M::mm_idx_gen::0.001*1.14] collected minimizers
[M::mm_idx_gen::0.002*1.09] sorted minimizers
[M::main::0.002*1.09] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*1.08] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*1.08] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.600*1.00] mapped 372 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/RtmpwBZQAu/file9c8881c7afa51/reference.bed --junc-bonus 1 /tmp/RtmpwBZQAu/file9c8881c7afa51/rps24.fa /tmp/RtmpwBZQAu/file9c8881c7afa51/sampleA_matched_reads.fastq.gz
[M::main] Real time: 0.601 sec; CPU: 0.601 sec; Peak RSS: 0.006 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpwBZQAu/file9c8881c7afa51/sample1_matched_reads.fastq.gz -> /tmp/RtmpwBZQAu/file9c8881c7afa51/sample1_align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
[M::mm_idx_gen::0.001*1.75] collected minimizers
[M::mm_idx_gen::0.002*1.49] sorted minimizers
[M::main::0.002*1.48] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*1.45] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*1.43] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.154*1.01] mapped 93 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/RtmpwBZQAu/file9c8881c7afa51/reference.bed --junc-bonus 1 /tmp/RtmpwBZQAu/file9c8881c7afa51/rps24.fa /tmp/RtmpwBZQAu/file9c8881c7afa51/sample1_matched_reads.fastq.gz
[M::main] Real time: 0.155 sec; CPU: 0.156 sec; Peak RSS: 0.005 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpwBZQAu/file9c8881c7afa51/sample2_matched_reads.fastq.gz -> /tmp/RtmpwBZQAu/file9c8881c7afa51/sample2_align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
[M::mm_idx_gen::0.001*1.32] collected minimizers
[M::mm_idx_gen::0.002*1.21] sorted minimizers
[M::main::0.002*1.21] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*1.20] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*1.19] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.156*1.00] mapped 96 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/RtmpwBZQAu/file9c8881c7afa51/reference.bed --junc-bonus 1 /tmp/RtmpwBZQAu/file9c8881c7afa51/rps24.fa /tmp/RtmpwBZQAu/file9c8881c7afa51/sample2_matched_reads.fastq.gz
[M::main] Real time: 0.157 sec; CPU: 0.157 sec; Peak RSS: 0.004 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpwBZQAu/file9c8881c7afa51/sample3_matched_reads.fastq.gz -> /tmp/RtmpwBZQAu/file9c8881c7afa51/sample3_align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
[M::mm_idx_gen::0.001*1.84] collected minimizers
[M::mm_idx_gen::0.002*1.56] sorted minimizers
[M::main::0.002*1.56] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*1.52] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*1.49] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.298*1.00] mapped 183 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/RtmpwBZQAu/file9c8881c7afa51/reference.bed --junc-bonus 1 /tmp/RtmpwBZQAu/file9c8881c7afa51/rps24.fa /tmp/RtmpwBZQAu/file9c8881c7afa51/sample3_matched_reads.fastq.gz
[M::main] Real time: 0.299 sec; CPU: 0.300 sec; Peak RSS: 0.006 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Fri Aug  8 22:49:20 2025 ----------------
22:49:20 Fri Aug 08 2025 quantify genes 
Using BAM(s): '/tmp/RtmpwBZQAu/file9c8881c7afa51/sampleA_align2genome.bam',
'/tmp/RtmpwBZQAu/file9c8881c7afa51/sample1_align2genome.bam',
'/tmp/RtmpwBZQAu/file9c8881c7afa51/sample2_align2genome.bam', and
'/tmp/RtmpwBZQAu/file9c8881c7afa51/sample3_align2genome.bam'
parsing /tmp/RtmpwBZQAu/file9c8881c7afa51/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 22.01gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 39313.88Read/s]
parsing /tmp/RtmpwBZQAu/file9c8881c7afa51/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 51.07gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 186304.21Read/s]
parsing /tmp/RtmpwBZQAu/file9c8881c7afa51/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 50.35gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 153736.62Read/s]
parsing /tmp/RtmpwBZQAu/file9c8881c7afa51/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 36.39gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 77052.45Read/s]
-- Running step: isoform_identification @ Fri Aug  8 22:49:21 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
-- Running step: read_realignment @ Fri Aug  8 22:49:22 2025 -------------------
Checking for fastq file(s) /tmp/RtmpwBZQAu/file9c8881c7afa51/fastq, /tmp/RtmpwBZQAu/file9c8881c7afa51/fastq/sample1.fq.gz, /tmp/RtmpwBZQAu/file9c8881c7afa51/fastq/sample2.fq.gz, /tmp/RtmpwBZQAu/file9c8881c7afa51/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpwBZQAu/file9c8881c7afa51/sampleA_matched_reads.fastq.gz, /tmp/RtmpwBZQAu/file9c8881c7afa51/sample1_matched_reads.fastq.gz, /tmp/RtmpwBZQAu/file9c8881c7afa51/sample2_matched_reads.fastq.gz, /tmp/RtmpwBZQAu/file9c8881c7afa51/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpwBZQAu/file9c8881c7afa51/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpwBZQAu/file9c8881c7afa51/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpwBZQAu/file9c8881c7afa51/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpwBZQAu/file9c8881c7afa51/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpwBZQAu/file9c8881c7afa51/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpwBZQAu/file9c8881c7afa51/sampleA_realign2transcript.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
[M::mm_idx_gen::0.001*1.02] collected minimizers
[M::mm_idx_gen::0.002*1.01] sorted minimizers
[M::main::0.002*1.01] loaded/built the index for 14 target sequence(s)
[M::mm_mapopt_update::0.002*1.01] mid_occ = 15
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 14
[M::mm_idx_stat::0.002*1.01] distinct minimizers: 632 (64.56% are singletons); average occurrences: 3.326; average spacing: 5.176; total length: 10880
[M::worker_pipeline::0.351*1.00] mapped 358 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax map-ont -p 0.9 --end-bonus 10 -N 3 -t 1 --seed 2022 /tmp/RtmpwBZQAu/file9c8881c7afa51/transcript_assembly.fa /tmp/RtmpwBZQAu/file9c8881c7afa51/sampleA_matched_reads_dedup.fastq.gz
[M::main] Real time: 0.351 sec; CPU: 0.351 sec; Peak RSS: 0.004 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample /tmp/RtmpwBZQAu/file9c8881c7afa51/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpwBZQAu/file9c8881c7afa51/sample1_realign2transcript.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
[M::mm_idx_gen::0.001*1.41] collected minimizers
[M::mm_idx_gen::0.002*1.31] sorted minimizers
[M::main::0.002*1.30] loaded/built the index for 14 target sequence(s)
[M::mm_mapopt_update::0.002*1.29] mid_occ = 15
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 14
[M::mm_idx_stat::0.002*1.28] distinct minimizers: 632 (64.56% are singletons); average occurrences: 3.326; average spacing: 5.176; total length: 10880
[M::worker_pipeline::0.091*1.01] mapped 91 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax map-ont -p 0.9 --end-bonus 10 -N 3 -t 1 --seed 2022 /tmp/RtmpwBZQAu/file9c8881c7afa51/transcript_assembly.fa /tmp/RtmpwBZQAu/file9c8881c7afa51/sample1_matched_reads_dedup.fastq.gz
[M::main] Real time: 0.092 sec; CPU: 0.092 sec; Peak RSS: 0.004 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample /tmp/RtmpwBZQAu/file9c8881c7afa51/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpwBZQAu/file9c8881c7afa51/sample2_realign2transcript.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
[M::mm_idx_gen::0.002*1.38] collected minimizers
[M::mm_idx_gen::0.002*1.32] sorted minimizers
[M::main::0.002*1.32] loaded/built the index for 14 target sequence(s)
[M::mm_mapopt_update::0.002*1.30] mid_occ = 15
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 14
[M::mm_idx_stat::0.002*1.29] distinct minimizers: 632 (64.56% are singletons); average occurrences: 3.326; average spacing: 5.176; total length: 10880
[M::worker_pipeline::0.093*1.01] mapped 95 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax map-ont -p 0.9 --end-bonus 10 -N 3 -t 1 --seed 2022 /tmp/RtmpwBZQAu/file9c8881c7afa51/transcript_assembly.fa /tmp/RtmpwBZQAu/file9c8881c7afa51/sample2_matched_reads_dedup.fastq.gz
[M::main] Real time: 0.094 sec; CPU: 0.094 sec; Peak RSS: 0.003 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample /tmp/RtmpwBZQAu/file9c8881c7afa51/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpwBZQAu/file9c8881c7afa51/sample3_realign2transcript.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
[M::mm_idx_gen::0.001*1.25] collected minimizers
[M::mm_idx_gen::0.002*1.20] sorted minimizers
[M::main::0.002*1.20] loaded/built the index for 14 target sequence(s)
[M::mm_mapopt_update::0.002*1.19] mid_occ = 15
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 14
[M::mm_idx_stat::0.002*1.18] distinct minimizers: 632 (64.56% are singletons); average occurrences: 3.326; average spacing: 5.176; total length: 10880
[M::worker_pipeline::0.177*1.00] mapped 176 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax map-ont -p 0.9 --end-bonus 10 -N 3 -t 1 --seed 2022 /tmp/RtmpwBZQAu/file9c8881c7afa51/transcript_assembly.fa /tmp/RtmpwBZQAu/file9c8881c7afa51/sample3_matched_reads_dedup.fastq.gz
[M::main] Real time: 0.178 sec; CPU: 0.178 sec; Peak RSS: 0.005 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Fri Aug  8 22:49:23 2025 ----------
22:49:23 Fri Aug 08 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpwBZQAu/file9c8881c7afa51/sampleA_realign2transcript.bam...
parsing /tmp/RtmpwBZQAu/file9c8881c7afa51/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpwBZQAu/file9c8881c7afa51/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpwBZQAu/file9c8881c7afa51/sample1_realign2transcript.bam...
parsing /tmp/RtmpwBZQAu/file9c8881c7afa51/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpwBZQAu/file9c8881c7afa51/sample1_realign2transcript.bamdone
parsing /tmp/RtmpwBZQAu/file9c8881c7afa51/sample2_realign2transcript.bam...
parsing /tmp/RtmpwBZQAu/file9c8881c7afa51/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpwBZQAu/file9c8881c7afa51/sample2_realign2transcript.bamdone
parsing /tmp/RtmpwBZQAu/file9c8881c7afa51/sample3_realign2transcript.bam...
parsing /tmp/RtmpwBZQAu/file9c8881c7afa51/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpwBZQAu/file9c8881c7afa51/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Error in x$.self$finalize() : attempt to apply non-function
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpwBZQAu/file9c88830720a28/config_file_641160.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Fri Aug  8 22:49:26 2025 ----------------
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpwBZQAu/file9c88830720a28/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpwBZQAu/file9c88830720a28/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpwBZQAu/file9c88830720a28/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpwBZQAu/file9c88830720a28/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpwBZQAu/file9c88830720a28/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpwBZQAu/file9c88830720a28/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 92
Number of reads with exactly one barcode match: 91
Number of chimera reads: 1
All done!
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpwBZQAu/file9c88830720a28/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpwBZQAu/file9c88830720a28/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 95
Number of reads with exactly one barcode match: 94
Number of chimera reads: 0
All done!
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpwBZQAu/file9c88830720a28/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpwBZQAu/file9c88830720a28/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
-- Running step: genome_alignment @ Fri Aug  8 22:49:27 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpwBZQAu/file9c88830720a28/sampleA_matched_reads.fastq.gz ->/tmp/RtmpwBZQAu/file9c88830720a28/sampleA_align2genome.bam
/tmp/RtmpwBZQAu/file9c88830720a28/sample1_matched_reads.fastq.gz ->/tmp/RtmpwBZQAu/file9c88830720a28/sample1_align2genome.bam
/tmp/RtmpwBZQAu/file9c88830720a28/sample2_matched_reads.fastq.gz ->/tmp/RtmpwBZQAu/file9c88830720a28/sample2_align2genome.bam
/tmp/RtmpwBZQAu/file9c88830720a28/sample3_matched_reads.fastq.gz ->/tmp/RtmpwBZQAu/file9c88830720a28/sample3_align2genome.bam
-- Running step: gene_quantification @ Fri Aug  8 22:49:48 2025 ----------------
22:49:48 Fri Aug 08 2025 quantify genes 
Using BAM(s): '/tmp/RtmpwBZQAu/file9c88830720a28/sampleA_align2genome.bam',
'/tmp/RtmpwBZQAu/file9c88830720a28/sample1_align2genome.bam',
'/tmp/RtmpwBZQAu/file9c88830720a28/sample2_align2genome.bam', and
'/tmp/RtmpwBZQAu/file9c88830720a28/sample3_align2genome.bam'
	Counter({'counted_reads': 357, 'not_enough_coverage': 1})
	Counter({'counted_reads': 91})
	Counter({'counted_reads': 95})
	Counter({'counted_reads': 175, 'not_enough_coverage': 1})
parsing /tmp/RtmpwBZQAu/file9c88830720a28/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 17.14gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 39346.78Read/s]
parsing /tmp/RtmpwBZQAu/file9c88830720a28/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 52.06gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 191321.55Read/s]
parsing /tmp/RtmpwBZQAu/file9c88830720a28/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 50.84gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 154926.86Read/s]
parsing /tmp/RtmpwBZQAu/file9c88830720a28/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 34.30gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 77164.72Read/s]
-- Running step: isoform_identification @ Fri Aug  8 22:49:49 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
-- Running step: read_realignment @ Fri Aug  8 22:49:50 2025 -------------------
Checking for fastq file(s) /tmp/RtmpwBZQAu/file9c88830720a28/fastq, /tmp/RtmpwBZQAu/file9c88830720a28/fastq/sample1.fq.gz, /tmp/RtmpwBZQAu/file9c88830720a28/fastq/sample2.fq.gz, /tmp/RtmpwBZQAu/file9c88830720a28/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpwBZQAu/file9c88830720a28/sampleA_matched_reads.fastq.gz, /tmp/RtmpwBZQAu/file9c88830720a28/sample1_matched_reads.fastq.gz, /tmp/RtmpwBZQAu/file9c88830720a28/sample2_matched_reads.fastq.gz, /tmp/RtmpwBZQAu/file9c88830720a28/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpwBZQAu/file9c88830720a28/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpwBZQAu/file9c88830720a28/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpwBZQAu/file9c88830720a28/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpwBZQAu/file9c88830720a28/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpwBZQAu/file9c88830720a28/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpwBZQAu/file9c88830720a28/sampleA_realign2transcript.bam
/tmp/RtmpwBZQAu/file9c88830720a28/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpwBZQAu/file9c88830720a28/sample1_realign2transcript.bam
/tmp/RtmpwBZQAu/file9c88830720a28/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpwBZQAu/file9c88830720a28/sample2_realign2transcript.bam
/tmp/RtmpwBZQAu/file9c88830720a28/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpwBZQAu/file9c88830720a28/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Fri Aug  8 22:50:12 2025 ----------
22:50:12 Fri Aug 08 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpwBZQAu/file9c88830720a28/sampleA_realign2transcript.bam...
parsing /tmp/RtmpwBZQAu/file9c88830720a28/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpwBZQAu/file9c88830720a28/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpwBZQAu/file9c88830720a28/sample1_realign2transcript.bam...
parsing /tmp/RtmpwBZQAu/file9c88830720a28/sample1_realign2transcript.bamdone
	Counter({'counted_reads': 357, 'not_enough_coverage': 1})
	Counter({'counted_reads': 91})
wrt_tr_to_csv for/tmp/RtmpwBZQAu/file9c88830720a28/sample1_realign2transcript.bamdone
parsing /tmp/RtmpwBZQAu/file9c88830720a28/sample2_realign2transcript.bam...
parsing /tmp/RtmpwBZQAu/file9c88830720a28/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpwBZQAu/file9c88830720a28/sample2_realign2transcript.bamdone
parsing /tmp/RtmpwBZQAu/file9c88830720a28/sample3_realign2transcript.bam...
parsing /tmp/RtmpwBZQAu/file9c88830720a28/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpwBZQAu/file9c88830720a28/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
[ FAIL 0 | WARN 235 | SKIP 0 | PASS 37 ]

[ FAIL 0 | WARN 235 | SKIP 0 | PASS 37 ]
	Counter({'counted_reads': 95})
	Counter({'counted_reads': 175, 'not_enough_coverage': 1})
> 
> proc.time()
   user  system elapsed 
817.457  61.087 877.487