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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_2.3.3.tar.gz
###
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##############################################################################


* using log directory ‘/home/biocbuild/bbs-3.22-bioc/meat/FLAMES.Rcheck’
* using R version 4.5.0 (2025-04-11)
* 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 ‘2.3.3’
* 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:
  'barcode_demultiplex' 'gene_quantification' 'genome_alignment'
  'isoform_identification' 'minimap2_align' 'read_realignment'
  '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
  ‘CellBarcode’
plot_demultiplex_raw: no visible binding for global variable ‘Sample’
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’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘j’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘config’
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 ‘config’
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 capture.output cell_total_reads
  chisq.test config count counts_no_ins cumpct demultiplexed reads
  download.file everything expr 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                         5.001 18.412  13.807
plot_isoform_reduced_dim     22.071  0.599  22.670
find_variants                20.023  0.830  20.081
sc_long_multisample_pipeline 12.476  5.226  12.039
bulk_long_pipeline            2.801 13.869   3.044
MultiSampleSCPipeline        13.749  0.915  15.014
sc_DTU_analysis               8.884  1.756   8.718
create_sce_from_dir           6.182  3.358   7.175
plot_isoform_heatmap          6.790  0.243   7.032
SingleCellPipeline            4.927  1.617   4.312
sc_long_pipeline              4.480  1.505   4.041
BulkPipeline                  5.291  0.366   5.514
* 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 ‘2.3.3’
** 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 4.5.0 (2025-04-11) -- "How About a Twenty-Six"
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
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> # 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/Rtmp7HpXG6/file10a9136fb71b7e/config_file_1091859.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/Rtmp7HpXG6/file10a91360c1e318/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/Rtmp7HpXG6/file10a9135b810c8d/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/Rtmp7HpXG6/file10a9135b810c8d/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp7HpXG6/file10a9133f2136b2/musc_rps24_1.fastq
Searching for barcodes...
Processing file: /tmp/Rtmp7HpXG6/file10a9133f2136b2/musc_rps24_2.fastq
Searching for barcodes...
Processing file: /tmp/Rtmp7HpXG6/file10a9133f2136b2/musc_rps24_3.fastq
Searching for barcodes...
Processing file: /tmp/Rtmp7HpXG6/file10a9133f2136b2/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/Rtmp7HpXG6/file10a9137be90273/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/Rtmp7HpXG6/file10a9137be90273/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp7HpXG6/file10a913a9ff32a/musc_rps24_1.fastq
Searching for barcodes...
Processing file: /tmp/Rtmp7HpXG6/file10a913a9ff32a/musc_rps24_2.fastq
Searching for barcodes...
Processing file: /tmp/Rtmp7HpXG6/file10a913a9ff32a/musc_rps24_3.fastq
Searching for barcodes...
Processing file: /tmp/Rtmp7HpXG6/file10a913a9ff32a/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/Rtmp7HpXG6/file10a9137be90273/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/Rtmp7HpXG6/file10a91315705f86/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/Rtmp7HpXG6/file10a9132ad4b4fb/config_file_1091859.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 @ Wed Jun 18 23:21:37 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/Rtmp7HpXG6/file10a9132ad4b4fb/sample1_align2genome.bam
[M::mm_idx_gen::0.003*1.04] collected minimizers
[M::mm_idx_gen::0.004*1.03] sorted minimizers
[M::main::0.004*1.03] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.004*1.03] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.004*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::0.172*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 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/Rtmp7HpXG6/file10a9132ad4b4fb/reference.bed --junc-bonus 1 /tmp/Rtmp7HpXG6/file10a9132ad4b4fb/rps24.fa /tmp/Rtmp7HpXG6/file10a9132ad4b4fb/fastq/sample1.fq.gz
[M::main] Real time: 0.172 sec; CPU: 0.172 sec; Peak RSS: 0.005 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/Rtmp7HpXG6/file10a9132ad4b4fb/sample2_align2genome.bam
[M::mm_idx_gen::0.002*0.90] collected minimizers
[M::mm_idx_gen::0.003*0.92] sorted minimizers
[M::main::0.003*0.92] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.003*0.93] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.003*0.93] 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::0.172*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 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/Rtmp7HpXG6/file10a9132ad4b4fb/reference.bed --junc-bonus 1 /tmp/Rtmp7HpXG6/file10a9132ad4b4fb/rps24.fa /tmp/Rtmp7HpXG6/file10a9132ad4b4fb/fastq/sample2.fq.gz
[M::main] Real time: 0.173 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/Rtmp7HpXG6/file10a9132ad4b4fb/sample3_align2genome.bam
[M::mm_idx_gen::0.001*1.29] collected minimizers
[M::mm_idx_gen::0.002*1.18] sorted minimizers
[M::main::0.002*1.18] 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::0.334*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/Rtmp7HpXG6/file10a9132ad4b4fb/reference.bed --junc-bonus 1 /tmp/Rtmp7HpXG6/file10a9132ad4b4fb/rps24.fa /tmp/Rtmp7HpXG6/file10a9132ad4b4fb/fastq/sample3.fq.gz
[M::main] Real time: 0.335 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 @ Wed Jun 18 23:21:39 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

  |                                                                            
  |                                                                      |   0%[23:21:47] 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.

[23:21:47] 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.

[23:21:47] 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.

[23:21:47] 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.

[23:21:49] 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.

[23:21:49] 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 @ Wed Jun 18 23:22:06 2025 -------------------
Realigning sample sample1 -> /tmp/Rtmp7HpXG6/file10a9132ad4b4fb/sample1_realign2transcript.bam
[M::mm_idx_gen::0.001*1.75] collected minimizers
[M::mm_idx_gen::0.001*1.57] sorted minimizers
[M::main::0.001*1.56] loaded/built the index for 5 target sequence(s)
[M::mm_mapopt_update::0.001*1.54] mid_occ = 10
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 5
[M::mm_idx_stat::0.001*1.52] distinct minimizers: 198 (33.33% are singletons); average occurrences: 2.884; average spacing: 5.196; total length: 2967
[M::worker_pipeline::0.082*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/Rtmp7HpXG6/file10a9132ad4b4fb/transcript_assembly.fa /tmp/Rtmp7HpXG6/file10a9132ad4b4fb/fastq/sample1.fq.gz
[M::main] Real time: 0.082 sec; CPU: 0.083 sec; Peak RSS: 0.004 GB
Skipped sorting BAM files.

Realigning sample sample2 -> /tmp/Rtmp7HpXG6/file10a9132ad4b4fb/sample2_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::0.087*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/Rtmp7HpXG6/file10a9132ad4b4fb/transcript_assembly.fa /tmp/Rtmp7HpXG6/file10a9132ad4b4fb/fastq/sample2.fq.gz
[M::main] Real time: 0.087 sec; CPU: 0.087 sec; Peak RSS: 0.005 GB
Skipped sorting BAM files.

Realigning sample sample3 -> /tmp/Rtmp7HpXG6/file10a9132ad4b4fb/sample3_realign2transcript.bam
[M::mm_idx_gen::0.001*1.50] collected minimizers
[M::mm_idx_gen::0.001*1.41] sorted minimizers
[M::main::0.001*1.41] loaded/built the index for 5 target sequence(s)
[M::mm_mapopt_update::0.001*1.39] mid_occ = 10
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 5
[M::mm_idx_stat::0.001*1.38] distinct minimizers: 198 (33.33% are singletons); average occurrences: 2.884; average spacing: 5.196; total length: 2967
[M::worker_pipeline::0.162*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/Rtmp7HpXG6/file10a9132ad4b4fb/transcript_assembly.fa /tmp/Rtmp7HpXG6/file10a9132ad4b4fb/fastq/sample3.fq.gz
[M::main] Real time: 0.163 sec; CPU: 0.163 sec; Peak RSS: 0.004 GB
Skipped sorting BAM files.

-- Running step: transcript_quantification @ Wed Jun 18 23:22:06 2025 ----------
2025-06-19T03:22:06.967541Z  INFO oarfish: setting user-provided filter parameters.
2025-06-19T03:22:06.967886Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp7HpXG6/file10a9132ad4b4fb/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-06-19T03:22:06.967894Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-06-19T03:22:06.967897Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-06-19T03:22:06.967958Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-06-19T03:22:06.967964Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-06-19T03:22:06.969492Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-06-19T03:22:06.969611Z  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    │
╰─────────────────────────────────┴───────╯

2025-06-19T03:22:06.969631Z  INFO oarfish::bulk: Total number of alignment records : 116
2025-06-19T03:22:06.969633Z  INFO oarfish::bulk: number of aligned reads : 96
2025-06-19T03:22:06.969636Z  INFO oarfish::bulk: number of unique alignments : 86
2025-06-19T03:22:06.970220Z  INFO oarfish: oarfish completed successfully.
2025-06-19T03:22:06.976958Z  INFO oarfish: setting user-provided filter parameters.
2025-06-19T03:22:06.977494Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp7HpXG6/file10a9132ad4b4fb/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-06-19T03:22:06.977505Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-06-19T03:22:06.977516Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-06-19T03:22:06.977566Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-06-19T03:22:06.977571Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-06-19T03:22:06.979516Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-06-19T03:22:06.979635Z  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    │
╰─────────────────────────────────┴───────╯

2025-06-19T03:22:06.979655Z  INFO oarfish::bulk: Total number of alignment records : 114
2025-06-19T03:22:06.979658Z  INFO oarfish::bulk: number of aligned reads : 95
2025-06-19T03:22:06.979660Z  INFO oarfish::bulk: number of unique alignments : 82
2025-06-19T03:22:06.980232Z  INFO oarfish: oarfish completed successfully.
2025-06-19T03:22:06.987112Z  INFO oarfish: setting user-provided filter parameters.
2025-06-19T03:22:06.987464Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp7HpXG6/file10a9132ad4b4fb/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-06-19T03:22:06.987472Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-06-19T03:22:06.987475Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-06-19T03:22:06.987537Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-06-19T03:22:06.987542Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-06-19T03:22:06.990190Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2025-06-19T03:22:06.990342Z  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   │
╰─────────────────────────────────┴───────╯

2025-06-19T03:22:06.990370Z  INFO oarfish::bulk: Total number of alignment records : 239
2025-06-19T03:22:06.990373Z  INFO oarfish::bulk: number of aligned reads : 179
2025-06-19T03:22:06.990384Z  INFO oarfish::bulk: number of unique alignments : 143
2025-06-19T03:22:06.991044Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp7HpXG6/file10a91312c57d6/config_file_1091859.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 @ Wed Jun 18 23:22:07 2025 -------------------
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp7HpXG6/file10a91322251b2d/config_file_1091859.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 @ Wed Jun 18 23:22:29 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/Rtmp7HpXG6/file10a91322251b2d/sample1_align2genome.bam
[M::mm_idx_gen::0.001*1.46] collected minimizers
[M::mm_idx_gen::0.002*1.31] sorted minimizers
[M::main::0.002*1.31] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*1.29] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*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::0.169*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/Rtmp7HpXG6/file10a91322251b2d/reference.bed --junc-bonus 1 /tmp/Rtmp7HpXG6/file10a91322251b2d/rps24.fa /tmp/Rtmp7HpXG6/file10a91322251b2d/fastq/sample1.fq.gz
[M::main] Real time: 0.170 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/Rtmp7HpXG6/file10a91322251b2d/sample2_align2genome.bam
[M::mm_idx_gen::0.001*1.68] collected minimizers
[M::mm_idx_gen::0.002*1.44] sorted minimizers
[M::main::0.002*1.43] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*1.40] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*1.38] 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/Rtmp7HpXG6/file10a91322251b2d/reference.bed --junc-bonus 1 /tmp/Rtmp7HpXG6/file10a91322251b2d/rps24.fa /tmp/Rtmp7HpXG6/file10a91322251b2d/fastq/sample2.fq.gz
[M::main] Real time: 0.170 sec; CPU: 0.171 sec; Peak RSS: 0.006 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/Rtmp7HpXG6/file10a91322251b2d/sample3_align2genome.bam
[M::mm_idx_gen::0.001*1.12] collected minimizers
[M::mm_idx_gen::0.002*1.08] sorted minimizers
[M::main::0.002*1.08] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*1.07] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*1.07] 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::0.338*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/Rtmp7HpXG6/file10a91322251b2d/reference.bed --junc-bonus 1 /tmp/Rtmp7HpXG6/file10a91322251b2d/rps24.fa /tmp/Rtmp7HpXG6/file10a91322251b2d/fastq/sample3.fq.gz
[M::main] Real time: 0.339 sec; CPU: 0.339 sec; Peak RSS: 0.006 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Wed Jun 18 23:22:30 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 @ Wed Jun 18 23:22:49 2025 -------------------
Realigning sample sample1 -> /tmp/Rtmp7HpXG6/file10a91322251b2d/sample1_realign2transcript.bam
[M::mm_idx_gen::0.002*1.30] collected minimizers
[M::mm_idx_gen::0.002*1.27] sorted minimizers
[M::main::0.002*1.27] loaded/built the index for 5 target sequence(s)
[M::mm_mapopt_update::0.002*1.26] mid_occ = 10
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 5
[M::mm_idx_stat::0.002*1.25] 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/Rtmp7HpXG6/file10a91322251b2d/transcript_assembly.fa /tmp/Rtmp7HpXG6/file10a91322251b2d/fastq/sample1.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 sample2 -> /tmp/Rtmp7HpXG6/file10a91322251b2d/sample2_realign2transcript.bam
[M::mm_idx_gen::0.001*2.12] collected minimizers
[M::mm_idx_gen::0.001*1.89] sorted minimizers
[M::main::0.001*1.88] loaded/built the index for 5 target sequence(s)
[M::mm_mapopt_update::0.001*1.84] mid_occ = 10
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 5
[M::mm_idx_stat::0.001*1.81] distinct minimizers: 198 (33.33% are singletons); average occurrences: 2.884; average spacing: 5.196; total length: 2967
[M::worker_pipeline::0.070*1.02] 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/Rtmp7HpXG6/file10a91322251b2d/transcript_assembly.fa /tmp/Rtmp7HpXG6/file10a91322251b2d/fastq/sample2.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 sample3 -> /tmp/Rtmp7HpXG6/file10a91322251b2d/sample3_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::0.136*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/Rtmp7HpXG6/file10a91322251b2d/transcript_assembly.fa /tmp/Rtmp7HpXG6/file10a91322251b2d/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 @ Wed Jun 18 23:22:49 2025 ----------
23:22:49 Wed Jun 18 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/Rtmp7HpXG6/file10a9131e705628/config_file_1091859.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 @ Wed Jun 18 23:22:50 2025 -------------------
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp7HpXG6/file10a9135ccc6b74/config_file_1091859.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 @ Wed Jun 18 23:23:12 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/Rtmp7HpXG6/file10a9135ccc6b74/sample1_align2genome.bam
[M::mm_idx_gen::0.003*1.22] collected minimizers
[M::mm_idx_gen::0.004*1.18] sorted minimizers
[M::main::0.004*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::0.171*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/Rtmp7HpXG6/file10a9135ccc6b74/reference.bed --junc-bonus 1 /tmp/Rtmp7HpXG6/file10a9135ccc6b74/rps24.fa /tmp/Rtmp7HpXG6/file10a9135ccc6b74/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/Rtmp7HpXG6/file10a9135ccc6b74/sample2_align2genome.bam
[M::mm_idx_gen::0.001*1.21] 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*1.13] 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::0.172*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 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/Rtmp7HpXG6/file10a9135ccc6b74/reference.bed --junc-bonus 1 /tmp/Rtmp7HpXG6/file10a9135ccc6b74/rps24.fa /tmp/Rtmp7HpXG6/file10a9135ccc6b74/fastq/sample2.fq.gz
[M::main] Real time: 0.173 sec; CPU: 0.173 sec; Peak RSS: 0.004 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/Rtmp7HpXG6/file10a9135ccc6b74/sample3_align2genome.bam
[M::mm_idx_gen::0.001*1.17] collected minimizers
[M::mm_idx_gen::0.002*1.11] sorted minimizers
[M::main::0.002*1.11] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*1.11] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*1.10] 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::0.330*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/Rtmp7HpXG6/file10a9135ccc6b74/reference.bed --junc-bonus 1 /tmp/Rtmp7HpXG6/file10a9135ccc6b74/rps24.fa /tmp/Rtmp7HpXG6/file10a9135ccc6b74/fastq/sample3.fq.gz
[M::main] Real time: 0.331 sec; CPU: 0.331 sec; Peak RSS: 0.007 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Wed Jun 18 23:23:13 2025 -------------
Inputs:  ['/tmp/Rtmp7HpXG6/file10a91322251b2d/sample1_realign2transcript.bam', '/tmp/Rtmp7HpXG6/file10a91322251b2d/sample2_realign2transcript.bam', '/tmp/Rtmp7HpXG6/file10a91322251b2d/sample3_realign2transcript.bam'] /tmp/Rtmp7HpXG6/file10a91322251b2d/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 @ Wed Jun 18 23:23:14 2025 -------------------
Realigning sample sample1 -> /tmp/Rtmp7HpXG6/file10a9135ccc6b74/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::0.175*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/Rtmp7HpXG6/file10a9135ccc6b74/transcript_assembly.fa /tmp/Rtmp7HpXG6/file10a9135ccc6b74/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/Rtmp7HpXG6/file10a9135ccc6b74/sample2_realign2transcript.bam
[M::mm_idx_gen::0.001*1.39] collected minimizers
[M::mm_idx_gen::0.002*1.31] sorted minimizers
[M::main::0.002*1.31] loaded/built the index for 10 target sequence(s)
[M::mm_mapopt_update::0.002*1.29] mid_occ = 11
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 10
[M::mm_idx_stat::0.002*1.28] distinct minimizers: 611 (72.01% are singletons); average occurrences: 2.597; average spacing: 5.171; total length: 8207
[M::worker_pipeline::0.174*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/Rtmp7HpXG6/file10a9135ccc6b74/transcript_assembly.fa /tmp/Rtmp7HpXG6/file10a9135ccc6b74/fastq/sample2.fq.gz
[M::main] Real time: 0.174 sec; CPU: 0.175 sec; Peak RSS: 0.004 GB
Skipped sorting BAM files.

Realigning sample sample3 -> /tmp/Rtmp7HpXG6/file10a9135ccc6b74/sample3_realign2transcript.bam
[M::mm_idx_gen::0.001*0.86] collected minimizers
[M::mm_idx_gen::0.002*0.90] sorted minimizers
[M::main::0.002*0.90] loaded/built the index for 10 target sequence(s)
[M::mm_mapopt_update::0.002*0.91] mid_occ = 11
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 10
[M::mm_idx_stat::0.002*0.91] distinct minimizers: 611 (72.01% are singletons); average occurrences: 2.597; average spacing: 5.171; total length: 8207
[M::worker_pipeline::0.306*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/Rtmp7HpXG6/file10a9135ccc6b74/transcript_assembly.fa /tmp/Rtmp7HpXG6/file10a9135ccc6b74/fastq/sample3.fq.gz
[M::main] Real time: 0.306 sec; CPU: 0.306 sec; Peak RSS: 0.004 GB
Skipped sorting BAM files.

-- Running step: transcript_quantification @ Wed Jun 18 23:23:15 2025 ----------
2025-06-19T03:23:15.028753Z  INFO oarfish: setting user-provided filter parameters.
2025-06-19T03:23:15.029146Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp7HpXG6/file10a9135ccc6b74/sample1_realign2transcript.bam, contains 10 reference sequences.
2025-06-19T03:23:15.029158Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-06-19T03:23:15.029161Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-06-19T03:23:15.029234Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-06-19T03:23:15.029241Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-06-19T03:23:15.031848Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-06-19T03:23:15.031994Z  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    │
╰─────────────────────────────────┴───────╯

2025-06-19T03:23:15.032029Z  INFO oarfish::bulk: Total number of alignment records : 125
2025-06-19T03:23:15.032032Z  INFO oarfish::bulk: number of aligned reads : 98
2025-06-19T03:23:15.032035Z  INFO oarfish::bulk: number of unique alignments : 86
2025-06-19T03:23:15.032639Z  INFO oarfish: oarfish completed successfully.
2025-06-19T03:23:15.040516Z  INFO oarfish: setting user-provided filter parameters.
2025-06-19T03:23:15.040983Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp7HpXG6/file10a9135ccc6b74/sample2_realign2transcript.bam, contains 10 reference sequences.
2025-06-19T03:23:15.040991Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-06-19T03:23:15.040994Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-06-19T03:23:15.041084Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-06-19T03:23:15.041094Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-06-19T03:23:15.043924Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-06-19T03:23:15.044069Z  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    │
╰─────────────────────────────────┴───────╯

2025-06-19T03:23:15.044097Z  INFO oarfish::bulk: Total number of alignment records : 136
2025-06-19T03:23:15.044099Z  INFO oarfish::bulk: number of aligned reads : 97
2025-06-19T03:23:15.044102Z  INFO oarfish::bulk: number of unique alignments : 79
2025-06-19T03:23:15.044723Z  INFO oarfish: oarfish completed successfully.
2025-06-19T03:23:15.052072Z  INFO oarfish: setting user-provided filter parameters.
2025-06-19T03:23:15.052539Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp7HpXG6/file10a9135ccc6b74/sample3_realign2transcript.bam, contains 10 reference sequences.
2025-06-19T03:23:15.052548Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-06-19T03:23:15.052550Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-06-19T03:23:15.052616Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-06-19T03:23:15.052622Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-06-19T03:23:15.056766Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-06-19T03:23:15.056935Z  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   │
╰─────────────────────────────────┴───────╯

2025-06-19T03:23:15.056961Z  INFO oarfish::bulk: Total number of alignment records : 272
2025-06-19T03:23:15.056964Z  INFO oarfish::bulk: number of aligned reads : 187
2025-06-19T03:23:15.056966Z  INFO oarfish::bulk: number of unique alignments : 140
2025-06-19T03:23:15.057657Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp7HpXG6/file10a9134dcb0231/config_file_1091859.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 @ Wed Jun 18 23:23:15 2025 -------------------
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp7HpXG6/file10a9131f36f978/config_file_1091859.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 @ Wed Jun 18 23:23:36 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/Rtmp7HpXG6/file10a9131f36f978/sample1_align2genome.bam
[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::0.003*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::0.172*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/Rtmp7HpXG6/file10a9131f36f978/reference.bed --junc-bonus 1 /tmp/Rtmp7HpXG6/file10a9131f36f978/rps24.fa /tmp/Rtmp7HpXG6/file10a9131f36f978/fastq/sample1.fq.gz
[M::main] Real time: 0.172 sec; CPU: 0.174 sec; Peak RSS: 0.006 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/Rtmp7HpXG6/file10a9131f36f978/sample2_align2genome.bam
[M::mm_idx_gen::0.001*0.96] collected minimizers
[M::mm_idx_gen::0.002*0.97] sorted minimizers
[M::main::0.002*0.97] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*0.98] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*0.98] 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/Rtmp7HpXG6/file10a9131f36f978/reference.bed --junc-bonus 1 /tmp/Rtmp7HpXG6/file10a9131f36f978/rps24.fa /tmp/Rtmp7HpXG6/file10a9131f36f978/fastq/sample2.fq.gz
[M::main] Real time: 0.171 sec; CPU: 0.171 sec; Peak RSS: 0.004 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/Rtmp7HpXG6/file10a9131f36f978/sample3_align2genome.bam
[M::mm_idx_gen::0.003*1.19] collected minimizers
[M::mm_idx_gen::0.003*1.15] sorted minimizers
[M::main::0.003*1.15] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.004*1.15] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.004*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::0.334*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/Rtmp7HpXG6/file10a9131f36f978/reference.bed --junc-bonus 1 /tmp/Rtmp7HpXG6/file10a9131f36f978/rps24.fa /tmp/Rtmp7HpXG6/file10a9131f36f978/fastq/sample3.fq.gz
[M::main] Real time: 0.334 sec; CPU: 0.335 sec; Peak RSS: 0.006 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Wed Jun 18 23:23:37 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 @ Wed Jun 18 23:23:37 2025 -------------------
Realigning sample sample1 -> /tmp/Rtmp7HpXG6/file10a9131f36f978/sample1_realign2transcript.bam
[M::mm_idx_gen::0.001*1.39] collected minimizers
[M::mm_idx_gen::0.002*1.28] sorted minimizers
[M::main::0.002*1.27] loaded/built the index for 10 target sequence(s)
[M::mm_mapopt_update::0.002*1.26] mid_occ = 11
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 10
[M::mm_idx_stat::0.002*1.25] distinct minimizers: 611 (72.01% are singletons); average occurrences: 2.597; average spacing: 5.171; total length: 8207
[M::worker_pipeline::0.094*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/Rtmp7HpXG6/file10a9131f36f978/transcript_assembly.fa /tmp/Rtmp7HpXG6/file10a9131f36f978/fastq/sample1.fq.gz
[M::main] Real time: 0.094 sec; CPU: 0.095 sec; Peak RSS: 0.003 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample2 -> /tmp/Rtmp7HpXG6/file10a9131f36f978/sample2_realign2transcript.bam
[M::mm_idx_gen::0.002*1.14] collected minimizers
[M::mm_idx_gen::0.003*1.12] sorted minimizers
[M::main::0.003*1.11] loaded/built the index for 10 target sequence(s)
[M::mm_mapopt_update::0.003*1.11] mid_occ = 11
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 10
[M::mm_idx_stat::0.003*1.11] 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/Rtmp7HpXG6/file10a9131f36f978/transcript_assembly.fa /tmp/Rtmp7HpXG6/file10a9131f36f978/fastq/sample2.fq.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 sample3 -> /tmp/Rtmp7HpXG6/file10a9131f36f978/sample3_realign2transcript.bam
[M::mm_idx_gen::0.001*1.18] collected minimizers
[M::mm_idx_gen::0.002*1.14] sorted minimizers
[M::main::0.002*1.14] loaded/built the index for 10 target sequence(s)
[M::mm_mapopt_update::0.002*1.13] mid_occ = 11
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 10
[M::mm_idx_stat::0.002*1.12] distinct minimizers: 611 (72.01% are singletons); average occurrences: 2.597; average spacing: 5.171; total length: 8207
[M::worker_pipeline::0.174*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/Rtmp7HpXG6/file10a9131f36f978/transcript_assembly.fa /tmp/Rtmp7HpXG6/file10a9131f36f978/fastq/sample3.fq.gz
[M::main] Real time: 0.175 sec; CPU: 0.175 sec; Peak RSS: 0.004 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Wed Jun 18 23:23:38 2025 ----------
23:23:38 Wed Jun 18 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Inputs:  ['/tmp/Rtmp7HpXG6/file10a9131f36f978/sample1_realign2transcript.bam', '/tmp/Rtmp7HpXG6/file10a9131f36f978/sample2_realign2transcript.bam', '/tmp/Rtmp7HpXG6/file10a9131f36f978/sample3_realign2transcript.bam'] /tmp/Rtmp7HpXG6/file10a9131f36f978/transcript_assembly.fa.fai 5 0.4 0.4
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp7HpXG6/file10a91362f1e6db/config_file_1091859.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 @ Wed Jun 18 23:23:39 2025 -------------------
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp7HpXG6/file10a91321480703/config_file_1091859.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 @ Wed Jun 18 23:24:01 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/Rtmp7HpXG6/file10a91321480703/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 @ Wed Jun 18 23:24:01 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmp7HpXG6/file10a91321480703/matched_reads.fastq.gz -> /tmp/Rtmp7HpXG6/file10a91321480703/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*1.60] collected minimizers
[M::mm_idx_gen::0.003*1.64] sorted minimizers
[M::main::0.003*1.64] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.003*1.59] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.003*1.56] 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::0.125*7.34] 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/Rtmp7HpXG6/file10a91321480703/reference.bed --junc-bonus 1 /tmp/Rtmp7HpXG6/file10a91321480703/rps24.fa /tmp/Rtmp7HpXG6/file10a91321480703/matched_reads.fastq.gz
[M::main] Real time: 0.126 sec; CPU: 0.921 sec; Peak RSS: 0.020 GB
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: gene_quantification @ Wed Jun 18 23:24:01 2025 ----------------
23:24:01 Wed Jun 18 2025 quantify genes 
Using BAM(s): '/tmp/Rtmp7HpXG6/file10a91321480703/align2genome.bam'
	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,  2.20gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.20gene_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, 39524.01Read/s]
-- Running step: isoform_identification @ Wed Jun 18 23:24:03 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 @ Wed Jun 18 23:24:13 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/Rtmp7HpXG6/file10a91321480703/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp7HpXG6/file10a91321480703/matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/Rtmp7HpXG6/file10a91321480703/matched_reads_dedup.fastq.gz -> /tmp/Rtmp7HpXG6/file10a91321480703/realign2transcript.bam
[M::mm_idx_gen::0.001*2.01] collected minimizers
[M::mm_idx_gen::0.002*2.30] sorted minimizers
[M::main::0.002*2.28] loaded/built the index for 5 target sequence(s)
[M::mm_mapopt_update::0.002*2.22] mid_occ = 10
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 5
[M::mm_idx_stat::0.002*2.19] distinct minimizers: 198 (33.33% are singletons); average occurrences: 2.884; average spacing: 5.196; total length: 2967
[M::worker_pipeline::0.054*6.45] 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/Rtmp7HpXG6/file10a91321480703/transcript_assembly.fa /tmp/Rtmp7HpXG6/file10a91321480703/matched_reads_dedup.fastq.gz
[M::main] Real time: 0.054 sec; CPU: 0.349 sec; Peak RSS: 0.008 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 @ Wed Jun 18 23:24:13 2025 ----------
2025-06-19T03:24:13.729899Z  INFO oarfish: setting user-provided filter parameters.
2025-06-19T03:24:13.730457Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp7HpXG6/file10a91321480703/realign2transcript.bam, contains 5 reference sequences.
2025-06-19T03:24:13.730468Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-06-19T03:24:13.730472Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-06-19T03:24:13.730541Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-06-19T03:24:13.730548Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-06-19T03:24:13.737315Z  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/Rtmp7HpXG6/file10a91368a2e272/config_file_1091859.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 @ Wed Jun 18 23:24:15 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/Rtmp7HpXG6/file10a91368a2e272/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 @ Wed Jun 18 23:24:15 2025 -------------------
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp7HpXG6/file10a9134fbbfe00/config_file_1091859.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 @ Wed Jun 18 23:24:37 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/Rtmp7HpXG6/file10a9134fbbfe00/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 @ Wed Jun 18 23:24:38 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmp7HpXG6/file10a9134fbbfe00/matched_reads.fastq.gz -> /tmp/Rtmp7HpXG6/file10a9134fbbfe00/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*1.34] collected minimizers
[M::mm_idx_gen::0.002*2.38] sorted minimizers
[M::main::0.002*2.36] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*2.28] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*2.21] 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::0.095*7.21] 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/Rtmp7HpXG6/file10a9134fbbfe00/reference.bed --junc-bonus 1 /tmp/Rtmp7HpXG6/file10a9134fbbfe00/rps24.fa /tmp/Rtmp7HpXG6/file10a9134fbbfe00/matched_reads.fastq.gz
[M::main] Real time: 0.096 sec; CPU: 0.687 sec; Peak RSS: 0.019 GB
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: gene_quantification @ Wed Jun 18 23:24:38 2025 ----------------
23:24:38 Wed Jun 18 2025 quantify genes 
Using BAM(s): '/tmp/Rtmp7HpXG6/file10a9134fbbfe00/align2genome.bam'
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  1.93gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  1.93gene_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, 38654.04Read/s]
-- Running step: isoform_identification @ Wed Jun 18 23:24:39 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 @ Wed Jun 18 23:24:49 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/Rtmp7HpXG6/file10a9134fbbfe00/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp7HpXG6/file10a9134fbbfe00/matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/Rtmp7HpXG6/file10a9134fbbfe00/matched_reads_dedup.fastq.gz -> /tmp/Rtmp7HpXG6/file10a9134fbbfe00/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*1.55] collected minimizers
[M::mm_idx_gen::0.002*3.21] sorted minimizers
[M::main::0.002*3.19] loaded/built the index for 5 target sequence(s)
[M::mm_mapopt_update::0.002*3.11] mid_occ = 10
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 5
[M::mm_idx_stat::0.002*3.06] distinct minimizers: 198 (33.33% are singletons); average occurrences: 2.884; average spacing: 5.196; total length: 2967
[M::worker_pipeline::0.042*6.35] 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/Rtmp7HpXG6/file10a9134fbbfe00/transcript_assembly.fa /tmp/Rtmp7HpXG6/file10a9134fbbfe00/matched_reads_dedup.fastq.gz
[M::main] Real time: 0.042 sec; CPU: 0.265 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 @ Wed Jun 18 23:24:49 2025 ----------
23:24:49 Wed Jun 18 2025 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp7HpXG6/file10a91346cbceea/config_file_1091859.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 @ Wed Jun 18 23:24:50 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/Rtmp7HpXG6/file10a91346cbceea/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 @ Wed Jun 18 23:24:50 2025 -------------------
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp7HpXG6/file10a91346240a99/config_file_1091859.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 @ Wed Jun 18 23:25:12 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/Rtmp7HpXG6/file10a91346240a99/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 @ Wed Jun 18 23:25:12 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmp7HpXG6/file10a91346240a99/matched_reads.fastq.gz -> /tmp/Rtmp7HpXG6/file10a91346240a99/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*0.96] collected minimizers
[M::mm_idx_gen::0.002*0.72] sorted minimizers
[M::main::0.002*0.72] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*0.74] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*0.76] 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*7.12] 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/Rtmp7HpXG6/file10a91346240a99/reference.bed --junc-bonus 1 /tmp/Rtmp7HpXG6/file10a91346240a99/rps24.fa /tmp/Rtmp7HpXG6/file10a91346240a99/matched_reads.fastq.gz
[M::main] Real time: 0.096 sec; CPU: 0.683 sec; Peak RSS: 0.019 GB
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: gene_quantification @ Wed Jun 18 23:25:12 2025 ----------------
23:25:12 Wed Jun 18 2025 quantify genes 
Using BAM(s): '/tmp/Rtmp7HpXG6/file10a91346240a99/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,  2.35gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.35gene_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, 39236.95Read/s]
-- Running step: isoform_identification @ Wed Jun 18 23:25:13 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 @ Wed Jun 18 23:25:14 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/Rtmp7HpXG6/file10a91346240a99/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp7HpXG6/file10a91346240a99/matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/Rtmp7HpXG6/file10a91346240a99/matched_reads_dedup.fastq.gz -> /tmp/Rtmp7HpXG6/file10a91346240a99/realign2transcript.bam
[M::mm_idx_gen::0.001*0.95] collected minimizers
[M::mm_idx_gen::0.002*2.75] sorted minimizers
[M::main::0.002*2.73] loaded/built the index for 10 target sequence(s)
[M::mm_mapopt_update::0.002*2.66] mid_occ = 11
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 10
[M::mm_idx_stat::0.002*2.60] distinct minimizers: 611 (72.01% are singletons); average occurrences: 2.597; average spacing: 5.171; total length: 8207
[M::worker_pipeline::0.082*6.52] 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/Rtmp7HpXG6/file10a91346240a99/transcript_assembly.fa /tmp/Rtmp7HpXG6/file10a91346240a99/matched_reads_dedup.fastq.gz
[M::main] Real time: 0.083 sec; CPU: 0.538 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 @ Wed Jun 18 23:25:14 2025 ----------
2025-06-19T03:25:14.368769Z  INFO oarfish: setting user-provided filter parameters.
2025-06-19T03:25:14.369177Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp7HpXG6/file10a91346240a99/realign2transcript.bam, contains 10 reference sequences.
2025-06-19T03:25:14.369189Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-06-19T03:25:14.369192Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-06-19T03:25:14.369259Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-06-19T03:25:14.369267Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-06-19T03:25:14.378396Z  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/Rtmp7HpXG6/file10a91368d8d33b/config_file_1091859.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 @ Wed Jun 18 23:25:16 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/Rtmp7HpXG6/file10a91368d8d33b/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 @ Wed Jun 18 23:25:16 2025 -------------------
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp7HpXG6/file10a913556169e6/config_file_1091859.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 @ Wed Jun 18 23:25:37 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/Rtmp7HpXG6/file10a913556169e6/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 @ Wed Jun 18 23:25:38 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmp7HpXG6/file10a913556169e6/matched_reads.fastq.gz -> /tmp/Rtmp7HpXG6/file10a913556169e6/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*1.26] collected minimizers
[M::mm_idx_gen::0.002*1.32] sorted minimizers
[M::main::0.002*1.32] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*1.30] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*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::0.095*7.16] 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/Rtmp7HpXG6/file10a913556169e6/reference.bed --junc-bonus 1 /tmp/Rtmp7HpXG6/file10a913556169e6/rps24.fa /tmp/Rtmp7HpXG6/file10a913556169e6/matched_reads.fastq.gz
[M::main] Real time: 0.096 sec; CPU: 0.682 sec; Peak RSS: 0.019 GB
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: gene_quantification @ Wed Jun 18 23:25:38 2025 ----------------
23:25:38 Wed Jun 18 2025 quantify genes 
Using BAM(s): '/tmp/Rtmp7HpXG6/file10a913556169e6/align2genome.bam'
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.10gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.10gene_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, 38535.15Read/s]
-- Running step: isoform_identification @ Wed Jun 18 23:25:39 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 @ Wed Jun 18 23:25:39 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/Rtmp7HpXG6/file10a913556169e6/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp7HpXG6/file10a913556169e6/matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/Rtmp7HpXG6/file10a913556169e6/matched_reads_dedup.fastq.gz -> /tmp/Rtmp7HpXG6/file10a913556169e6/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*1.77] collected minimizers
[M::mm_idx_gen::0.002*2.88] sorted minimizers
[M::main::0.002*2.86] loaded/built the index for 10 target sequence(s)
[M::mm_mapopt_update::0.002*2.78] mid_occ = 11
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 10
[M::mm_idx_stat::0.002*2.72] distinct minimizers: 611 (72.01% are singletons); average occurrences: 2.597; average spacing: 5.171; total length: 8207
[M::worker_pipeline::0.053*6.52] 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/Rtmp7HpXG6/file10a913556169e6/transcript_assembly.fa /tmp/Rtmp7HpXG6/file10a913556169e6/matched_reads_dedup.fastq.gz
[M::main] Real time: 0.054 sec; CPU: 0.349 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 @ Wed Jun 18 23:25:40 2025 ----------
23:25:40 Wed Jun 18 2025 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp7HpXG6/file10a9131096149a/config_file_1091859.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 @ Wed Jun 18 23:25:41 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/Rtmp7HpXG6/file10a9131096149a/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 @ Wed Jun 18 23:25:41 2025 -------------------
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp7HpXG6/file10a913f551ac0/config_file_1091859.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 @ Wed Jun 18 23:26:03 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/Rtmp7HpXG6/file10a913f551ac0/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp7HpXG6/file10a913f551ac0/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmp7HpXG6/file10a913f551ac0/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmp7HpXG6/file10a913f551ac0/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/Rtmp7HpXG6/file10a913f551ac0/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp7HpXG6/file10a913f551ac0/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/Rtmp7HpXG6/file10a913f551ac0/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp7HpXG6/file10a913f551ac0/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/Rtmp7HpXG6/file10a913f551ac0/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp7HpXG6/file10a913f551ac0/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 @ Wed Jun 18 23:26:04 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmp7HpXG6/file10a913f551ac0/sampleA_matched_reads.fastq.gz -> /tmp/Rtmp7HpXG6/file10a913f551ac0/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*0.93] collected minimizers
[M::mm_idx_gen::0.002*0.96] sorted minimizers
[M::main::0.002*0.96] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*0.96] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*0.96] 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::0.602*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/Rtmp7HpXG6/file10a913f551ac0/reference.bed --junc-bonus 1 /tmp/Rtmp7HpXG6/file10a913f551ac0/rps24.fa /tmp/Rtmp7HpXG6/file10a913f551ac0/sampleA_matched_reads.fastq.gz
[M::main] Real time: 0.603 sec; CPU: 0.603 sec; Peak RSS: 0.007 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmp7HpXG6/file10a913f551ac0/sample1_matched_reads.fastq.gz -> /tmp/Rtmp7HpXG6/file10a913f551ac0/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*0.86] collected minimizers
[M::mm_idx_gen::0.002*0.90] sorted minimizers
[M::main::0.002*0.91] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*0.91] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*0.92] 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/Rtmp7HpXG6/file10a913f551ac0/reference.bed --junc-bonus 1 /tmp/Rtmp7HpXG6/file10a913f551ac0/rps24.fa /tmp/Rtmp7HpXG6/file10a913f551ac0/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/Rtmp7HpXG6/file10a913f551ac0/sample2_matched_reads.fastq.gz -> /tmp/Rtmp7HpXG6/file10a913f551ac0/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*1.53] collected minimizers
[M::mm_idx_gen::0.002*1.35] sorted minimizers
[M::main::0.002*1.34] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*1.32] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*1.31] 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/Rtmp7HpXG6/file10a913f551ac0/reference.bed --junc-bonus 1 /tmp/Rtmp7HpXG6/file10a913f551ac0/rps24.fa /tmp/Rtmp7HpXG6/file10a913f551ac0/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/Rtmp7HpXG6/file10a913f551ac0/sample3_matched_reads.fastq.gz -> /tmp/Rtmp7HpXG6/file10a913f551ac0/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::0.002*1.05] collected minimizers
[M::mm_idx_gen::0.003*1.04] sorted minimizers
[M::main::0.003*1.04] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.003*1.04] 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::0.303*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/Rtmp7HpXG6/file10a913f551ac0/reference.bed --junc-bonus 1 /tmp/Rtmp7HpXG6/file10a913f551ac0/rps24.fa /tmp/Rtmp7HpXG6/file10a913f551ac0/sample3_matched_reads.fastq.gz
[M::main] Real time: 0.304 sec; CPU: 0.304 sec; Peak RSS: 0.006 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Wed Jun 18 23:26:05 2025 ----------------
23:26:05 Wed Jun 18 2025 quantify genes 
Using BAM(s): '/tmp/Rtmp7HpXG6/file10a913f551ac0/sampleA_align2genome.bam',
'/tmp/Rtmp7HpXG6/file10a913f551ac0/sample1_align2genome.bam',
'/tmp/Rtmp7HpXG6/file10a913f551ac0/sample2_align2genome.bam', and
'/tmp/Rtmp7HpXG6/file10a913f551ac0/sample3_align2genome.bam'
	Counter({'counted_reads': 358})
parsing /tmp/Rtmp7HpXG6/file10a913f551ac0/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 18.94gene_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, 39155.19Read/s]
parsing /tmp/Rtmp7HpXG6/file10a913f551ac0/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 48.37gene_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, 188636.91Read/s]
parsing /tmp/Rtmp7HpXG6/file10a913f551ac0/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 45.54gene_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, 152882.62Read/s]
parsing /tmp/Rtmp7HpXG6/file10a913f551ac0/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 34.25gene_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, 76874.51Read/s]
-- Running step: isoform_identification @ Wed Jun 18 23:26:06 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 @ Wed Jun 18 23:26:30 2025 -------------------
Checking for fastq file(s) /tmp/Rtmp7HpXG6/file10a913f551ac0/fastq, /tmp/Rtmp7HpXG6/file10a913f551ac0/fastq/sample1.fq.gz, /tmp/Rtmp7HpXG6/file10a913f551ac0/fastq/sample2.fq.gz, /tmp/Rtmp7HpXG6/file10a913f551ac0/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp7HpXG6/file10a913f551ac0/sampleA_matched_reads.fastq.gz, /tmp/Rtmp7HpXG6/file10a913f551ac0/sample1_matched_reads.fastq.gz, /tmp/Rtmp7HpXG6/file10a913f551ac0/sample2_matched_reads.fastq.gz, /tmp/Rtmp7HpXG6/file10a913f551ac0/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp7HpXG6/file10a913f551ac0/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmp7HpXG6/file10a913f551ac0/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmp7HpXG6/file10a913f551ac0/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmp7HpXG6/file10a913f551ac0/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/Rtmp7HpXG6/file10a913f551ac0/sampleA_matched_reads_dedup.fastq.gz -> /tmp/Rtmp7HpXG6/file10a913f551ac0/sampleA_realign2transcript.bam
[M::mm_idx_gen::0.001*2.43] collected minimizers
[M::mm_idx_gen::0.001*2.08] sorted minimizers
[M::main::0.001*2.07] loaded/built the index for 5 target sequence(s)
[M::mm_mapopt_update::0.001*2.03] mid_occ = 10
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 5
[M::mm_idx_stat::0.001*1.99] distinct minimizers: 198 (33.33% are singletons); average occurrences: 2.884; average spacing: 5.196; total length: 2967
[M::worker_pipeline::0.270*1.01] 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/Rtmp7HpXG6/file10a913f551ac0/transcript_assembly.fa /tmp/Rtmp7HpXG6/file10a913f551ac0/sampleA_matched_reads_dedup.fastq.gz
[M::main] Real time: 0.270 sec; CPU: 0.272 sec; Peak RSS: 0.005 GB
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/Rtmp7HpXG6/file10a913f551ac0/sample1_matched_reads_dedup.fastq.gz -> /tmp/Rtmp7HpXG6/file10a913f551ac0/sample1_realign2transcript.bam
[M::mm_idx_gen::0.001*1.71] collected minimizers
[M::mm_idx_gen::0.001*1.56] sorted minimizers
[M::main::0.001*1.56] loaded/built the index for 5 target sequence(s)
[M::mm_mapopt_update::0.001*1.53] mid_occ = 10
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 5
[M::mm_idx_stat::0.001*1.51] 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 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/Rtmp7HpXG6/file10a913f551ac0/transcript_assembly.fa /tmp/Rtmp7HpXG6/file10a913f551ac0/sample1_matched_reads_dedup.fastq.gz
[M::main] Real time: 0.072 sec; CPU: 0.072 sec; Peak RSS: 0.003 GB
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/Rtmp7HpXG6/file10a913f551ac0/sample2_matched_reads_dedup.fastq.gz -> /tmp/Rtmp7HpXG6/file10a913f551ac0/sample2_realign2transcript.bam
[M::mm_idx_gen::0.001*2.18] collected minimizers
[M::mm_idx_gen::0.002*1.84] sorted minimizers
[M::main::0.002*1.83] loaded/built the index for 5 target sequence(s)
[M::mm_mapopt_update::0.002*1.81] mid_occ = 10
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 5
[M::mm_idx_stat::0.002*1.78] distinct minimizers: 198 (33.33% are singletons); average occurrences: 2.884; average spacing: 5.196; total length: 2967
[M::worker_pipeline::0.079*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 --eqx -N 100 -ax map-ont -t 1 --seed 2022 -y /tmp/Rtmp7HpXG6/file10a913f551ac0/transcript_assembly.fa /tmp/Rtmp7HpXG6/file10a913f551ac0/sample2_matched_reads_dedup.fastq.gz
[M::main] Real time: 0.079 sec; CPU: 0.080 sec; Peak RSS: 0.004 GB
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/Rtmp7HpXG6/file10a913f551ac0/sample3_matched_reads_dedup.fastq.gz -> /tmp/Rtmp7HpXG6/file10a913f551ac0/sample3_realign2transcript.bam
[M::mm_idx_gen::0.001*1.80] collected minimizers
[M::mm_idx_gen::0.001*1.59] sorted minimizers
[M::main::0.001*1.58] loaded/built the index for 5 target sequence(s)
[M::mm_mapopt_update::0.001*1.56] mid_occ = 10
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 5
[M::mm_idx_stat::0.001*1.54] distinct minimizers: 198 (33.33% are singletons); average occurrences: 2.884; average spacing: 5.196; total length: 2967
[M::worker_pipeline::0.135*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/Rtmp7HpXG6/file10a913f551ac0/transcript_assembly.fa /tmp/Rtmp7HpXG6/file10a913f551ac0/sample3_matched_reads_dedup.fastq.gz
[M::main] Real time: 0.136 sec; CPU: 0.136 sec; Peak RSS: 0.004 GB
Sorting BAM files by 1 with CB threads...

-- Running step: transcript_quantification @ Wed Jun 18 23:26:31 2025 ----------
2025-06-19T03:26:31.657825Z  INFO oarfish: setting user-provided filter parameters.
2025-06-19T03:26:31.658363Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp7HpXG6/file10a913f551ac0/sampleA_realign2transcript.bam, contains 5 reference sequences.
2025-06-19T03:26:31.658376Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-06-19T03:26:31.658379Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-06-19T03:26:31.658444Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-06-19T03:26:31.658461Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-06-19T03:26:31.664296Z  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
2025-06-19T03:26:33.018270Z  INFO oarfish: setting user-provided filter parameters.
2025-06-19T03:26:33.018612Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp7HpXG6/file10a913f551ac0/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-06-19T03:26:33.018620Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-06-19T03:26:33.018622Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-06-19T03:26:33.018678Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-06-19T03:26:33.018684Z  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
2025-06-19T03:26:34.365368Z  INFO oarfish: setting user-provided filter parameters.
2025-06-19T03:26:34.365745Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp7HpXG6/file10a913f551ac0/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-06-19T03:26:34.365754Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-06-19T03:26:34.365757Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-06-19T03:26:34.365825Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-06-19T03:26:34.365831Z  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
2025-06-19T03:26:35.684753Z  INFO oarfish: setting user-provided filter parameters.
2025-06-19T03:26:35.685099Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp7HpXG6/file10a913f551ac0/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-06-19T03:26:35.685109Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-06-19T03:26:35.685112Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-06-19T03:26:35.685164Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-06-19T03:26:35.685169Z  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/Rtmp7HpXG6/file10a91317ebbbff/config_file_1091859.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 @ Wed Jun 18 23:26:37 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/Rtmp7HpXG6/file10a91317ebbbff/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp7HpXG6/file10a91317ebbbff/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmp7HpXG6/file10a91317ebbbff/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmp7HpXG6/file10a91317ebbbff/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/Rtmp7HpXG6/file10a91317ebbbff/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp7HpXG6/file10a91317ebbbff/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/Rtmp7HpXG6/file10a91317ebbbff/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp7HpXG6/file10a91317ebbbff/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/Rtmp7HpXG6/file10a91317ebbbff/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp7HpXG6/file10a91317ebbbff/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 @ Wed Jun 18 23:26:38 2025 -------------------
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp7HpXG6/file10a9133010532d/config_file_1091859.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 @ Wed Jun 18 23:26:58 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 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmp7HpXG6/file10a9133010532d/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp7HpXG6/file10a9133010532d/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmp7HpXG6/file10a9133010532d/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmp7HpXG6/file10a9133010532d/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/Rtmp7HpXG6/file10a9133010532d/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp7HpXG6/file10a9133010532d/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/Rtmp7HpXG6/file10a9133010532d/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp7HpXG6/file10a9133010532d/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/Rtmp7HpXG6/file10a9133010532d/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp7HpXG6/file10a9133010532d/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 @ Wed Jun 18 23:26:59 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmp7HpXG6/file10a9133010532d/sampleA_matched_reads.fastq.gz -> /tmp/Rtmp7HpXG6/file10a9133010532d/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*1.81] collected minimizers
[M::mm_idx_gen::0.002*1.54] sorted minimizers
[M::main::0.002*1.54] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*1.50] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*1.47] 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::0.626*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/Rtmp7HpXG6/file10a9133010532d/reference.bed --junc-bonus 1 /tmp/Rtmp7HpXG6/file10a9133010532d/rps24.fa /tmp/Rtmp7HpXG6/file10a9133010532d/sampleA_matched_reads.fastq.gz
[M::main] Real time: 0.627 sec; CPU: 0.627 sec; Peak RSS: 0.006 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmp7HpXG6/file10a9133010532d/sample1_matched_reads.fastq.gz -> /tmp/Rtmp7HpXG6/file10a9133010532d/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*0.97] collected minimizers
[M::mm_idx_gen::0.002*0.98] sorted minimizers
[M::main::0.002*0.98] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*0.98] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*0.98] 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::0.153*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/Rtmp7HpXG6/file10a9133010532d/reference.bed --junc-bonus 1 /tmp/Rtmp7HpXG6/file10a9133010532d/rps24.fa /tmp/Rtmp7HpXG6/file10a9133010532d/sample1_matched_reads.fastq.gz
[M::main] Real time: 0.154 sec; CPU: 0.154 sec; Peak RSS: 0.005 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmp7HpXG6/file10a9133010532d/sample2_matched_reads.fastq.gz -> /tmp/Rtmp7HpXG6/file10a9133010532d/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*1.17] collected minimizers
[M::mm_idx_gen::0.002*1.12] sorted minimizers
[M::main::0.002*1.12] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*1.11] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*1.10] 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/Rtmp7HpXG6/file10a9133010532d/reference.bed --junc-bonus 1 /tmp/Rtmp7HpXG6/file10a9133010532d/rps24.fa /tmp/Rtmp7HpXG6/file10a9133010532d/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/Rtmp7HpXG6/file10a9133010532d/sample3_matched_reads.fastq.gz -> /tmp/Rtmp7HpXG6/file10a9133010532d/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*0.93] collected minimizers
[M::mm_idx_gen::0.002*0.96] sorted minimizers
[M::main::0.002*0.96] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*0.96] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*0.96] 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/Rtmp7HpXG6/file10a9133010532d/reference.bed --junc-bonus 1 /tmp/Rtmp7HpXG6/file10a9133010532d/rps24.fa /tmp/Rtmp7HpXG6/file10a9133010532d/sample3_matched_reads.fastq.gz
[M::main] Real time: 0.299 sec; CPU: 0.299 sec; Peak RSS: 0.006 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Wed Jun 18 23:27:01 2025 ----------------
23:27:01 Wed Jun 18 2025 quantify genes 
Using BAM(s): '/tmp/Rtmp7HpXG6/file10a9133010532d/sampleA_align2genome.bam',
'/tmp/Rtmp7HpXG6/file10a9133010532d/sample1_align2genome.bam',
'/tmp/Rtmp7HpXG6/file10a9133010532d/sample2_align2genome.bam', and
'/tmp/Rtmp7HpXG6/file10a9133010532d/sample3_align2genome.bam'
parsing /tmp/Rtmp7HpXG6/file10a9133010532d/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 22.75gene_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, 39431.42Read/s]
parsing /tmp/Rtmp7HpXG6/file10a9133010532d/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 53.35gene_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, 191440.31Read/s]
parsing /tmp/Rtmp7HpXG6/file10a9133010532d/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 51.55gene_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, 155103.32Read/s]
parsing /tmp/Rtmp7HpXG6/file10a9133010532d/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 34.82gene_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, 77555.68Read/s]
-- Running step: isoform_identification @ Wed Jun 18 23:27:02 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 @ Wed Jun 18 23:27:24 2025 -------------------
Checking for fastq file(s) /tmp/Rtmp7HpXG6/file10a9133010532d/fastq, /tmp/Rtmp7HpXG6/file10a9133010532d/fastq/sample1.fq.gz, /tmp/Rtmp7HpXG6/file10a9133010532d/fastq/sample2.fq.gz, /tmp/Rtmp7HpXG6/file10a9133010532d/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp7HpXG6/file10a9133010532d/sampleA_matched_reads.fastq.gz, /tmp/Rtmp7HpXG6/file10a9133010532d/sample1_matched_reads.fastq.gz, /tmp/Rtmp7HpXG6/file10a9133010532d/sample2_matched_reads.fastq.gz, /tmp/Rtmp7HpXG6/file10a9133010532d/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp7HpXG6/file10a9133010532d/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmp7HpXG6/file10a9133010532d/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmp7HpXG6/file10a9133010532d/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmp7HpXG6/file10a9133010532d/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/Rtmp7HpXG6/file10a9133010532d/sampleA_matched_reads_dedup.fastq.gz -> /tmp/Rtmp7HpXG6/file10a9133010532d/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::0.002*1.46] collected minimizers
[M::mm_idx_gen::0.002*1.41] sorted minimizers
[M::main::0.002*1.40] loaded/built the index for 5 target sequence(s)
[M::mm_mapopt_update::0.002*1.39] mid_occ = 10
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 5
[M::mm_idx_stat::0.002*1.38] distinct minimizers: 198 (33.33% are singletons); average occurrences: 2.884; average spacing: 5.196; total length: 2967
[M::worker_pipeline::0.232*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/Rtmp7HpXG6/file10a9133010532d/transcript_assembly.fa /tmp/Rtmp7HpXG6/file10a9133010532d/sampleA_matched_reads_dedup.fastq.gz
[M::main] Real time: 0.233 sec; CPU: 0.234 sec; Peak RSS: 0.004 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample /tmp/Rtmp7HpXG6/file10a9133010532d/sample1_matched_reads_dedup.fastq.gz -> /tmp/Rtmp7HpXG6/file10a9133010532d/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*1.84] collected minimizers
[M::mm_idx_gen::0.001*1.68] sorted minimizers
[M::main::0.001*1.67] loaded/built the index for 5 target sequence(s)
[M::mm_mapopt_update::0.001*1.64] 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::0.059*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/Rtmp7HpXG6/file10a9133010532d/transcript_assembly.fa /tmp/Rtmp7HpXG6/file10a9133010532d/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/Rtmp7HpXG6/file10a9133010532d/sample2_matched_reads_dedup.fastq.gz -> /tmp/Rtmp7HpXG6/file10a9133010532d/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*1.24] collected minimizers
[M::mm_idx_gen::0.001*1.19] sorted minimizers
[M::main::0.001*1.19] loaded/built the index for 5 target sequence(s)
[M::mm_mapopt_update::0.001*1.18] mid_occ = 10
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 5
[M::mm_idx_stat::0.001*1.17] distinct minimizers: 198 (33.33% are singletons); average occurrences: 2.884; average spacing: 5.196; total length: 2967
[M::worker_pipeline::0.063*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 -ax map-ont -p 0.9 --end-bonus 10 -N 3 -t 1 --seed 2022 /tmp/Rtmp7HpXG6/file10a9133010532d/transcript_assembly.fa /tmp/Rtmp7HpXG6/file10a9133010532d/sample2_matched_reads_dedup.fastq.gz
[M::main] Real time: 0.064 sec; CPU: 0.064 sec; Peak RSS: 0.003 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample /tmp/Rtmp7HpXG6/file10a9133010532d/sample3_matched_reads_dedup.fastq.gz -> /tmp/Rtmp7HpXG6/file10a9133010532d/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*1.70] collected minimizers
[M::mm_idx_gen::0.001*1.56] sorted minimizers
[M::main::0.001*1.55] loaded/built the index for 5 target sequence(s)
[M::mm_mapopt_update::0.001*1.53] mid_occ = 10
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 5
[M::mm_idx_stat::0.001*1.51] distinct minimizers: 198 (33.33% are singletons); average occurrences: 2.884; average spacing: 5.196; total length: 2967
[M::worker_pipeline::0.116*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/Rtmp7HpXG6/file10a9133010532d/transcript_assembly.fa /tmp/Rtmp7HpXG6/file10a9133010532d/sample3_matched_reads_dedup.fastq.gz
[M::main] Real time: 0.116 sec; CPU: 0.117 sec; Peak RSS: 0.003 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Wed Jun 18 23:27:25 2025 ----------
23:27:25 Wed Jun 18 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/Rtmp7HpXG6/file10a9133010532d/sampleA_realign2transcript.bam...
parsing /tmp/Rtmp7HpXG6/file10a9133010532d/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmp7HpXG6/file10a9133010532d/sampleA_realign2transcript.bamdone
parsing /tmp/Rtmp7HpXG6/file10a9133010532d/sample1_realign2transcript.bam...
parsing /tmp/Rtmp7HpXG6/file10a9133010532d/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmp7HpXG6/file10a9133010532d/sample1_realign2transcript.bamdone
parsing /tmp/Rtmp7HpXG6/file10a9133010532d/sample2_realign2transcript.bam...
parsing /tmp/Rtmp7HpXG6/file10a9133010532d/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmp7HpXG6/file10a9133010532d/sample2_realign2transcript.bamdone
parsing /tmp/Rtmp7HpXG6/file10a9133010532d/sample3_realign2transcript.bam...
parsing /tmp/Rtmp7HpXG6/file10a9133010532d/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmp7HpXG6/file10a9133010532d/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp7HpXG6/file10a91376b6a558/config_file_1091859.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 @ Wed Jun 18 23:27:27 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/Rtmp7HpXG6/file10a91376b6a558/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp7HpXG6/file10a91376b6a558/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmp7HpXG6/file10a91376b6a558/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmp7HpXG6/file10a91376b6a558/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/Rtmp7HpXG6/file10a91376b6a558/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp7HpXG6/file10a91376b6a558/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/Rtmp7HpXG6/file10a91376b6a558/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp7HpXG6/file10a91376b6a558/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/Rtmp7HpXG6/file10a91376b6a558/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp7HpXG6/file10a91376b6a558/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 @ Wed Jun 18 23:27:28 2025 -------------------
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp7HpXG6/file10a913707d9c91/config_file_1091859.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 @ Wed Jun 18 23:27:50 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/Rtmp7HpXG6/file10a913707d9c91/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp7HpXG6/file10a913707d9c91/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmp7HpXG6/file10a913707d9c91/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmp7HpXG6/file10a913707d9c91/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/Rtmp7HpXG6/file10a913707d9c91/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp7HpXG6/file10a913707d9c91/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/Rtmp7HpXG6/file10a913707d9c91/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp7HpXG6/file10a913707d9c91/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/Rtmp7HpXG6/file10a913707d9c91/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp7HpXG6/file10a913707d9c91/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 @ Wed Jun 18 23:27:51 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmp7HpXG6/file10a913707d9c91/sampleA_matched_reads.fastq.gz -> /tmp/Rtmp7HpXG6/file10a913707d9c91/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*1.41] collected minimizers
[M::mm_idx_gen::0.002*1.27] sorted minimizers
[M::main::0.002*1.27] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*1.25] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*1.24] 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/Rtmp7HpXG6/file10a913707d9c91/reference.bed --junc-bonus 1 /tmp/Rtmp7HpXG6/file10a913707d9c91/rps24.fa /tmp/Rtmp7HpXG6/file10a913707d9c91/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/Rtmp7HpXG6/file10a913707d9c91/sample1_matched_reads.fastq.gz -> /tmp/Rtmp7HpXG6/file10a913707d9c91/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*0.96] collected minimizers
[M::mm_idx_gen::0.003*0.97] sorted minimizers
[M::main::0.003*0.97] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.003*0.97] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.003*0.97] 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/Rtmp7HpXG6/file10a913707d9c91/reference.bed --junc-bonus 1 /tmp/Rtmp7HpXG6/file10a913707d9c91/rps24.fa /tmp/Rtmp7HpXG6/file10a913707d9c91/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/Rtmp7HpXG6/file10a913707d9c91/sample2_matched_reads.fastq.gz -> /tmp/Rtmp7HpXG6/file10a913707d9c91/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*1.49] collected minimizers
[M::mm_idx_gen::0.002*1.32] sorted minimizers
[M::main::0.002*1.32] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*1.29] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*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*1.00] 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/Rtmp7HpXG6/file10a913707d9c91/reference.bed --junc-bonus 1 /tmp/Rtmp7HpXG6/file10a913707d9c91/rps24.fa /tmp/Rtmp7HpXG6/file10a913707d9c91/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/Rtmp7HpXG6/file10a913707d9c91/sample3_matched_reads.fastq.gz -> /tmp/Rtmp7HpXG6/file10a913707d9c91/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*1.56] 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::0.299*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/Rtmp7HpXG6/file10a913707d9c91/reference.bed --junc-bonus 1 /tmp/Rtmp7HpXG6/file10a913707d9c91/rps24.fa /tmp/Rtmp7HpXG6/file10a913707d9c91/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 @ Wed Jun 18 23:27:52 2025 ----------------
23:27:52 Wed Jun 18 2025 quantify genes 
Using BAM(s): '/tmp/Rtmp7HpXG6/file10a913707d9c91/sampleA_align2genome.bam',
'/tmp/Rtmp7HpXG6/file10a913707d9c91/sample1_align2genome.bam',
'/tmp/Rtmp7HpXG6/file10a913707d9c91/sample2_align2genome.bam', and
'/tmp/Rtmp7HpXG6/file10a913707d9c91/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/Rtmp7HpXG6/file10a913707d9c91/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 22.87gene_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, 39550.85Read/s]
parsing /tmp/Rtmp7HpXG6/file10a913707d9c91/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 52.89gene_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, 186945.27Read/s]
parsing /tmp/Rtmp7HpXG6/file10a913707d9c91/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 48.33gene_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, 151585.28Read/s]
parsing /tmp/Rtmp7HpXG6/file10a913707d9c91/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 36.58gene_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, 77875.93Read/s]
-- Running step: isoform_identification @ Wed Jun 18 23:27:53 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 @ Wed Jun 18 23:27:54 2025 -------------------
Checking for fastq file(s) /tmp/Rtmp7HpXG6/file10a913707d9c91/fastq, /tmp/Rtmp7HpXG6/file10a913707d9c91/fastq/sample1.fq.gz, /tmp/Rtmp7HpXG6/file10a913707d9c91/fastq/sample2.fq.gz, /tmp/Rtmp7HpXG6/file10a913707d9c91/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp7HpXG6/file10a913707d9c91/sampleA_matched_reads.fastq.gz, /tmp/Rtmp7HpXG6/file10a913707d9c91/sample1_matched_reads.fastq.gz, /tmp/Rtmp7HpXG6/file10a913707d9c91/sample2_matched_reads.fastq.gz, /tmp/Rtmp7HpXG6/file10a913707d9c91/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp7HpXG6/file10a913707d9c91/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmp7HpXG6/file10a913707d9c91/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmp7HpXG6/file10a913707d9c91/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmp7HpXG6/file10a913707d9c91/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/Rtmp7HpXG6/file10a913707d9c91/sampleA_matched_reads_dedup.fastq.gz -> /tmp/Rtmp7HpXG6/file10a913707d9c91/sampleA_realign2transcript.bam
[M::mm_idx_gen::0.001*1.28] collected minimizers
[M::mm_idx_gen::0.002*1.22] sorted minimizers
[M::main::0.002*1.22] loaded/built the index for 14 target sequence(s)
[M::mm_mapopt_update::0.002*1.21] mid_occ = 15
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 14
[M::mm_idx_stat::0.002*1.20] distinct minimizers: 632 (64.56% are singletons); average occurrences: 3.326; average spacing: 5.176; total length: 10880
[M::worker_pipeline::0.726*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/Rtmp7HpXG6/file10a913707d9c91/transcript_assembly.fa /tmp/Rtmp7HpXG6/file10a913707d9c91/sampleA_matched_reads_dedup.fastq.gz
[M::main] Real time: 0.727 sec; CPU: 0.727 sec; Peak RSS: 0.007 GB
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/Rtmp7HpXG6/file10a913707d9c91/sample1_matched_reads_dedup.fastq.gz -> /tmp/Rtmp7HpXG6/file10a913707d9c91/sample1_realign2transcript.bam
[M::mm_idx_gen::0.001*0.89] collected minimizers
[M::mm_idx_gen::0.002*0.91] sorted minimizers
[M::main::0.002*0.91] loaded/built the index for 14 target sequence(s)
[M::mm_mapopt_update::0.002*0.92] mid_occ = 15
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 14
[M::mm_idx_stat::0.002*0.92] distinct minimizers: 632 (64.56% are singletons); average occurrences: 3.326; average spacing: 5.176; total length: 10880
[M::worker_pipeline::0.197*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 --eqx -N 100 -ax map-ont -t 1 --seed 2022 -y /tmp/Rtmp7HpXG6/file10a913707d9c91/transcript_assembly.fa /tmp/Rtmp7HpXG6/file10a913707d9c91/sample1_matched_reads_dedup.fastq.gz
[M::main] Real time: 0.198 sec; CPU: 0.198 sec; Peak RSS: 0.004 GB
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/Rtmp7HpXG6/file10a913707d9c91/sample2_matched_reads_dedup.fastq.gz -> /tmp/Rtmp7HpXG6/file10a913707d9c91/sample2_realign2transcript.bam
[M::mm_idx_gen::0.001*1.41] 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::0.202*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/Rtmp7HpXG6/file10a913707d9c91/transcript_assembly.fa /tmp/Rtmp7HpXG6/file10a913707d9c91/sample2_matched_reads_dedup.fastq.gz
[M::main] Real time: 0.202 sec; CPU: 0.203 sec; Peak RSS: 0.003 GB
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/Rtmp7HpXG6/file10a913707d9c91/sample3_matched_reads_dedup.fastq.gz -> /tmp/Rtmp7HpXG6/file10a913707d9c91/sample3_realign2transcript.bam
[M::mm_idx_gen::0.001*0.98] collected minimizers
[M::mm_idx_gen::0.002*0.98] sorted minimizers
[M::main::0.002*0.98] loaded/built the index for 14 target sequence(s)
[M::mm_mapopt_update::0.002*0.99] mid_occ = 15
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 14
[M::mm_idx_stat::0.002*0.99] 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/Rtmp7HpXG6/file10a913707d9c91/transcript_assembly.fa /tmp/Rtmp7HpXG6/file10a913707d9c91/sample3_matched_reads_dedup.fastq.gz
[M::main] Real time: 0.344 sec; CPU: 0.344 sec; Peak RSS: 0.006 GB
Sorting BAM files by 1 with CB threads...

-- Running step: transcript_quantification @ Wed Jun 18 23:27:56 2025 ----------
2025-06-19T03:27:56.350085Z  INFO oarfish: setting user-provided filter parameters.
2025-06-19T03:27:56.350541Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp7HpXG6/file10a913707d9c91/sampleA_realign2transcript.bam, contains 14 reference sequences.
2025-06-19T03:27:56.350550Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-06-19T03:27:56.350553Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-06-19T03:27:56.350646Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-06-19T03:27:56.350653Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-06-19T03:27:56.362520Z  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
2025-06-19T03:27:57.997532Z  INFO oarfish: setting user-provided filter parameters.
2025-06-19T03:27:57.998090Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp7HpXG6/file10a913707d9c91/sample1_realign2transcript.bam, contains 14 reference sequences.
2025-06-19T03:27:57.998101Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-06-19T03:27:57.998104Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-06-19T03:27:57.998179Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-06-19T03:27:57.998186Z  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
2025-06-19T03:27:59.592840Z  INFO oarfish: setting user-provided filter parameters.
2025-06-19T03:27:59.593211Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp7HpXG6/file10a913707d9c91/sample2_realign2transcript.bam, contains 14 reference sequences.
2025-06-19T03:27:59.593222Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-06-19T03:27:59.593225Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-06-19T03:27:59.593305Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-06-19T03:27:59.593312Z  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
2025-06-19T03:28:01.179394Z  INFO oarfish: setting user-provided filter parameters.
2025-06-19T03:28:01.179913Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp7HpXG6/file10a913707d9c91/sample3_realign2transcript.bam, contains 14 reference sequences.
2025-06-19T03:28:01.179922Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-06-19T03:28:01.179925Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-06-19T03:28:01.180028Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-06-19T03:28:01.180040Z  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/Rtmp7HpXG6/file10a9137da4fffe/config_file_1091859.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 @ Wed Jun 18 23:28:03 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/Rtmp7HpXG6/file10a9137da4fffe/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp7HpXG6/file10a9137da4fffe/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmp7HpXG6/file10a9137da4fffe/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmp7HpXG6/file10a9137da4fffe/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/Rtmp7HpXG6/file10a9137da4fffe/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp7HpXG6/file10a9137da4fffe/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/Rtmp7HpXG6/file10a9137da4fffe/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp7HpXG6/file10a9137da4fffe/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/Rtmp7HpXG6/file10a9137da4fffe/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp7HpXG6/file10a9137da4fffe/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 @ Wed Jun 18 23:28:03 2025 -------------------
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp7HpXG6/file10a91334bb141e/config_file_1091859.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 @ Wed Jun 18 23:28:24 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/Rtmp7HpXG6/file10a91334bb141e/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp7HpXG6/file10a91334bb141e/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmp7HpXG6/file10a91334bb141e/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmp7HpXG6/file10a91334bb141e/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/Rtmp7HpXG6/file10a91334bb141e/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp7HpXG6/file10a91334bb141e/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/Rtmp7HpXG6/file10a91334bb141e/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp7HpXG6/file10a91334bb141e/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/Rtmp7HpXG6/file10a91334bb141e/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp7HpXG6/file10a91334bb141e/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 @ Wed Jun 18 23:28:25 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmp7HpXG6/file10a91334bb141e/sampleA_matched_reads.fastq.gz -> /tmp/Rtmp7HpXG6/file10a91334bb141e/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*1.34] collected minimizers
[M::mm_idx_gen::0.003*1.26] sorted minimizers
[M::main::0.003*1.26] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.003*1.25] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.003*1.24] 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::0.599*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/Rtmp7HpXG6/file10a91334bb141e/reference.bed --junc-bonus 1 /tmp/Rtmp7HpXG6/file10a91334bb141e/rps24.fa /tmp/Rtmp7HpXG6/file10a91334bb141e/sampleA_matched_reads.fastq.gz
[M::main] Real time: 0.601 sec; CPU: 0.600 sec; Peak RSS: 0.006 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmp7HpXG6/file10a91334bb141e/sample1_matched_reads.fastq.gz -> /tmp/Rtmp7HpXG6/file10a91334bb141e/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*1.46] collected minimizers
[M::mm_idx_gen::0.002*1.32] sorted minimizers
[M::main::0.002*1.32] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*1.30] 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::0.154*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/Rtmp7HpXG6/file10a91334bb141e/reference.bed --junc-bonus 1 /tmp/Rtmp7HpXG6/file10a91334bb141e/rps24.fa /tmp/Rtmp7HpXG6/file10a91334bb141e/sample1_matched_reads.fastq.gz
[M::main] Real time: 0.154 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/Rtmp7HpXG6/file10a91334bb141e/sample2_matched_reads.fastq.gz -> /tmp/Rtmp7HpXG6/file10a91334bb141e/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::0.002*1.21] collected minimizers
[M::mm_idx_gen::0.003*1.16] sorted minimizers
[M::main::0.003*1.16] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.003*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::0.157*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/Rtmp7HpXG6/file10a91334bb141e/reference.bed --junc-bonus 1 /tmp/Rtmp7HpXG6/file10a91334bb141e/rps24.fa /tmp/Rtmp7HpXG6/file10a91334bb141e/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/Rtmp7HpXG6/file10a91334bb141e/sample3_matched_reads.fastq.gz -> /tmp/Rtmp7HpXG6/file10a91334bb141e/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::0.002*0.86] collected minimizers
[M::mm_idx_gen::0.002*0.91] sorted minimizers
[M::main::0.002*0.91] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.003*0.91] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.003*0.92] 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::0.301*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/Rtmp7HpXG6/file10a91334bb141e/reference.bed --junc-bonus 1 /tmp/Rtmp7HpXG6/file10a91334bb141e/rps24.fa /tmp/Rtmp7HpXG6/file10a91334bb141e/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 @ Wed Jun 18 23:28:27 2025 ----------------
23:28:27 Wed Jun 18 2025 quantify genes 
Using BAM(s): '/tmp/Rtmp7HpXG6/file10a91334bb141e/sampleA_align2genome.bam',
'/tmp/Rtmp7HpXG6/file10a91334bb141e/sample1_align2genome.bam',
'/tmp/Rtmp7HpXG6/file10a91334bb141e/sample2_align2genome.bam', and
'/tmp/Rtmp7HpXG6/file10a91334bb141e/sample3_align2genome.bam'
parsing /tmp/Rtmp7HpXG6/file10a91334bb141e/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 23.03gene_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, 39210.54Read/s]
parsing /tmp/Rtmp7HpXG6/file10a91334bb141e/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 50.87gene_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, 188328.60Read/s]
parsing /tmp/Rtmp7HpXG6/file10a91334bb141e/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 52.48gene_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, 153204.27Read/s]
parsing /tmp/Rtmp7HpXG6/file10a91334bb141e/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 37.06gene_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, 76844.09Read/s]
-- Running step: isoform_identification @ Wed Jun 18 23:28:28 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 @ Wed Jun 18 23:28:28 2025 -------------------
Checking for fastq file(s) /tmp/Rtmp7HpXG6/file10a91334bb141e/fastq, /tmp/Rtmp7HpXG6/file10a91334bb141e/fastq/sample1.fq.gz, /tmp/Rtmp7HpXG6/file10a91334bb141e/fastq/sample2.fq.gz, /tmp/Rtmp7HpXG6/file10a91334bb141e/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp7HpXG6/file10a91334bb141e/sampleA_matched_reads.fastq.gz, /tmp/Rtmp7HpXG6/file10a91334bb141e/sample1_matched_reads.fastq.gz, /tmp/Rtmp7HpXG6/file10a91334bb141e/sample2_matched_reads.fastq.gz, /tmp/Rtmp7HpXG6/file10a91334bb141e/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp7HpXG6/file10a91334bb141e/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmp7HpXG6/file10a91334bb141e/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmp7HpXG6/file10a91334bb141e/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmp7HpXG6/file10a91334bb141e/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/Rtmp7HpXG6/file10a91334bb141e/sampleA_matched_reads_dedup.fastq.gz -> /tmp/Rtmp7HpXG6/file10a91334bb141e/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*1.54] collected minimizers
[M::mm_idx_gen::0.002*1.40] sorted minimizers
[M::main::0.002*1.40] loaded/built the index for 14 target sequence(s)
[M::mm_mapopt_update::0.002*1.38] mid_occ = 15
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 14
[M::mm_idx_stat::0.002*1.36] distinct minimizers: 632 (64.56% are singletons); average occurrences: 3.326; average spacing: 5.176; total length: 10880
[M::worker_pipeline::0.350*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/Rtmp7HpXG6/file10a91334bb141e/transcript_assembly.fa /tmp/Rtmp7HpXG6/file10a91334bb141e/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/Rtmp7HpXG6/file10a91334bb141e/sample1_matched_reads_dedup.fastq.gz -> /tmp/Rtmp7HpXG6/file10a91334bb141e/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*1.13] collected minimizers
[M::mm_idx_gen::0.002*1.10] sorted minimizers
[M::main::0.002*1.10] loaded/built the index for 14 target sequence(s)
[M::mm_mapopt_update::0.002*1.10] mid_occ = 15
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 14
[M::mm_idx_stat::0.002*1.09] distinct minimizers: 632 (64.56% are singletons); average occurrences: 3.326; average spacing: 5.176; total length: 10880
[M::worker_pipeline::0.091*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/Rtmp7HpXG6/file10a91334bb141e/transcript_assembly.fa /tmp/Rtmp7HpXG6/file10a91334bb141e/sample1_matched_reads_dedup.fastq.gz
[M::main] Real time: 0.091 sec; CPU: 0.092 sec; Peak RSS: 0.003 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample /tmp/Rtmp7HpXG6/file10a91334bb141e/sample2_matched_reads_dedup.fastq.gz -> /tmp/Rtmp7HpXG6/file10a91334bb141e/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*1.37] collected minimizers
[M::mm_idx_gen::0.002*1.28] sorted minimizers
[M::main::0.002*1.28] loaded/built the index for 14 target sequence(s)
[M::mm_mapopt_update::0.002*1.26] mid_occ = 15
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 14
[M::mm_idx_stat::0.002*1.25] 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/Rtmp7HpXG6/file10a91334bb141e/transcript_assembly.fa /tmp/Rtmp7HpXG6/file10a91334bb141e/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/Rtmp7HpXG6/file10a91334bb141e/sample3_matched_reads_dedup.fastq.gz -> /tmp/Rtmp7HpXG6/file10a91334bb141e/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*1.15] collected minimizers
[M::mm_idx_gen::0.002*1.12] sorted minimizers
[M::main::0.002*1.12] 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::0.179*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/Rtmp7HpXG6/file10a91334bb141e/transcript_assembly.fa /tmp/Rtmp7HpXG6/file10a91334bb141e/sample3_matched_reads_dedup.fastq.gz
[M::main] Real time: 0.180 sec; CPU: 0.180 sec; Peak RSS: 0.004 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Wed Jun 18 23:28:29 2025 ----------
23:28:29 Wed Jun 18 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/Rtmp7HpXG6/file10a91334bb141e/sampleA_realign2transcript.bam...
parsing /tmp/Rtmp7HpXG6/file10a91334bb141e/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmp7HpXG6/file10a91334bb141e/sampleA_realign2transcript.bamdone
parsing /tmp/Rtmp7HpXG6/file10a91334bb141e/sample1_realign2transcript.bam...
parsing /tmp/Rtmp7HpXG6/file10a91334bb141e/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmp7HpXG6/file10a91334bb141e/sample1_realign2transcript.bamdone
parsing /tmp/Rtmp7HpXG6/file10a91334bb141e/sample2_realign2transcript.bam...
parsing /tmp/Rtmp7HpXG6/file10a91334bb141e/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmp7HpXG6/file10a91334bb141e/sample2_realign2transcript.bamdone
parsing /tmp/Rtmp7HpXG6/file10a91334bb141e/sample3_realign2transcript.bam...
parsing /tmp/Rtmp7HpXG6/file10a91334bb141e/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmp7HpXG6/file10a91334bb141e/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp7HpXG6/file10a9132ca7f809/config_file_1091859.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 @ Wed Jun 18 23:28:32 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/Rtmp7HpXG6/file10a9132ca7f809/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp7HpXG6/file10a9132ca7f809/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmp7HpXG6/file10a9132ca7f809/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmp7HpXG6/file10a9132ca7f809/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/Rtmp7HpXG6/file10a9132ca7f809/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp7HpXG6/file10a9132ca7f809/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/Rtmp7HpXG6/file10a9132ca7f809/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp7HpXG6/file10a9132ca7f809/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/Rtmp7HpXG6/file10a9132ca7f809/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp7HpXG6/file10a9132ca7f809/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 @ Wed Jun 18 23:28:33 2025 -------------------
[ FAIL 0 | WARN 150 | SKIP 0 | PASS 37 ]

[ FAIL 0 | WARN 150 | SKIP 0 | PASS 37 ]
	Counter({'counted_reads': 357, 'not_enough_coverage': 1})
	Counter({'counted_reads': 91})
	Counter({'counted_reads': 95})
	Counter({'counted_reads': 175, 'not_enough_coverage': 1})
> 
> proc.time()
   user  system elapsed 
430.911  29.008 459.004