Back to Multiple platform build/check report for BioC 3.22:   simplified   long
ABCDE[F]GHIJKLMNOPQRSTUVWXYZ

This page was generated on 2025-10-18 12:04 -0400 (Sat, 18 Oct 2025).

HostnameOSArch (*)R versionInstalled pkgs
nebbiolo2Linux (Ubuntu 24.04.3 LTS)x86_644.5.1 Patched (2025-08-23 r88802) -- "Great Square Root" 4887
lconwaymacOS 12.7.6 Montereyx86_644.5.1 Patched (2025-09-10 r88807) -- "Great Square Root" 4677
kjohnson3macOS 13.7.7 Venturaarm644.5.1 Patched (2025-09-10 r88807) -- "Great Square Root" 4622
taishanLinux (openEuler 24.03 LTS)aarch644.5.0 (2025-04-11) -- "How About a Twenty-Six" 4632
Click on any hostname to see more info about the system (e.g. compilers)      (*) as reported by 'uname -p', except on Windows and Mac OS X

Package 740/2353HostnameOS / ArchINSTALLBUILDCHECKBUILD BIN
FLAMES 2.3.5  (landing page)
Changqing Wang
Snapshot Date: 2025-10-17 13:45 -0400 (Fri, 17 Oct 2025)
git_url: https://git.bioconductor.org/packages/FLAMES
git_branch: devel
git_last_commit: 5771592
git_last_commit_date: 2025-09-12 02:43:50 -0400 (Fri, 12 Sep 2025)
nebbiolo2Linux (Ubuntu 24.04.3 LTS) / x86_64  OK    OK    OK  UNNEEDED, same version is already published
lconwaymacOS 12.7.6 Monterey / x86_64  OK    OK    OK    OK  UNNEEDED, same version is already published
kjohnson3macOS 13.7.7 Ventura / arm64  OK    OK    OK    OK  UNNEEDED, same version is already published
taishanLinux (openEuler 24.03 LTS) / aarch64  ERROR    ERROR  skipped


CHECK results for FLAMES on lconway

To the developers/maintainers of the FLAMES package:
- Allow up to 24 hours (and sometimes 48 hours) for your latest push to git@git.bioconductor.org:packages/FLAMES.git to reflect on this report. See Troubleshooting Build Report for more information.
- Use the following Renviron settings to reproduce errors and warnings.
- If 'R CMD check' started to fail recently on the Linux builder(s) over a missing dependency, add the missing dependency to 'Suggests:' in your DESCRIPTION file. See Renviron.bioc for more information.

raw results


Summary

Package: FLAMES
Version: 2.3.5
Command: /Library/Frameworks/R.framework/Resources/bin/R CMD check --install=check:FLAMES.install-out.txt --library=/Library/Frameworks/R.framework/Resources/library --no-vignettes --timings FLAMES_2.3.5.tar.gz
StartedAt: 2025-10-17 22:10:07 -0400 (Fri, 17 Oct 2025)
EndedAt: 2025-10-17 22:42:22 -0400 (Fri, 17 Oct 2025)
EllapsedTime: 1935.1 seconds
RetCode: 0
Status:   OK  
CheckDir: FLAMES.Rcheck
Warnings: 0

Command output

##############################################################################
##############################################################################
###
### Running command:
###
###   /Library/Frameworks/R.framework/Resources/bin/R CMD check --install=check:FLAMES.install-out.txt --library=/Library/Frameworks/R.framework/Resources/library --no-vignettes --timings FLAMES_2.3.5.tar.gz
###
##############################################################################
##############################################################################


* using log directory ‘/Users/biocbuild/bbs-3.22-bioc/meat/FLAMES.Rcheck’
* using R version 4.5.1 Patched (2025-09-10 r88807)
* using platform: x86_64-apple-darwin20
* R was compiled by
    Apple clang version 14.0.0 (clang-1400.0.29.202)
    GNU Fortran (GCC) 14.2.0
* running under: macOS Monterey 12.7.6
* using session charset: UTF-8
* using option ‘--no-vignettes’
* checking for file ‘FLAMES/DESCRIPTION’ ... OK
* this is package ‘FLAMES’ version ‘2.3.5’
* package encoding: UTF-8
* checking package namespace information ... OK
* checking package dependencies ... INFO
Imports includes 47 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 ... NOTE
Found the following hidden files and directories:
  .dockerignore
These were most likely included in error. See section ‘Package
structure’ in the ‘Writing R Extensions’ manual.
* 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 ‘/Users/biocbuild/bbs-3.22-bioc/meat/FLAMES.Rcheck/00install.out’ for details.
* used C++ compiler: ‘Apple clang version 14.0.0 (clang-1400.0.29.202)’
* used SDK: ‘MacOSX11.3.1.sdk’
* checking C++ specification ... OK
* checking installed package size ... INFO
  installed size is  5.5Mb
  sub-directories of 1Mb or more:
    data   1.8Mb
    libs   1.8Mb
* 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 dependencies in R code ... NOTE
There are ::: calls to the package's namespace in its code. A package
  almost never needs to use ::: for its own objects:
  'blaze' 'find_barcode' 'gene_quantification' 'isoform_identification'
  'minimap2_align' 'transcript_quantification'
* checking S3 generic/method consistency ... OK
* checking replacement functions ... OK
* checking foreign function calls ... OK
* checking R code for possible problems ... NOTE
BulkPipeline: no visible global function definition for ‘new’
BulkPipeline: no visible global function definition for ‘setNames’
MultiSampleSCPipeline: no visible global function definition for ‘new’
MultiSampleSCPipeline: no visible global function definition for
  ‘setNames’
SingleCellPipeline: no visible global function definition for ‘new’
SingleCellPipeline: no visible global function definition for
  ‘setNames’
addRowRanges: no visible global function definition for ‘head’
addRowRanges: no visible global function definition for ‘as’
add_gene_counts: 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_sce_from_dir: no visible global function definition for
  ‘setNames’
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_variants: no visible global function definition for ‘setNames’
find_variants_grange: no visible binding for global variable
  ‘which_label’
find_variants_grange: no visible binding for global variable
  ‘nucleotide’
find_variants_grange: no visible binding for global variable ‘pos’
find_variants_grange: no visible binding for global variable ‘count’
find_variants_grange: no visible binding for global variable
  ‘counts_no_ins’
find_variants_grange: no visible binding for global variable ‘ref’
generate_sc_sce: no visible binding for global variable ‘FSM_match’
get_coverage: no visible binding for global variable ‘Freq’
homopolymer_pct : <anonymous>: no visible binding for global variable
  ‘Freq’
homopolymer_pct : <anonymous>: no visible binding for global variable
  ‘pct’
plot_coverage: no visible binding for global variable ‘tr_length’
plot_coverage: no visible binding for global variable ‘read_counts’
plot_coverage: no visible binding for global variable ‘total_counts’
plot_coverage: no visible binding for global variable ‘cumpct’
plot_coverage: no visible binding for global variable ‘length_bin’
plot_coverage: no visible binding for global variable ‘min_length’
plot_coverage: no visible binding for global variable ‘max_length’
plot_coverage: no visible global function definition for ‘head’
plot_coverage: no visible binding for global variable ‘transcript’
plot_demultiplex_raw: no visible binding for global variable ‘Sample’
plot_demultiplex_raw: no visible binding for global variable
  ‘CellBarcode’
plot_demultiplex_raw: no visible binding for global variable ‘UMI’
plot_demultiplex_raw: no visible binding for global variable
  ‘UMI_count’
plot_demultiplex_raw: no visible binding for global variable
  ‘barcode_rank’
plot_demultiplex_raw: no visible binding for global variable
  ‘FlankEditDist’
plot_demultiplex_raw: no visible binding for global variable ‘n_reads’
plot_demultiplex_raw: no visible binding for global variable
  ‘BarcodeEditDist’
plot_demultiplex_raw: no visible binding for global variable ‘total
  reads’
plot_demultiplex_raw: no visible binding for global variable
  ‘demultiplexed reads’
plot_demultiplex_raw: no visible binding for global variable ‘single
  match reads’
plot_demultiplex_raw: no visible binding for global variable
  ‘undemultiplexted reads’
plot_demultiplex_raw: no visible binding for global variable
  ‘multi-matching reads’
plot_demultiplex_raw: no visible binding for global variable ‘Type’
plot_demultiplex_raw: no visible binding for global variable ‘Reads’
plot_demultiplex_raw: no visible binding for global variable ‘input’
plot_demultiplex_raw: no visible binding for global variable ‘output’
plot_demultiplex_raw: no visible binding for global variable
  ‘read1_with_adapter’
plot_demultiplex_raw: no visible binding for global variable ‘Count’
plot_flagstat: no visible global function definition for ‘everything’
plot_flagstat: no visible binding for global variable ‘name’
plot_flagstat: no visible binding for global variable ‘value’
plot_isoform_reduced_dim: no visible binding for global variable ‘x’
plot_isoform_reduced_dim: no visible binding for global variable ‘y’
plot_isoform_reduced_dim: no visible binding for global variable ‘expr’
plot_spatial: no visible binding for global variable ‘imageX’
plot_spatial: no visible binding for global variable ‘imageY’
plot_spatial_feature: no visible binding for global variable ‘imageX’
plot_spatial_feature: no visible binding for global variable ‘imageY’
plot_spatial_feature: no visible binding for global variable ‘x’
plot_spatial_feature: no visible binding for global variable ‘y’
plot_spatial_feature: no visible global function definition for
  ‘scale_alpha_continuous’
plot_spatial_feature: no visible global function definition for
  ‘scale_colour_gradient’
plot_spatial_isoform: no visible global function definition for ‘head’
plot_spatial_pie: no visible global function definition for ‘setNames’
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_gene_entropy: no visible global function definition for ‘as’
sc_genotype: no visible binding for global variable ‘allele’
sc_genotype: no visible binding for global variable ‘allele_count’
sc_genotype: no visible binding for global variable ‘barcode’
sc_genotype: no visible binding for global variable ‘pct’
sc_mutations: no visible binding for global variable ‘mutation_index’
sc_mutations: no visible binding for global variable ‘bam_index’
sc_plot_genotype: no visible global function definition for ‘setNames’
sc_plot_genotype: no visible binding for global variable ‘barcode’
sc_plot_genotype: no visible binding for global variable ‘genotype’
sc_plot_genotype: no visible binding for global variable ‘x’
sc_plot_genotype: no visible binding for global variable ‘y’
sc_transcript_usage_chisq: no visible global function definition for
  ‘as’
sc_transcript_usage_chisq: no visible binding for global variable
  ‘p.value’
sc_transcript_usage_chisq: no visible binding for global variable
  ‘adj.p.value’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘total’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘test’
sc_transcript_usage_permutation: no visible global function definition
  for ‘as’
sc_transcript_usage_permutation : <anonymous>: no visible global
  function definition for ‘as’
sc_transcript_usage_permutation : <anonymous> : <anonymous>: no visible
  global function definition for ‘na.omit’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘transcript’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘p.value’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘adj.p.value’
variant_count_tb: no visible binding for global variable ‘barcode’
variant_count_tb: no visible binding for global variable ‘allele_count’
variant_count_tb: no visible binding for global variable
  ‘cell_total_reads’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘expect_cell_number’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘fastq’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘demultiplexed_fastq’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘barcodes_file’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘outdir’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible global
  function definition for ‘setNames’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline : <anonymous>: no
  visible global function definition for ‘setNames’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘expect_cell_number’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘fastq’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘demultiplexed_fastq’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘barcodes_file’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘outdir’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible global
  function definition for ‘setNames’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘j’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘genome_bam’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘minimap2’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘samtools’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘threads’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘outdir’
plot_durations,FLAMES.Pipeline: no visible binding for global variable
  ‘step’
plot_durations,FLAMES.Pipeline: no visible binding for global variable
  ‘duration’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘j’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘transcriptome_assembly’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘transcriptome_bam’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘minimap2’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘samtools’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘outdir’
resume_FLAMES,FLAMES.Pipeline : <anonymous>: no visible global function
  definition for ‘capture.output’
run_FLAMES,FLAMES.Pipeline : <anonymous>: no visible global function
  definition for ‘capture.output’
Undefined global functions or variables:
  BarcodeEditDist CellBarcode Count FSM_match FlankEditDist Freq Reads
  Sample Type UMI UMI_count adj.p.value allele allele_count as
  bam_index barcode barcode_rank barcodes_file capture.output
  cell_total_reads chisq.test count counts_no_ins cumpct demultiplexed
  reads demultiplexed_fastq download.file duration everything
  expect_cell_number expr fastq filter_res genome_bam genotype 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 setNames single match reads starts_with step
  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", "setNames", "step")
  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 ‘/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/FLAMES/libs/FLAMES.so’:
  Found ‘___assert_rtn’, possibly from ‘assert’ (C)
  Found ‘___stderrp’, possibly from ‘stderr’ (C)
  Found ‘___stdoutp’, possibly from ‘stdout’ (C)
  Found ‘_abort’, possibly from ‘abort’ (C)
  Found ‘_exit’, possibly from ‘exit’ (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
plot_isoform_reduced_dim     27.442  0.601  29.832
find_variants                25.456  0.505  25.852
sc_long_multisample_pipeline 17.205  3.998  25.200
MultiSampleSCPipeline        13.128  2.698  30.323
sc_plot_genotype             11.301  0.455  11.752
sc_DTU_analysis               9.664  1.750  12.639
plot_isoform_heatmap          9.688  0.607  10.505
create_sce_from_dir           7.759  2.203  10.216
blaze                         7.659  1.404  15.498
sc_long_pipeline              5.483  1.018   5.687
sc_genotype                   4.064  1.211   4.339
BulkPipeline                  4.525  0.734   5.915
bulk_long_pipeline            3.871  1.087   5.833
create_se_from_dir            3.700  0.890   5.056
resume_FLAMES                 2.863  0.889   9.762
experiment                    2.865  0.528   6.396
* 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 running R code from vignettes ... SKIPPED
* checking re-building of vignette outputs ... SKIPPED
* checking PDF version of manual ... OK
* DONE

Status: 5 NOTEs
See
  ‘/Users/biocbuild/bbs-3.22-bioc/meat/FLAMES.Rcheck/00check.log’
for details.


Installation output

FLAMES.Rcheck/00install.out

##############################################################################
##############################################################################
###
### Running command:
###
###   /Library/Frameworks/R.framework/Resources/bin/R CMD INSTALL FLAMES
###
##############################################################################
##############################################################################


* installing to library ‘/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library’
* installing *source* package ‘FLAMES’ ...
** this is package ‘FLAMES’ version ‘2.3.5’
** using non-staged installation via StagedInstall field
** libs
/bin/sh: rustc: command not found
using C++ compiler: ‘Apple clang version 14.0.0 (clang-1400.0.29.202)’
using C++17
using SDK: ‘MacOSX11.3.1.sdk’
/bin/sh: rustc: command not found
clang++ -arch x86_64 -std=gnu++17 -I"/Library/Frameworks/R.framework/Resources/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/Rcpp/include' -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/Rhtslib/include' -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/testthat/include' -I/opt/R/x86_64/include    -fPIC  -falign-functions=64 -Wall -g -O2   -c RcppExports.cpp -o RcppExports.o
clang++ -arch x86_64 -std=gnu++17 -I"/Library/Frameworks/R.framework/Resources/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/Rcpp/include' -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/Rhtslib/include' -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/testthat/include' -I/opt/R/x86_64/include    -fPIC  -falign-functions=64 -Wall -g -O2   -c RcppFunctions.cpp -o RcppFunctions.o
clang++ -arch x86_64 -std=gnu++17 -I"/Library/Frameworks/R.framework/Resources/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/Rcpp/include' -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/Rhtslib/include' -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/testthat/include' -I/opt/R/x86_64/include    -fPIC  -falign-functions=64 -Wall -g -O2   -c classes/BamRecord.cpp -o classes/BamRecord.o
clang++ -arch x86_64 -std=gnu++17 -I"/Library/Frameworks/R.framework/Resources/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/Rcpp/include' -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/Rhtslib/include' -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/testthat/include' -I/opt/R/x86_64/include    -fPIC  -falign-functions=64 -Wall -g -O2   -c classes/GFFRecord.cpp -o classes/GFFRecord.o
clang++ -arch x86_64 -std=gnu++17 -I"/Library/Frameworks/R.framework/Resources/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/Rcpp/include' -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/Rhtslib/include' -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/testthat/include' -I/opt/R/x86_64/include    -fPIC  -falign-functions=64 -Wall -g -O2   -c classes/GeneAnnotationParser.cpp -o classes/GeneAnnotationParser.o
clang++ -arch x86_64 -std=gnu++17 -I"/Library/Frameworks/R.framework/Resources/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/Rcpp/include' -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/Rhtslib/include' -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/testthat/include' -I/opt/R/x86_64/include    -fPIC  -falign-functions=64 -Wall -g -O2   -c classes/Isoforms.cpp -o classes/Isoforms.o
clang++ -arch x86_64 -std=gnu++17 -I"/Library/Frameworks/R.framework/Resources/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/Rcpp/include' -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/Rhtslib/include' -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/testthat/include' -I/opt/R/x86_64/include    -fPIC  -falign-functions=64 -Wall -g -O2   -c classes/junctions.cpp -o classes/junctions.o
clang++ -arch x86_64 -std=gnu++17 -I"/Library/Frameworks/R.framework/Resources/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/Rcpp/include' -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/Rhtslib/include' -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/testthat/include' -I/opt/R/x86_64/include    -fPIC  -falign-functions=64 -Wall -g -O2   -c main-functions/find_isoform.cpp -o main-functions/find_isoform.o
clang++ -arch x86_64 -std=gnu++17 -I"/Library/Frameworks/R.framework/Resources/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/Rcpp/include' -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/Rhtslib/include' -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/testthat/include' -I/opt/R/x86_64/include    -fPIC  -falign-functions=64 -Wall -g -O2   -c main-functions/flexiplex.cpp -o main-functions/flexiplex.o
clang++ -arch x86_64 -std=gnu++17 -I"/Library/Frameworks/R.framework/Resources/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/Rcpp/include' -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/Rhtslib/include' -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/testthat/include' -I/opt/R/x86_64/include    -fPIC  -falign-functions=64 -Wall -g -O2   -c main-functions/get_transcript_seq.cpp -o main-functions/get_transcript_seq.o
clang++ -arch x86_64 -std=gnu++17 -I"/Library/Frameworks/R.framework/Resources/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/Rcpp/include' -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/Rhtslib/include' -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/testthat/include' -I/opt/R/x86_64/include    -fPIC  -falign-functions=64 -Wall -g -O2   -c main-functions/group_bam2isoform.cpp -o main-functions/group_bam2isoform.o
clang++ -arch x86_64 -std=gnu++17 -I"/Library/Frameworks/R.framework/Resources/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/Rcpp/include' -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/Rhtslib/include' -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/testthat/include' -I/opt/R/x86_64/include    -fPIC  -falign-functions=64 -Wall -g -O2   -c main-functions/pileup_readid.cpp -o main-functions/pileup_readid.o
main-functions/pileup_readid.cpp:86:16: warning: unused variable 'end' [-Wunused-variable]
  unsigned int end;
               ^
1 warning generated.
clang++ -arch x86_64 -std=gnu++17 -I"/Library/Frameworks/R.framework/Resources/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/Rcpp/include' -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/Rhtslib/include' -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/testthat/include' -I/opt/R/x86_64/include    -fPIC  -falign-functions=64 -Wall -g -O2   -c tests/test-junctions.cpp -o tests/test-junctions.o
clang++ -arch x86_64 -std=gnu++17 -I"/Library/Frameworks/R.framework/Resources/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/Rcpp/include' -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/Rhtslib/include' -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/testthat/include' -I/opt/R/x86_64/include    -fPIC  -falign-functions=64 -Wall -g -O2   -c tests/test-parsing.cpp -o tests/test-parsing.o
clang++ -arch x86_64 -std=gnu++17 -I"/Library/Frameworks/R.framework/Resources/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/Rcpp/include' -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/Rhtslib/include' -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/testthat/include' -I/opt/R/x86_64/include    -fPIC  -falign-functions=64 -Wall -g -O2   -c utility/cigars.cpp -o utility/cigars.o
clang++ -arch x86_64 -std=gnu++17 -I"/Library/Frameworks/R.framework/Resources/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/Rcpp/include' -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/Rhtslib/include' -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/testthat/include' -I/opt/R/x86_64/include    -fPIC  -falign-functions=64 -Wall -g -O2   -c utility/edlib-1.2.7/edlib.cpp -o utility/edlib-1.2.7/edlib.o
clang -arch x86_64 -I"/Library/Frameworks/R.framework/Resources/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/Rcpp/include' -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/Rhtslib/include' -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/testthat/include' -I/opt/R/x86_64/include    -fPIC  -falign-functions=64 -Wall -g -O2  -c utility/bam.c -o utility/bam.o
clang++ -arch x86_64 -std=gnu++17 -dynamiclib -Wl,-headerpad_max_install_names -undefined dynamic_lookup -L/Library/Frameworks/R.framework/Resources/lib -L/opt/R/x86_64/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 /Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/Rhtslib/usrlib/libhts.a -lcurl -lbz2 -llzma -lz -F/Library/Frameworks/R.framework/.. -framework R
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="-falign-functions=64 -Wall -g -O2  -Wno-unused-result" minimap2)
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  main.c -o main.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  kthread.c -o kthread.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  kalloc.c -o kalloc.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  misc.c -o misc.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  bseq.c -o bseq.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  sketch.c -o sketch.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  sdust.c -o sdust.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  options.c -o options.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  index.c -o index.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  lchain.c -o lchain.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  align.c -o align.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  hit.c -o hit.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  seed.c -o seed.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  jump.c -o jump.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  map.c -o map.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  format.c -o format.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  pe.c -o pe.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  esterr.c -o esterr.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  splitidx.c -o splitidx.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -msse2 -DHAVE_KALLOC  ksw2_ll_sse.c -o ksw2_ll_sse.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -msse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH  ksw2_extz2_sse.c -o ksw2_extz2_sse41.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -msse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH  ksw2_extd2_sse.c -o ksw2_extd2_sse41.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -msse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH  ksw2_exts2_sse.c -o ksw2_exts2_sse41.o
cc -c -falign-functions=64 -Wall -g -O2  -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 -falign-functions=64 -Wall -g -O2  -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 -falign-functions=64 -Wall -g -O2  -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 -falign-functions=64 -Wall -g -O2  -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
cc -falign-functions=64 -Wall -g -O2  -Wno-unused-result main.o -o minimap2 -L. -lminimap2 -lm -lz -lpthread
echo "Installing binary to /Users/biocbuild/bbs-3.22-bioc/meat/FLAMES/src/../inst/bin"
Installing binary to /Users/biocbuild/bbs-3.22-bioc/meat/FLAMES/src/../inst/bin
mkdir -p ../inst/bin
cp submodule/minimap2/minimap2 ../inst/bin/
Cargo not found, skipping oarfish installation.
installing to /Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/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)

Tests output

FLAMES.Rcheck/tests/testthat.Rout


R version 4.5.1 Patched (2025-09-10 r88807) -- "Great Square Root"
Copyright (C) 2025 The R Foundation for Statistical Computing
Platform: x86_64-apple-darwin20

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.

R is a collaborative project with many contributors.
Type 'contributors()' for more information and
'citation()' on how to cite R or R packages in publications.

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

> # This file is part of the standard setup for testthat.
> # It is recommended that you do not modify it.
> #
> # Where should you do additional test configuration?
> # Learn more about the roles of various files in:
> # * https://r-pkgs.org/tests.html
> # * https://testthat.r-lib.org/reference/test_package.html#special-files
> 
> library(testthat)
> library(FLAMES)
> 
> test_check("FLAMES")
Writing configuration parameters to:  /tmp/RtmpjNKYVT/file129c58bca08/config_file_4764.json 
Writing configuration parameters to:  /tmp/RtmpjNKYVT/file129c58bca08/config_file_4764.json 
Writing configuration parameters to:  /tmp/RtmpjNKYVT/file129c58bca08/config_file_4764.json 
Writing configuration parameters to:  /tmp/RtmpjNKYVT/file129c17360cdf/config_file_4764.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/RtmpjNKYVT/file129c30f3b931/bc_allow.tsv
Number of known barcodes: 143
Processing file: /Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/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!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
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/RtmpjNKYVT/file129c21c92655/bc_allow.tsv
Number of known barcodes: 143
Processing file: /Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/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!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
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/RtmpjNKYVT/file129c21c92655/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpjNKYVT/file129c408c08aa/musc_rps24_1.fastq
Searching for barcodes...
Processing file: /tmp/RtmpjNKYVT/file129c408c08aa/musc_rps24_2.fastq
Searching for barcodes...
Processing file: /tmp/RtmpjNKYVT/file129c408c08aa/musc_rps24_3.fastq
Searching for barcodes...
Processing file: /tmp/RtmpjNKYVT/file129c408c08aa/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!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
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/RtmpjNKYVT/file129c18fc5962/bc_allow.tsv
Number of known barcodes: 143
Processing file: /Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/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!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
Skipping TSO trimming...
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpjNKYVT/file129c9a6ea95/config_file_4764.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Fri Oct 17 22:24:36 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpjNKYVT/file129c9a6ea95/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpjNKYVT/file129c9a6ea95/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpjNKYVT/file129c9a6ea95/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Fri Oct 17 22:24:38 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

  |                                                                            
  |                                                                      |   0%[22:24:48] WARNING: src/learner.cc:553: 
  If you are loading a serialized model (like pickle in Python, RDS in R) generated by
  older XGBoost, please export the model by calling `Booster.save_model` from that version
  first, then load it back in current version. See:

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

  for more details about differences between saving model and serializing.

[22:24:48] WARNING: src/learner.cc:553: 
  If you are loading a serialized model (like pickle in Python, RDS in R) generated by
  older XGBoost, please export the model by calling `Booster.save_model` from that version
  first, then load it back in current version. See:

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

  for more details about differences between saving model and serializing.

[22:24:48] WARNING: src/learner.cc:553: 
  If you are loading a serialized model (like pickle in Python, RDS in R) generated by
  older XGBoost, please export the model by calling `Booster.save_model` from that version
  first, then load it back in current version. See:

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

  for more details about differences between saving model and serializing.

[22:24:48] WARNING: src/learner.cc:553: 
  If you are loading a serialized model (like pickle in Python, RDS in R) generated by
  older XGBoost, please export the model by calling `Booster.save_model` from that version
  first, then load it back in current version. See:

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

  for more details about differences between saving model and serializing.

[22:24:51] WARNING: src/learner.cc:553: 
  If you are loading a serialized model (like pickle in Python, RDS in R) generated by
  older XGBoost, please export the model by calling `Booster.save_model` from that version
  first, then load it back in current version. See:

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

  for more details about differences between saving model and serializing.

[22:24:51] 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 ---
-- Running step: read_realignment @ Fri Oct 17 22:25:09 2025 -------------------
Realigning sample sample1 -> /tmp/RtmpjNKYVT/file129c9a6ea95/sample1_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample2 -> /tmp/RtmpjNKYVT/file129c9a6ea95/sample2_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample3 -> /tmp/RtmpjNKYVT/file129c9a6ea95/sample3_realign2transcript.bam
Skipped sorting BAM files.

-- Running step: transcript_quantification @ Fri Oct 17 22:25:11 2025 ----------
2025-10-18T02:25:11.388574Z  INFO oarfish: setting user-provided filter parameters.
2025-10-18T02:25:11.426074Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpjNKYVT/file129c9a6ea95/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-10-18T02:25:11.428568Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-18T02:25:11.428980Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-18T02:25:11.445161Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-18T02:25:11.445269Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-18T02:25:11.463307Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-18T02:25:11.467169Z  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    │
╰─────────────────────────────────┴───────╯

2025-10-18T02:25:11.467308Z  INFO oarfish::bulk: Total number of alignment records : 116
2025-10-18T02:25:11.467556Z  INFO oarfish::bulk: number of aligned reads : 96
2025-10-18T02:25:11.467581Z  INFO oarfish::bulk: number of unique alignments : 86
2025-10-18T02:25:11.476826Z  INFO oarfish: oarfish completed successfully.
2025-10-18T02:25:11.595140Z  INFO oarfish: setting user-provided filter parameters.
2025-10-18T02:25:11.596245Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpjNKYVT/file129c9a6ea95/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-10-18T02:25:11.596292Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-18T02:25:11.596306Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-18T02:25:11.596450Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-18T02:25:11.596474Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-18T02:25:11.599490Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-18T02:25:11.599958Z  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    │
╰─────────────────────────────────┴───────╯

2025-10-18T02:25:11.600029Z  INFO oarfish::bulk: Total number of alignment records : 114
2025-10-18T02:25:11.600042Z  INFO oarfish::bulk: number of aligned reads : 95
2025-10-18T02:25:11.600049Z  INFO oarfish::bulk: number of unique alignments : 82
2025-10-18T02:25:11.604974Z  INFO oarfish: oarfish completed successfully.
2025-10-18T02:25:11.700849Z  INFO oarfish: setting user-provided filter parameters.
2025-10-18T02:25:11.701401Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpjNKYVT/file129c9a6ea95/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-10-18T02:25:11.701436Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-18T02:25:11.701447Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-18T02:25:11.701600Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-18T02:25:11.701634Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-18T02:25:11.704967Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2025-10-18T02:25:11.705360Z  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   │
╰─────────────────────────────────┴───────╯

2025-10-18T02:25:11.705429Z  INFO oarfish::bulk: Total number of alignment records : 239
2025-10-18T02:25:11.705439Z  INFO oarfish::bulk: number of aligned reads : 179
2025-10-18T02:25:11.705447Z  INFO oarfish::bulk: number of unique alignments : 143
2025-10-18T02:25:11.710578Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpjNKYVT/file129c23fe2894/config_file_4764.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Fri Oct 17 22:25:15 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpjNKYVT/file129c23fe2894/sample1_align2genome.bam
sample2 ->/tmp/RtmpjNKYVT/file129c23fe2894/sample2_align2genome.bam
sample3 ->/tmp/RtmpjNKYVT/file129c23fe2894/sample3_align2genome.bam
-- Running step: isoform_identification @ Fri Oct 17 22:25:43 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 4 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Fri Oct 17 22:26:07 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpjNKYVT/file129c23fe2894/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpjNKYVT/file129c23fe2894/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpjNKYVT/file129c23fe2894/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Fri Oct 17 22:26:33 2025 ----------
2025-10-18T02:26:33.767709Z  INFO oarfish: setting user-provided filter parameters.
2025-10-18T02:26:33.772220Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpjNKYVT/file129c23fe2894/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-10-18T02:26:33.772418Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-18T02:26:33.772485Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-18T02:26:33.773951Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-18T02:26:33.774062Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-18T02:26:33.778186Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-18T02:26:33.781097Z  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    │
╰─────────────────────────────────┴───────╯

2025-10-18T02:26:33.781361Z  INFO oarfish::bulk: Total number of alignment records : 116
2025-10-18T02:26:33.781377Z  INFO oarfish::bulk: number of aligned reads : 96
2025-10-18T02:26:33.781385Z  INFO oarfish::bulk: number of unique alignments : 86
2025-10-18T02:26:33.789336Z  INFO oarfish: oarfish completed successfully.
2025-10-18T02:26:33.891363Z  INFO oarfish: setting user-provided filter parameters.
2025-10-18T02:26:33.893540Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpjNKYVT/file129c23fe2894/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-10-18T02:26:33.893592Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-18T02:26:33.893681Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-18T02:26:33.893917Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-18T02:26:33.893945Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-18T02:26:33.898228Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-18T02:26:33.898531Z  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    │
╰─────────────────────────────────┴───────╯

2025-10-18T02:26:33.898582Z  INFO oarfish::bulk: Total number of alignment records : 114
2025-10-18T02:26:33.898613Z  INFO oarfish::bulk: number of aligned reads : 95
2025-10-18T02:26:33.898619Z  INFO oarfish::bulk: number of unique alignments : 82
2025-10-18T02:26:33.903884Z  INFO oarfish: oarfish completed successfully.
2025-10-18T02:26:34.016766Z  INFO oarfish: setting user-provided filter parameters.
2025-10-18T02:26:34.017515Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpjNKYVT/file129c23fe2894/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-10-18T02:26:34.017564Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-18T02:26:34.017586Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-18T02:26:34.017714Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-18T02:26:34.017738Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-18T02:26:34.023019Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2025-10-18T02:26:34.023606Z  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   │
╰─────────────────────────────────┴───────╯

2025-10-18T02:26:34.023690Z  INFO oarfish::bulk: Total number of alignment records : 239
2025-10-18T02:26:34.023703Z  INFO oarfish::bulk: number of aligned reads : 179
2025-10-18T02:26:34.023710Z  INFO oarfish::bulk: number of unique alignments : 143
2025-10-18T02:26:34.029368Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpjNKYVT/file129c31853e6b/config_file_4764.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Fri Oct 17 22:26:35 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpjNKYVT/file129c31853e6b/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpjNKYVT/file129c31853e6b/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpjNKYVT/file129c31853e6b/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Fri Oct 17 22:26:36 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 4 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Fri Oct 17 22:26:58 2025 -------------------
Realigning sample sample1 -> /tmp/RtmpjNKYVT/file129c31853e6b/sample1_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample2 -> /tmp/RtmpjNKYVT/file129c31853e6b/sample2_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample3 -> /tmp/RtmpjNKYVT/file129c31853e6b/sample3_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Fri Oct 17 22:26:59 2025 ----------
22:26:59 Fri Oct 17 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Inputs:  ['/tmp/RtmpjNKYVT/file129c31853e6b/sample3_realign2transcript.bam', '/tmp/RtmpjNKYVT/file129c31853e6b/sample2_realign2transcript.bam', '/tmp/RtmpjNKYVT/file129c31853e6b/sample1_realign2transcript.bam'] /tmp/RtmpjNKYVT/file129c31853e6b/transcript_assembly.fa.fai 5 0.4 0.4
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpjNKYVT/file129c23a90153/config_file_4764.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Fri Oct 17 22:27:02 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpjNKYVT/file129c23a90153/sample1_align2genome.bam
sample2 ->/tmp/RtmpjNKYVT/file129c23a90153/sample2_align2genome.bam
sample3 ->/tmp/RtmpjNKYVT/file129c23a90153/sample3_align2genome.bam
-- Running step: isoform_identification @ Fri Oct 17 22:27:25 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 4 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Fri Oct 17 22:27:47 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpjNKYVT/file129c23a90153/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpjNKYVT/file129c23a90153/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpjNKYVT/file129c23a90153/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Fri Oct 17 22:28:09 2025 ----------
22:28:09 Fri Oct 17 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	Counter({'counted_reads': 375, 'not_enough_coverage': 16, 'unmapped': 2})
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpjNKYVT/file129c594d7272/config_file_4764.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Fri Oct 17 22:28:13 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpjNKYVT/file129c594d7272/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpjNKYVT/file129c594d7272/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpjNKYVT/file129c594d7272/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Fri Oct 17 22:28:14 2025 -------------
Inputs:  ['/tmp/RtmpjNKYVT/file129c23a90153/sample3_realign2transcript.bam', '/tmp/RtmpjNKYVT/file129c23a90153/sample2_realign2transcript.bam', '/tmp/RtmpjNKYVT/file129c23a90153/sample1_realign2transcript.bam'] /tmp/RtmpjNKYVT/file129c23a90153/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
-- Running step: read_realignment @ Fri Oct 17 22:28:15 2025 -------------------
Realigning sample sample1 -> /tmp/RtmpjNKYVT/file129c594d7272/sample1_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample2 -> /tmp/RtmpjNKYVT/file129c594d7272/sample2_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample3 -> /tmp/RtmpjNKYVT/file129c594d7272/sample3_realign2transcript.bam
Skipped sorting BAM files.

-- Running step: transcript_quantification @ Fri Oct 17 22:28:17 2025 ----------
2025-10-18T02:28:18.418505Z  INFO oarfish: setting user-provided filter parameters.
2025-10-18T02:28:18.734480Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpjNKYVT/file129c594d7272/sample1_realign2transcript.bam, contains 10 reference sequences.
2025-10-18T02:28:18.745481Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-18T02:28:18.745684Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-18T02:28:18.792851Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-18T02:28:18.792938Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-18T02:28:18.831646Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-18T02:28:18.870129Z  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    │
╰─────────────────────────────────┴───────╯

2025-10-18T02:28:18.912717Z  INFO oarfish::bulk: Total number of alignment records : 125
2025-10-18T02:28:18.912805Z  INFO oarfish::bulk: number of aligned reads : 98
2025-10-18T02:28:18.912821Z  INFO oarfish::bulk: number of unique alignments : 86
2025-10-18T02:28:18.919705Z  INFO oarfish: oarfish completed successfully.
2025-10-18T02:28:19.368840Z  INFO oarfish: setting user-provided filter parameters.
2025-10-18T02:28:19.369636Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpjNKYVT/file129c594d7272/sample2_realign2transcript.bam, contains 10 reference sequences.
2025-10-18T02:28:19.369688Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-18T02:28:19.369719Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-18T02:28:19.370079Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-18T02:28:19.370124Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-18T02:28:19.405014Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-18T02:28:19.419415Z  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    │
╰─────────────────────────────────┴───────╯

2025-10-18T02:28:19.419505Z  INFO oarfish::bulk: Total number of alignment records : 136
2025-10-18T02:28:19.419515Z  INFO oarfish::bulk: number of aligned reads : 97
2025-10-18T02:28:19.419521Z  INFO oarfish::bulk: number of unique alignments : 79
2025-10-18T02:28:19.435021Z  INFO oarfish: oarfish completed successfully.
2025-10-18T02:28:19.616832Z  INFO oarfish: setting user-provided filter parameters.
2025-10-18T02:28:19.618778Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpjNKYVT/file129c594d7272/sample3_realign2transcript.bam, contains 10 reference sequences.
2025-10-18T02:28:19.618909Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-18T02:28:19.618948Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-18T02:28:19.619095Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-18T02:28:19.619111Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-18T02:28:19.627868Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-18T02:28:19.628814Z  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   │
╰─────────────────────────────────┴───────╯

2025-10-18T02:28:19.628880Z  INFO oarfish::bulk: Total number of alignment records : 272
2025-10-18T02:28:19.628890Z  INFO oarfish::bulk: number of aligned reads : 187
2025-10-18T02:28:19.628899Z  INFO oarfish::bulk: number of unique alignments : 140
2025-10-18T02:28:19.635093Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpjNKYVT/file129c266c466b/config_file_4764.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Fri Oct 17 22:28:20 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpjNKYVT/file129c266c466b/sample1_align2genome.bam
sample2 ->/tmp/RtmpjNKYVT/file129c266c466b/sample2_align2genome.bam
sample3 ->/tmp/RtmpjNKYVT/file129c266c466b/sample3_align2genome.bam
-- Running step: isoform_identification @ Fri Oct 17 22:28:43 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri Oct 17 22:28:44 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpjNKYVT/file129c266c466b/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpjNKYVT/file129c266c466b/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpjNKYVT/file129c266c466b/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Fri Oct 17 22:29:11 2025 ----------
2025-10-18T02:29:11.853530Z  INFO oarfish: setting user-provided filter parameters.
2025-10-18T02:29:11.859920Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpjNKYVT/file129c266c466b/sample1_realign2transcript.bam, contains 10 reference sequences.
2025-10-18T02:29:11.860189Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-18T02:29:11.860269Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-18T02:29:11.862185Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-18T02:29:11.862220Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-18T02:29:11.871590Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-18T02:29:11.873970Z  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    │
╰─────────────────────────────────┴───────╯

2025-10-18T02:29:11.874240Z  INFO oarfish::bulk: Total number of alignment records : 125
2025-10-18T02:29:11.874260Z  INFO oarfish::bulk: number of aligned reads : 98
2025-10-18T02:29:11.874269Z  INFO oarfish::bulk: number of unique alignments : 86
2025-10-18T02:29:11.879192Z  INFO oarfish: oarfish completed successfully.
2025-10-18T02:29:12.021473Z  INFO oarfish: setting user-provided filter parameters.
2025-10-18T02:29:12.025358Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpjNKYVT/file129c266c466b/sample2_realign2transcript.bam, contains 10 reference sequences.
2025-10-18T02:29:12.025415Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-18T02:29:12.025428Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-18T02:29:12.025856Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-18T02:29:12.025899Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-18T02:29:12.034194Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-18T02:29:12.037080Z  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    │
╰─────────────────────────────────┴───────╯

2025-10-18T02:29:12.037226Z  INFO oarfish::bulk: Total number of alignment records : 136
2025-10-18T02:29:12.037260Z  INFO oarfish::bulk: number of aligned reads : 97
2025-10-18T02:29:12.037272Z  INFO oarfish::bulk: number of unique alignments : 79
2025-10-18T02:29:12.044726Z  INFO oarfish: oarfish completed successfully.
2025-10-18T02:29:12.123870Z  INFO oarfish: setting user-provided filter parameters.
2025-10-18T02:29:12.124615Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpjNKYVT/file129c266c466b/sample3_realign2transcript.bam, contains 10 reference sequences.
2025-10-18T02:29:12.124659Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-18T02:29:12.124674Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-18T02:29:12.124925Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-18T02:29:12.124950Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-18T02:29:12.131165Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-18T02:29:12.131697Z  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   │
╰─────────────────────────────────┴───────╯

2025-10-18T02:29:12.131784Z  INFO oarfish::bulk: Total number of alignment records : 272
2025-10-18T02:29:12.131795Z  INFO oarfish::bulk: number of aligned reads : 187
2025-10-18T02:29:12.131802Z  INFO oarfish::bulk: number of unique alignments : 140
2025-10-18T02:29:12.136009Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpjNKYVT/file129c3d20bfab/config_file_4764.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Fri Oct 17 22:29:13 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpjNKYVT/file129c3d20bfab/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpjNKYVT/file129c3d20bfab/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpjNKYVT/file129c3d20bfab/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Fri Oct 17 22:29:14 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri Oct 17 22:29:15 2025 -------------------
Realigning sample sample1 -> /tmp/RtmpjNKYVT/file129c3d20bfab/sample1_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample2 -> /tmp/RtmpjNKYVT/file129c3d20bfab/sample2_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample3 -> /tmp/RtmpjNKYVT/file129c3d20bfab/sample3_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Fri Oct 17 22:29:16 2025 ----------
22:29:16 Fri Oct 17 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpjNKYVT/file129c1f8dde0f/config_file_4764.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Fri Oct 17 22:29:17 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpjNKYVT/file129c1f8dde0f/sample1_align2genome.bam
sample2 ->/tmp/RtmpjNKYVT/file129c1f8dde0f/sample2_align2genome.bam
sample3 ->/tmp/RtmpjNKYVT/file129c1f8dde0f/sample3_align2genome.bam
-- Running step: isoform_identification @ Fri Oct 17 22:29:40 2025 -------------
Inputs:  ['/tmp/RtmpjNKYVT/file129c3d20bfab/sample3_realign2transcript.bam', '/tmp/RtmpjNKYVT/file129c3d20bfab/sample2_realign2transcript.bam', '/tmp/RtmpjNKYVT/file129c3d20bfab/sample1_realign2transcript.bam'] /tmp/RtmpjNKYVT/file129c3d20bfab/transcript_assembly.fa.fai 5 0.4 0.4
	Counter({'counted_reads': 391, 'not_enough_coverage': 2})
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri Oct 17 22:29:41 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpjNKYVT/file129c1f8dde0f/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpjNKYVT/file129c1f8dde0f/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpjNKYVT/file129c1f8dde0f/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Fri Oct 17 22:30:09 2025 ----------
22:30:09 Fri Oct 17 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Inputs:  ['/tmp/RtmpjNKYVT/file129c1f8dde0f/sample3_realign2transcript.bam', '/tmp/RtmpjNKYVT/file129c1f8dde0f/sample2_realign2transcript.bam', '/tmp/RtmpjNKYVT/file129c1f8dde0f/sample1_realign2transcript.bam'] /tmp/RtmpjNKYVT/file129c1f8dde0f/transcript_assembly.fa.fai 5 0.4 0.4
	Counter({'counted_reads': 391, 'not_enough_coverage': 2})
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpjNKYVT/file129cd3e59c9/config_file_4764.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Fri Oct 17 22:30:10 2025 ----------------
Using flexiplex for barcode demultiplexing.
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/RtmpjNKYVT/file129cd3e59c9/bc_allow.tsv
Number of known barcodes: 143
Processing file: /Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/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!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Fri Oct 17 22:30:11 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpjNKYVT/file129cd3e59c9/matched_reads.fastq.gz -> /tmp/RtmpjNKYVT/file129cd3e59c9/align2genome.bam
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: isoform_identification @ Fri Oct 17 22:30:11 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 ---
-- Running step: read_realignment @ Fri Oct 17 22:30:24 2025 -------------------
Checking for fastq file(s) /Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpjNKYVT/file129cd3e59c9/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpjNKYVT/file129cd3e59c9/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/RtmpjNKYVT/file129cd3e59c9/matched_reads.fastq.gz -> /tmp/RtmpjNKYVT/file129cd3e59c9/realign2transcript.bam
Sorting BAM files by 8 with CB threads...

[bam_sort_core] merging from 0 files and 8 in-memory blocks...
-- Running step: transcript_quantification @ Fri Oct 17 22:30:24 2025 ----------
2025-10-18T02:30:24.959114Z  INFO oarfish: setting user-provided filter parameters.
2025-10-18T02:30:24.997806Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpjNKYVT/file129cd3e59c9/realign2transcript.bam, contains 5 reference sequences.
2025-10-18T02:30:24.999851Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-18T02:30:25.000120Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-18T02:30:25.003635Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-18T02:30:25.003692Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-18T02:30:26.383631Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpjNKYVT/file129c10791fd7/config_file_4764.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Fri Oct 17 22:30:27 2025 ----------------
Using flexiplex for barcode demultiplexing.
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/RtmpjNKYVT/file129c10791fd7/bc_allow.tsv
Number of known barcodes: 143
Processing file: /Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/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!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Fri Oct 17 22:30:28 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpjNKYVT/file129c10791fd7/matched_reads.fastq.gz ->/tmp/RtmpjNKYVT/file129c10791fd7/align2genome.bam
-- Running step: isoform_identification @ Fri Oct 17 22:31:00 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 ---
-- Running step: read_realignment @ Fri Oct 17 22:31:12 2025 -------------------
Checking for fastq file(s) /Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpjNKYVT/file129c10791fd7/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpjNKYVT/file129c10791fd7/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/RtmpjNKYVT/file129c10791fd7/matched_reads.fastq.gz ->/tmp/RtmpjNKYVT/file129c10791fd7/realign2transcript.bam
-- Running step: transcript_quantification @ Fri Oct 17 22:31:39 2025 ----------
2025-10-18T02:31:40.007436Z  INFO oarfish: setting user-provided filter parameters.
2025-10-18T02:31:40.019180Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpjNKYVT/file129c10791fd7/realign2transcript.bam, contains 5 reference sequences.
2025-10-18T02:31:40.019608Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-18T02:31:40.019661Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-18T02:31:40.022694Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-18T02:31:40.022815Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-18T02:31:40.052567Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpjNKYVT/file129c30e52b1d/config_file_4764.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Fri Oct 17 22:31:40 2025 ----------------
Using flexiplex for barcode demultiplexing.
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/RtmpjNKYVT/file129c30e52b1d/bc_allow.tsv
Number of known barcodes: 143
Processing file: /Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/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!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Fri Oct 17 22:31:41 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpjNKYVT/file129c30e52b1d/matched_reads.fastq.gz -> /tmp/RtmpjNKYVT/file129c30e52b1d/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.
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: isoform_identification @ Fri Oct 17 22:31:41 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 ---
-- Running step: read_realignment @ Fri Oct 17 22:31:54 2025 -------------------
Checking for fastq file(s) /Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpjNKYVT/file129c30e52b1d/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpjNKYVT/file129c30e52b1d/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/RtmpjNKYVT/file129c30e52b1d/matched_reads.fastq.gz -> /tmp/RtmpjNKYVT/file129c30e52b1d/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.
Sorting BAM files by genome coordinates with 8 threads...

[bam_sort_core] merging from 0 files and 8 in-memory blocks...
Indexing bam files
-- Running step: transcript_quantification @ Fri Oct 17 22:31:54 2025 ----------
22:31:54 Fri Oct 17 2025 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpjNKYVT/file129c4c522f0c/config_file_4764.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Fri Oct 17 22:31:56 2025 ----------------
Using flexiplex for barcode demultiplexing.
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/RtmpjNKYVT/file129c4c522f0c/bc_allow.tsv
Number of known barcodes: 143
Processing file: /Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/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!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Fri Oct 17 22:31:56 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpjNKYVT/file129c4c522f0c/matched_reads.fastq.gz ->/tmp/RtmpjNKYVT/file129c4c522f0c/align2genome.bam
-- Running step: isoform_identification @ Fri Oct 17 22:32:25 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 ---
-- Running step: read_realignment @ Fri Oct 17 22:32:36 2025 -------------------
Checking for fastq file(s) /Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpjNKYVT/file129c4c522f0c/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpjNKYVT/file129c4c522f0c/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/RtmpjNKYVT/file129c4c522f0c/matched_reads.fastq.gz ->/tmp/RtmpjNKYVT/file129c4c522f0c/realign2transcript.bam
-- Running step: transcript_quantification @ Fri Oct 17 22:33:11 2025 ----------
22:33:11 Fri Oct 17 2025 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
	Counter({'counted_reads': 354, 'not_enough_coverage': 12, 'unmapped': 6})
	Counter({'counted_reads': 354, 'not_enough_coverage': 12, 'unmapped': 6})
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpjNKYVT/file129c3fcd9464/config_file_4764.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Fri Oct 17 22:33:15 2025 ----------------
Using flexiplex for barcode demultiplexing.
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/RtmpjNKYVT/file129c3fcd9464/bc_allow.tsv
Number of known barcodes: 143
Processing file: /Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/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!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Fri Oct 17 22:33:15 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpjNKYVT/file129c3fcd9464/matched_reads.fastq.gz -> /tmp/RtmpjNKYVT/file129c3fcd9464/align2genome.bam
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: isoform_identification @ Fri Oct 17 22:33:16 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri Oct 17 22:33:17 2025 -------------------
Checking for fastq file(s) /Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpjNKYVT/file129c3fcd9464/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpjNKYVT/file129c3fcd9464/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/RtmpjNKYVT/file129c3fcd9464/matched_reads.fastq.gz -> /tmp/RtmpjNKYVT/file129c3fcd9464/realign2transcript.bam
Sorting BAM files by 8 with CB threads...

[bam_sort_core] merging from 0 files and 8 in-memory blocks...
-- Running step: transcript_quantification @ Fri Oct 17 22:33:17 2025 ----------
2025-10-18T02:33:18.053594Z  INFO oarfish: setting user-provided filter parameters.
2025-10-18T02:33:18.066919Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpjNKYVT/file129c3fcd9464/realign2transcript.bam, contains 10 reference sequences.
2025-10-18T02:33:18.066969Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-18T02:33:18.067212Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-18T02:33:18.119701Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-18T02:33:18.120034Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-18T02:33:18.341470Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpjNKYVT/file129c39fad431/config_file_4764.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Fri Oct 17 22:33:20 2025 ----------------
Using flexiplex for barcode demultiplexing.
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/RtmpjNKYVT/file129c39fad431/bc_allow.tsv
Number of known barcodes: 143
Processing file: /Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/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!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Fri Oct 17 22:33:20 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpjNKYVT/file129c39fad431/matched_reads.fastq.gz ->/tmp/RtmpjNKYVT/file129c39fad431/align2genome.bam
-- Running step: isoform_identification @ Fri Oct 17 22:33:55 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri Oct 17 22:33:55 2025 -------------------
Checking for fastq file(s) /Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpjNKYVT/file129c39fad431/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpjNKYVT/file129c39fad431/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/RtmpjNKYVT/file129c39fad431/matched_reads.fastq.gz ->/tmp/RtmpjNKYVT/file129c39fad431/realign2transcript.bam
-- Running step: transcript_quantification @ Fri Oct 17 22:34:20 2025 ----------
2025-10-18T02:34:21.045269Z  INFO oarfish: setting user-provided filter parameters.
2025-10-18T02:34:21.060437Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpjNKYVT/file129c39fad431/realign2transcript.bam, contains 10 reference sequences.
2025-10-18T02:34:21.060552Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-18T02:34:21.060590Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-18T02:34:21.063040Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-18T02:34:21.063104Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-18T02:34:21.094743Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpjNKYVT/file129c205b77de/config_file_4764.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Fri Oct 17 22:34:23 2025 ----------------
Using flexiplex for barcode demultiplexing.
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/RtmpjNKYVT/file129c205b77de/bc_allow.tsv
Number of known barcodes: 143
Processing file: /Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/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!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Fri Oct 17 22:34:23 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpjNKYVT/file129c205b77de/matched_reads.fastq.gz -> /tmp/RtmpjNKYVT/file129c205b77de/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.
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: isoform_identification @ Fri Oct 17 22:34:24 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri Oct 17 22:34:24 2025 -------------------
Checking for fastq file(s) /Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpjNKYVT/file129c205b77de/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpjNKYVT/file129c205b77de/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/RtmpjNKYVT/file129c205b77de/matched_reads.fastq.gz -> /tmp/RtmpjNKYVT/file129c205b77de/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.
Sorting BAM files by genome coordinates with 8 threads...

[bam_sort_core] merging from 0 files and 8 in-memory blocks...
Indexing bam files
-- Running step: transcript_quantification @ Fri Oct 17 22:34:25 2025 ----------
22:34:25 Fri Oct 17 2025 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpjNKYVT/file129c527f48b8/config_file_4764.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Fri Oct 17 22:34:26 2025 ----------------
Using flexiplex for barcode demultiplexing.
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/RtmpjNKYVT/file129c527f48b8/bc_allow.tsv
Number of known barcodes: 143
Processing file: /Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/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!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Fri Oct 17 22:34:27 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpjNKYVT/file129c527f48b8/matched_reads.fastq.gz ->/tmp/RtmpjNKYVT/file129c527f48b8/align2genome.bam
-- Running step: isoform_identification @ Fri Oct 17 22:34:52 2025 -------------
	Counter({'counted_reads': 368, 'unmapped': 4})
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri Oct 17 22:34:52 2025 -------------------
Checking for fastq file(s) /Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpjNKYVT/file129c527f48b8/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpjNKYVT/file129c527f48b8/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/RtmpjNKYVT/file129c527f48b8/matched_reads.fastq.gz ->/tmp/RtmpjNKYVT/file129c527f48b8/realign2transcript.bam
-- Running step: transcript_quantification @ Fri Oct 17 22:35:15 2025 ----------
22:35:15 Fri Oct 17 2025 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpjNKYVT/file129c7e294af2/config_file_4764.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Fri Oct 17 22:35:17 2025 ----------------
Using flexiplex for barcode demultiplexing.
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/RtmpjNKYVT/file129c7e294af2/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpjNKYVT/file129c7e294af2/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpjNKYVT/file129c7e294af2/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpjNKYVT/file129c7e294af2/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!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
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/RtmpjNKYVT/file129c7e294af2/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpjNKYVT/file129c7e294af2/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!
Reads	Barcodes
4	1
3	9
2	9
1	44
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/RtmpjNKYVT/file129c7e294af2/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpjNKYVT/file129c7e294af2/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!
Reads	Barcodes
4	2
3	3
2	16
1	47
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/RtmpjNKYVT/file129c7e294af2/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpjNKYVT/file129c7e294af2/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!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Fri Oct 17 22:35:19 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpjNKYVT/file129c7e294af2/sampleA_matched_reads.fastq.gz -> /tmp/RtmpjNKYVT/file129c7e294af2/sampleA_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpjNKYVT/file129c7e294af2/sample1_matched_reads.fastq.gz -> /tmp/RtmpjNKYVT/file129c7e294af2/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpjNKYVT/file129c7e294af2/sample2_matched_reads.fastq.gz -> /tmp/RtmpjNKYVT/file129c7e294af2/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpjNKYVT/file129c7e294af2/sample3_matched_reads.fastq.gz -> /tmp/RtmpjNKYVT/file129c7e294af2/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Fri Oct 17 22:35:21 2025 ----------------
22:35:21 Fri Oct 17 2025 quantify genes 
Using BAM(s): '/tmp/RtmpjNKYVT/file129c7e294af2/sampleA_align2genome.bam',
'/tmp/RtmpjNKYVT/file129c7e294af2/sample1_align2genome.bam',
'/tmp/RtmpjNKYVT/file129c7e294af2/sample2_align2genome.bam', and
'/tmp/RtmpjNKYVT/file129c7e294af2/sample3_align2genome.bam'
	Counter({'counted_reads': 368, 'unmapped': 4})
parsing /tmp/RtmpjNKYVT/file129c7e294af2/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  3.67gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  3.66gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
2025-10-17 22:35:25.939 R[4764:573101443] XType: com.apple.fonts is not accessible.
2025-10-17 22:35:25.939 R[4764:573101443] XType: XTFontStaticRegistry is enabled.
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 55768.79Read/s]
parsing /tmp/RtmpjNKYVT/file129c7e294af2/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 35.03gene_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, 742039.49Read/s]
parsing /tmp/RtmpjNKYVT/file129c7e294af2/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 32.68gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 101588.48Read/s]
parsing /tmp/RtmpjNKYVT/file129c7e294af2/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 24.58gene_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, 460750.51Read/s]
-- Running step: isoform_identification @ Fri Oct 17 22:35:28 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

  |                                                                            
  |                                                                      |   0%
  |                                                                            
  |==================                                                    |  25%
  |                                                                            
  |===================================                                   |  50%
  |                                                                            
  |====================================================                  |  75%
  |                                                                            
  |======================================================================| 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 ---
-- Running step: read_realignment @ Fri Oct 17 22:35:56 2025 -------------------
Checking for fastq file(s) /tmp/RtmpjNKYVT/file129c7e294af2/fastq, /tmp/RtmpjNKYVT/file129c7e294af2/fastq/sample1.fq.gz, /tmp/RtmpjNKYVT/file129c7e294af2/fastq/sample2.fq.gz, /tmp/RtmpjNKYVT/file129c7e294af2/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpjNKYVT/file129c7e294af2/sampleA_matched_reads.fastq.gz, /tmp/RtmpjNKYVT/file129c7e294af2/sample1_matched_reads.fastq.gz, /tmp/RtmpjNKYVT/file129c7e294af2/sample2_matched_reads.fastq.gz, /tmp/RtmpjNKYVT/file129c7e294af2/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpjNKYVT/file129c7e294af2/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpjNKYVT/file129c7e294af2/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpjNKYVT/file129c7e294af2/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpjNKYVT/file129c7e294af2/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpjNKYVT/file129c7e294af2/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpjNKYVT/file129c7e294af2/sampleA_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpjNKYVT/file129c7e294af2/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpjNKYVT/file129c7e294af2/sample1_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpjNKYVT/file129c7e294af2/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpjNKYVT/file129c7e294af2/sample2_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpjNKYVT/file129c7e294af2/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpjNKYVT/file129c7e294af2/sample3_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

-- Running step: transcript_quantification @ Fri Oct 17 22:35:57 2025 ----------
2025-10-18T02:35:58.069923Z  INFO oarfish: setting user-provided filter parameters.
2025-10-18T02:35:58.073378Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpjNKYVT/file129c7e294af2/sampleA_realign2transcript.bam, contains 5 reference sequences.
2025-10-18T02:35:58.073435Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-18T02:35:58.073456Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-18T02:35:58.074481Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-18T02:35:58.074521Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-18T02:35:58.105597Z  INFO oarfish::single_cell: Processed 100 cells.
2025-10-18T02:35:58.509398Z  INFO oarfish: setting user-provided filter parameters.
2025-10-18T02:35:58.510042Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpjNKYVT/file129c7e294af2/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-10-18T02:35:58.510107Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-18T02:35:58.510144Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-18T02:35:58.510292Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-18T02:35:58.510338Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-18T02:35:58.913108Z  INFO oarfish: setting user-provided filter parameters.
2025-10-18T02:35:58.913773Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpjNKYVT/file129c7e294af2/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-10-18T02:35:58.913823Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-18T02:35:58.913841Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-18T02:35:58.913986Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-18T02:35:58.914010Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-18T02:35:59.361180Z  INFO oarfish: setting user-provided filter parameters.
2025-10-18T02:35:59.361858Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpjNKYVT/file129c7e294af2/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-10-18T02:35:59.361902Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-18T02:35:59.361919Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-18T02:35:59.362048Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-18T02:35:59.362067Z  INFO oarfish: parsed reference information for 5 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpjNKYVT/file129c6bd545/config_file_4764.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Fri Oct 17 22:36:00 2025 ----------------
Using flexiplex for barcode demultiplexing.
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/RtmpjNKYVT/file129c6bd545/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpjNKYVT/file129c6bd545/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpjNKYVT/file129c6bd545/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpjNKYVT/file129c6bd545/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!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
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/RtmpjNKYVT/file129c6bd545/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpjNKYVT/file129c6bd545/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!
Reads	Barcodes
4	1
3	9
2	9
1	44
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/RtmpjNKYVT/file129c6bd545/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpjNKYVT/file129c6bd545/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!
Reads	Barcodes
4	2
3	3
2	16
1	47
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/RtmpjNKYVT/file129c6bd545/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpjNKYVT/file129c6bd545/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!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Fri Oct 17 22:36:03 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpjNKYVT/file129c6bd545/sampleA_matched_reads.fastq.gz ->/tmp/RtmpjNKYVT/file129c6bd545/sampleA_align2genome.bam
/tmp/RtmpjNKYVT/file129c6bd545/sample1_matched_reads.fastq.gz ->/tmp/RtmpjNKYVT/file129c6bd545/sample1_align2genome.bam
/tmp/RtmpjNKYVT/file129c6bd545/sample2_matched_reads.fastq.gz ->/tmp/RtmpjNKYVT/file129c6bd545/sample2_align2genome.bam
/tmp/RtmpjNKYVT/file129c6bd545/sample3_matched_reads.fastq.gz ->/tmp/RtmpjNKYVT/file129c6bd545/sample3_align2genome.bam
-- Running step: gene_quantification @ Fri Oct 17 22:36:34 2025 ----------------
22:36:34 Fri Oct 17 2025 quantify genes 
Using BAM(s): '/tmp/RtmpjNKYVT/file129c6bd545/sampleA_align2genome.bam',
'/tmp/RtmpjNKYVT/file129c6bd545/sample1_align2genome.bam',
'/tmp/RtmpjNKYVT/file129c6bd545/sample2_align2genome.bam', and
'/tmp/RtmpjNKYVT/file129c6bd545/sample3_align2genome.bam'
parsing /tmp/RtmpjNKYVT/file129c6bd545/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 12.22gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 178544.84Read/s]
parsing /tmp/RtmpjNKYVT/file129c6bd545/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 33.62gene_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, 338163.05Read/s]
parsing /tmp/RtmpjNKYVT/file129c6bd545/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 34.36gene_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, 748127.85Read/s]
parsing /tmp/RtmpjNKYVT/file129c6bd545/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 26.28gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 309478.78Read/s]
-- Running step: isoform_identification @ Fri Oct 17 22:36:35 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

  |                                                                            
  |                                                                      |   0%
  |                                                                            
  |==================                                                    |  25%
  |                                                                            
  |===================================                                   |  50%
  |                                                                            
  |====================================================                  |  75%
  |                                                                            
  |======================================================================| 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 ---
-- Running step: read_realignment @ Fri Oct 17 22:37:00 2025 -------------------
Checking for fastq file(s) /tmp/RtmpjNKYVT/file129c6bd545/fastq, /tmp/RtmpjNKYVT/file129c6bd545/fastq/sample1.fq.gz, /tmp/RtmpjNKYVT/file129c6bd545/fastq/sample2.fq.gz, /tmp/RtmpjNKYVT/file129c6bd545/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpjNKYVT/file129c6bd545/sampleA_matched_reads.fastq.gz, /tmp/RtmpjNKYVT/file129c6bd545/sample1_matched_reads.fastq.gz, /tmp/RtmpjNKYVT/file129c6bd545/sample2_matched_reads.fastq.gz, /tmp/RtmpjNKYVT/file129c6bd545/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpjNKYVT/file129c6bd545/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpjNKYVT/file129c6bd545/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpjNKYVT/file129c6bd545/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpjNKYVT/file129c6bd545/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpjNKYVT/file129c6bd545/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpjNKYVT/file129c6bd545/sampleA_realign2transcript.bam
/tmp/RtmpjNKYVT/file129c6bd545/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpjNKYVT/file129c6bd545/sample1_realign2transcript.bam
/tmp/RtmpjNKYVT/file129c6bd545/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpjNKYVT/file129c6bd545/sample2_realign2transcript.bam
/tmp/RtmpjNKYVT/file129c6bd545/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpjNKYVT/file129c6bd545/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Fri Oct 17 22:37:30 2025 ----------
2025-10-18T02:37:30.552649Z  INFO oarfish: setting user-provided filter parameters.
2025-10-18T02:37:30.560613Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpjNKYVT/file129c6bd545/sampleA_realign2transcript.bam, contains 5 reference sequences.
2025-10-18T02:37:30.560949Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-18T02:37:30.560972Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-18T02:37:30.564242Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-18T02:37:30.564360Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-18T02:37:30.594377Z  INFO oarfish::single_cell: Processed 100 cells.
2025-10-18T02:37:31.111946Z  INFO oarfish: setting user-provided filter parameters.
2025-10-18T02:37:31.112707Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpjNKYVT/file129c6bd545/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-10-18T02:37:31.112745Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-18T02:37:31.112757Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-18T02:37:31.112869Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-18T02:37:31.112889Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-18T02:37:31.618447Z  INFO oarfish: setting user-provided filter parameters.
2025-10-18T02:37:31.619323Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpjNKYVT/file129c6bd545/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-10-18T02:37:31.619371Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-18T02:37:31.619384Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-18T02:37:31.619527Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-18T02:37:31.619548Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-18T02:37:32.087311Z  INFO oarfish: setting user-provided filter parameters.
2025-10-18T02:37:32.088155Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpjNKYVT/file129c6bd545/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-10-18T02:37:32.088200Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-18T02:37:32.088212Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-18T02:37:32.088357Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-18T02:37:32.088384Z  INFO oarfish: parsed reference information for 5 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpjNKYVT/file129c20c3f64c/config_file_4764.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Fri Oct 17 22:37:33 2025 ----------------
Using flexiplex for barcode demultiplexing.
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/RtmpjNKYVT/file129c20c3f64c/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpjNKYVT/file129c20c3f64c/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpjNKYVT/file129c20c3f64c/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpjNKYVT/file129c20c3f64c/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!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
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/RtmpjNKYVT/file129c20c3f64c/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpjNKYVT/file129c20c3f64c/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!
Reads	Barcodes
4	1
3	9
2	9
1	44
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/RtmpjNKYVT/file129c20c3f64c/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpjNKYVT/file129c20c3f64c/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!
Reads	Barcodes
4	2
3	3
2	16
1	47
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/RtmpjNKYVT/file129c20c3f64c/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpjNKYVT/file129c20c3f64c/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!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Fri Oct 17 22:37:35 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpjNKYVT/file129c20c3f64c/sampleA_matched_reads.fastq.gz -> /tmp/RtmpjNKYVT/file129c20c3f64c/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.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpjNKYVT/file129c20c3f64c/sample1_matched_reads.fastq.gz -> /tmp/RtmpjNKYVT/file129c20c3f64c/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.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpjNKYVT/file129c20c3f64c/sample2_matched_reads.fastq.gz -> /tmp/RtmpjNKYVT/file129c20c3f64c/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.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpjNKYVT/file129c20c3f64c/sample3_matched_reads.fastq.gz -> /tmp/RtmpjNKYVT/file129c20c3f64c/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.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Fri Oct 17 22:37:37 2025 ----------------
22:37:37 Fri Oct 17 2025 quantify genes 
Using BAM(s): '/tmp/RtmpjNKYVT/file129c20c3f64c/sampleA_align2genome.bam',
'/tmp/RtmpjNKYVT/file129c20c3f64c/sample1_align2genome.bam',
'/tmp/RtmpjNKYVT/file129c20c3f64c/sample2_align2genome.bam', and
'/tmp/RtmpjNKYVT/file129c20c3f64c/sample3_align2genome.bam'
parsing /tmp/RtmpjNKYVT/file129c20c3f64c/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 17.87gene_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, 348283.12Read/s]
parsing /tmp/RtmpjNKYVT/file129c20c3f64c/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 16.57gene_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, 1627718.10Read/s]
parsing /tmp/RtmpjNKYVT/file129c20c3f64c/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 44.25gene_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, 1460208.88Read/s]
parsing /tmp/RtmpjNKYVT/file129c20c3f64c/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 27.66gene_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, 116490.32Read/s]
-- Running step: isoform_identification @ Fri Oct 17 22:37:39 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

  |                                                                            
  |                                                                      |   0%
  |                                                                            
  |==================                                                    |  25%
  |                                                                            
  |===================================                                   |  50%
  |                                                                            
  |====================================================                  |  75%
  |                                                                            
  |======================================================================| 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 ---
-- Running step: read_realignment @ Fri Oct 17 22:38:14 2025 -------------------
Checking for fastq file(s) /tmp/RtmpjNKYVT/file129c20c3f64c/fastq, /tmp/RtmpjNKYVT/file129c20c3f64c/fastq/sample1.fq.gz, /tmp/RtmpjNKYVT/file129c20c3f64c/fastq/sample2.fq.gz, /tmp/RtmpjNKYVT/file129c20c3f64c/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpjNKYVT/file129c20c3f64c/sampleA_matched_reads.fastq.gz, /tmp/RtmpjNKYVT/file129c20c3f64c/sample1_matched_reads.fastq.gz, /tmp/RtmpjNKYVT/file129c20c3f64c/sample2_matched_reads.fastq.gz, /tmp/RtmpjNKYVT/file129c20c3f64c/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpjNKYVT/file129c20c3f64c/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpjNKYVT/file129c20c3f64c/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpjNKYVT/file129c20c3f64c/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpjNKYVT/file129c20c3f64c/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpjNKYVT/file129c20c3f64c/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpjNKYVT/file129c20c3f64c/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.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample /tmp/RtmpjNKYVT/file129c20c3f64c/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpjNKYVT/file129c20c3f64c/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.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample /tmp/RtmpjNKYVT/file129c20c3f64c/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpjNKYVT/file129c20c3f64c/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.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample /tmp/RtmpjNKYVT/file129c20c3f64c/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpjNKYVT/file129c20c3f64c/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.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Fri Oct 17 22:38:15 2025 ----------
22:38:15 Fri Oct 17 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpjNKYVT/file129c20c3f64c/sample3_realign2transcript.bam...
parsing /tmp/RtmpjNKYVT/file129c20c3f64c/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpjNKYVT/file129c20c3f64c/sample3_realign2transcript.bamdone
parsing /tmp/RtmpjNKYVT/file129c20c3f64c/sampleA_realign2transcript.bam...
parsing /tmp/RtmpjNKYVT/file129c20c3f64c/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpjNKYVT/file129c20c3f64c/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpjNKYVT/file129c20c3f64c/sample2_realign2transcript.bam...
parsing /tmp/RtmpjNKYVT/file129c20c3f64c/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpjNKYVT/file129c20c3f64c/sample2_realign2transcript.bamdone
parsing /tmp/RtmpjNKYVT/file129c20c3f64c/sample1_realign2transcript.bam...
	Counter({'counted_reads': 168, 'not_enough_coverage': 6, 'unmapped': 2})
	Counter({'counted_reads': 347, 'not_enough_coverage': 9, 'unmapped': 2})
	Counter({'counted_reads': 92, 'not_enough_coverage': 3})
parsing /tmp/RtmpjNKYVT/file129c20c3f64c/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpjNKYVT/file129c20c3f64c/sample1_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpjNKYVT/file129c7aa60e74/config_file_4764.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Fri Oct 17 22:38:18 2025 ----------------
Using flexiplex for barcode demultiplexing.
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/RtmpjNKYVT/file129c7aa60e74/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpjNKYVT/file129c7aa60e74/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpjNKYVT/file129c7aa60e74/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpjNKYVT/file129c7aa60e74/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!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
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/RtmpjNKYVT/file129c7aa60e74/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpjNKYVT/file129c7aa60e74/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!
Reads	Barcodes
4	1
3	9
2	9
1	44
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/RtmpjNKYVT/file129c7aa60e74/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpjNKYVT/file129c7aa60e74/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!
Reads	Barcodes
4	2
3	3
2	16
1	47
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/RtmpjNKYVT/file129c7aa60e74/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpjNKYVT/file129c7aa60e74/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!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Fri Oct 17 22:38:20 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpjNKYVT/file129c7aa60e74/sampleA_matched_reads.fastq.gz ->/tmp/RtmpjNKYVT/file129c7aa60e74/sampleA_align2genome.bam
/tmp/RtmpjNKYVT/file129c7aa60e74/sample1_matched_reads.fastq.gz ->/tmp/RtmpjNKYVT/file129c7aa60e74/sample1_align2genome.bam
/tmp/RtmpjNKYVT/file129c7aa60e74/sample2_matched_reads.fastq.gz ->/tmp/RtmpjNKYVT/file129c7aa60e74/sample2_align2genome.bam
/tmp/RtmpjNKYVT/file129c7aa60e74/sample3_matched_reads.fastq.gz ->/tmp/RtmpjNKYVT/file129c7aa60e74/sample3_align2genome.bam
-- Running step: gene_quantification @ Fri Oct 17 22:38:51 2025 ----------------
22:38:51 Fri Oct 17 2025 quantify genes 
Using BAM(s): '/tmp/RtmpjNKYVT/file129c7aa60e74/sampleA_align2genome.bam',
'/tmp/RtmpjNKYVT/file129c7aa60e74/sample1_align2genome.bam',
'/tmp/RtmpjNKYVT/file129c7aa60e74/sample2_align2genome.bam', and
'/tmp/RtmpjNKYVT/file129c7aa60e74/sample3_align2genome.bam'
	Counter({'counted_reads': 89, 'not_enough_coverage': 2})
parsing /tmp/RtmpjNKYVT/file129c7aa60e74/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 13.90gene_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, 268438.89Read/s]
parsing /tmp/RtmpjNKYVT/file129c7aa60e74/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 35.98gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 943303.35Read/s]
parsing /tmp/RtmpjNKYVT/file129c7aa60e74/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 37.80gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 94589.87Read/s]
parsing /tmp/RtmpjNKYVT/file129c7aa60e74/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 10.89gene_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, 180297.81Read/s]
-- Running step: isoform_identification @ Fri Oct 17 22:38:53 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

  |                                                                            
  |                                                                      |   0%
  |                                                                            
  |==================                                                    |  25%
  |                                                                            
  |===================================                                   |  50%
  |                                                                            
  |====================================================                  |  75%
  |                                                                            
  |======================================================================| 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 ---
-- Running step: read_realignment @ Fri Oct 17 22:39:20 2025 -------------------
Checking for fastq file(s) /tmp/RtmpjNKYVT/file129c7aa60e74/fastq, /tmp/RtmpjNKYVT/file129c7aa60e74/fastq/sample1.fq.gz, /tmp/RtmpjNKYVT/file129c7aa60e74/fastq/sample2.fq.gz, /tmp/RtmpjNKYVT/file129c7aa60e74/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpjNKYVT/file129c7aa60e74/sampleA_matched_reads.fastq.gz, /tmp/RtmpjNKYVT/file129c7aa60e74/sample1_matched_reads.fastq.gz, /tmp/RtmpjNKYVT/file129c7aa60e74/sample2_matched_reads.fastq.gz, /tmp/RtmpjNKYVT/file129c7aa60e74/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpjNKYVT/file129c7aa60e74/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpjNKYVT/file129c7aa60e74/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpjNKYVT/file129c7aa60e74/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpjNKYVT/file129c7aa60e74/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpjNKYVT/file129c7aa60e74/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpjNKYVT/file129c7aa60e74/sampleA_realign2transcript.bam
/tmp/RtmpjNKYVT/file129c7aa60e74/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpjNKYVT/file129c7aa60e74/sample1_realign2transcript.bam
/tmp/RtmpjNKYVT/file129c7aa60e74/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpjNKYVT/file129c7aa60e74/sample2_realign2transcript.bam
/tmp/RtmpjNKYVT/file129c7aa60e74/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpjNKYVT/file129c7aa60e74/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Fri Oct 17 22:39:45 2025 ----------
22:39:45 Fri Oct 17 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpjNKYVT/file129c7aa60e74/sample3_realign2transcript.bam...
parsing /tmp/RtmpjNKYVT/file129c7aa60e74/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpjNKYVT/file129c7aa60e74/sample3_realign2transcript.bamdone
parsing /tmp/RtmpjNKYVT/file129c7aa60e74/sampleA_realign2transcript.bam...
parsing /tmp/RtmpjNKYVT/file129c7aa60e74/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpjNKYVT/file129c7aa60e74/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpjNKYVT/file129c7aa60e74/sample2_realign2transcript.bam...
parsing /tmp/RtmpjNKYVT/file129c7aa60e74/sample2_realign2transcript.bamdone
	Counter({'counted_reads': 168, 'not_enough_coverage': 6, 'unmapped': 2})
	Counter({'counted_reads': 347, 'not_enough_coverage': 9, 'unmapped': 2})
	Counter({'counted_reads': 92, 'not_enough_coverage': 3})
wrt_tr_to_csv for/tmp/RtmpjNKYVT/file129c7aa60e74/sample2_realign2transcript.bamdone
parsing /tmp/RtmpjNKYVT/file129c7aa60e74/sample1_realign2transcript.bam...
parsing /tmp/RtmpjNKYVT/file129c7aa60e74/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpjNKYVT/file129c7aa60e74/sample1_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpjNKYVT/file129c48152a0c/config_file_4764.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Fri Oct 17 22:39:49 2025 ----------------
Using flexiplex for barcode demultiplexing.
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/RtmpjNKYVT/file129c48152a0c/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpjNKYVT/file129c48152a0c/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpjNKYVT/file129c48152a0c/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpjNKYVT/file129c48152a0c/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!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
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/RtmpjNKYVT/file129c48152a0c/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpjNKYVT/file129c48152a0c/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!
Reads	Barcodes
4	1
3	9
2	9
1	44
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/RtmpjNKYVT/file129c48152a0c/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpjNKYVT/file129c48152a0c/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!
Reads	Barcodes
4	2
3	3
2	16
1	47
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/RtmpjNKYVT/file129c48152a0c/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpjNKYVT/file129c48152a0c/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!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Fri Oct 17 22:39:51 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpjNKYVT/file129c48152a0c/sampleA_matched_reads.fastq.gz -> /tmp/RtmpjNKYVT/file129c48152a0c/sampleA_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpjNKYVT/file129c48152a0c/sample1_matched_reads.fastq.gz -> /tmp/RtmpjNKYVT/file129c48152a0c/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpjNKYVT/file129c48152a0c/sample2_matched_reads.fastq.gz -> /tmp/RtmpjNKYVT/file129c48152a0c/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpjNKYVT/file129c48152a0c/sample3_matched_reads.fastq.gz -> /tmp/RtmpjNKYVT/file129c48152a0c/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Fri Oct 17 22:39:53 2025 ----------------
22:39:53 Fri Oct 17 2025 quantify genes 
Using BAM(s): '/tmp/RtmpjNKYVT/file129c48152a0c/sampleA_align2genome.bam',
'/tmp/RtmpjNKYVT/file129c48152a0c/sample1_align2genome.bam',
'/tmp/RtmpjNKYVT/file129c48152a0c/sample2_align2genome.bam', and
'/tmp/RtmpjNKYVT/file129c48152a0c/sample3_align2genome.bam'
	Counter({'counted_reads': 89, 'not_enough_coverage': 2})
parsing /tmp/RtmpjNKYVT/file129c48152a0c/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 15.30gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 328779.36Read/s]
parsing /tmp/RtmpjNKYVT/file129c48152a0c/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 33.94gene_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, 1155328.34Read/s]
parsing /tmp/RtmpjNKYVT/file129c48152a0c/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 37.61gene_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, 717956.86Read/s]
parsing /tmp/RtmpjNKYVT/file129c48152a0c/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 24.14gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 562178.85Read/s]
-- Running step: isoform_identification @ Fri Oct 17 22:39:54 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri Oct 17 22:39:55 2025 -------------------
Checking for fastq file(s) /tmp/RtmpjNKYVT/file129c48152a0c/fastq, /tmp/RtmpjNKYVT/file129c48152a0c/fastq/sample1.fq.gz, /tmp/RtmpjNKYVT/file129c48152a0c/fastq/sample2.fq.gz, /tmp/RtmpjNKYVT/file129c48152a0c/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpjNKYVT/file129c48152a0c/sampleA_matched_reads.fastq.gz, /tmp/RtmpjNKYVT/file129c48152a0c/sample1_matched_reads.fastq.gz, /tmp/RtmpjNKYVT/file129c48152a0c/sample2_matched_reads.fastq.gz, /tmp/RtmpjNKYVT/file129c48152a0c/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpjNKYVT/file129c48152a0c/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpjNKYVT/file129c48152a0c/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpjNKYVT/file129c48152a0c/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpjNKYVT/file129c48152a0c/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpjNKYVT/file129c48152a0c/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpjNKYVT/file129c48152a0c/sampleA_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpjNKYVT/file129c48152a0c/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpjNKYVT/file129c48152a0c/sample1_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpjNKYVT/file129c48152a0c/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpjNKYVT/file129c48152a0c/sample2_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpjNKYVT/file129c48152a0c/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpjNKYVT/file129c48152a0c/sample3_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

-- Running step: transcript_quantification @ Fri Oct 17 22:39:57 2025 ----------
2025-10-18T02:39:57.844998Z  INFO oarfish: setting user-provided filter parameters.
2025-10-18T02:39:57.848022Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpjNKYVT/file129c48152a0c/sampleA_realign2transcript.bam, contains 14 reference sequences.
2025-10-18T02:39:57.848134Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-18T02:39:57.848157Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-18T02:39:57.849463Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-18T02:39:57.849528Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-10-18T02:39:57.874384Z  INFO oarfish::single_cell: Processed 100 cells.
2025-10-18T02:39:58.565157Z  INFO oarfish: setting user-provided filter parameters.
2025-10-18T02:39:58.565964Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpjNKYVT/file129c48152a0c/sample1_realign2transcript.bam, contains 14 reference sequences.
2025-10-18T02:39:58.566004Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-18T02:39:58.566022Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-18T02:39:58.566171Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-18T02:39:58.566189Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-10-18T02:39:59.355012Z  INFO oarfish: setting user-provided filter parameters.
2025-10-18T02:39:59.355632Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpjNKYVT/file129c48152a0c/sample2_realign2transcript.bam, contains 14 reference sequences.
2025-10-18T02:39:59.355675Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-18T02:39:59.355689Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-18T02:39:59.355834Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-18T02:39:59.355852Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-10-18T02:40:00.161326Z  INFO oarfish: setting user-provided filter parameters.
2025-10-18T02:40:00.162076Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpjNKYVT/file129c48152a0c/sample3_realign2transcript.bam, contains 14 reference sequences.
2025-10-18T02:40:00.162129Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-18T02:40:00.162148Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-18T02:40:00.162339Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-18T02:40:00.162371Z  INFO oarfish: parsed reference information for 14 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpjNKYVT/file129c3a6e9cb7/config_file_4764.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Fri Oct 17 22:40:01 2025 ----------------
Using flexiplex for barcode demultiplexing.
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/RtmpjNKYVT/file129c3a6e9cb7/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpjNKYVT/file129c3a6e9cb7/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpjNKYVT/file129c3a6e9cb7/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpjNKYVT/file129c3a6e9cb7/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!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
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/RtmpjNKYVT/file129c3a6e9cb7/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpjNKYVT/file129c3a6e9cb7/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!
Reads	Barcodes
4	1
3	9
2	9
1	44
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/RtmpjNKYVT/file129c3a6e9cb7/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpjNKYVT/file129c3a6e9cb7/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!
Reads	Barcodes
4	2
3	3
2	16
1	47
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/RtmpjNKYVT/file129c3a6e9cb7/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpjNKYVT/file129c3a6e9cb7/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!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Fri Oct 17 22:40:05 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpjNKYVT/file129c3a6e9cb7/sampleA_matched_reads.fastq.gz ->/tmp/RtmpjNKYVT/file129c3a6e9cb7/sampleA_align2genome.bam
/tmp/RtmpjNKYVT/file129c3a6e9cb7/sample1_matched_reads.fastq.gz ->/tmp/RtmpjNKYVT/file129c3a6e9cb7/sample1_align2genome.bam
/tmp/RtmpjNKYVT/file129c3a6e9cb7/sample2_matched_reads.fastq.gz ->/tmp/RtmpjNKYVT/file129c3a6e9cb7/sample2_align2genome.bam
/tmp/RtmpjNKYVT/file129c3a6e9cb7/sample3_matched_reads.fastq.gz ->/tmp/RtmpjNKYVT/file129c3a6e9cb7/sample3_align2genome.bam
-- Running step: gene_quantification @ Fri Oct 17 22:40:32 2025 ----------------
22:40:32 Fri Oct 17 2025 quantify genes 
Using BAM(s): '/tmp/RtmpjNKYVT/file129c3a6e9cb7/sampleA_align2genome.bam',
'/tmp/RtmpjNKYVT/file129c3a6e9cb7/sample1_align2genome.bam',
'/tmp/RtmpjNKYVT/file129c3a6e9cb7/sample2_align2genome.bam', and
'/tmp/RtmpjNKYVT/file129c3a6e9cb7/sample3_align2genome.bam'
parsing /tmp/RtmpjNKYVT/file129c3a6e9cb7/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 16.10gene_group/s]
/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/FLAMES/python/count_gene.py:705: RuntimeWarning: More than 20 figures have been opened. Figures created through the pyplot interface (`matplotlib.pyplot.figure`) are retained until explicitly closed and may consume too much memory. (To control this warning, see the rcParam `figure.max_open_warning`). Consider using `matplotlib.pyplot.close()`.
  plt.figure(figsize=(8, 6))
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 305440.14Read/s]
parsing /tmp/RtmpjNKYVT/file129c3a6e9cb7/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 36.15gene_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, 1096378.08Read/s]
parsing /tmp/RtmpjNKYVT/file129c3a6e9cb7/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 35.16gene_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, 582348.11Read/s]
parsing /tmp/RtmpjNKYVT/file129c3a6e9cb7/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 28.96gene_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, 607517.96Read/s]
-- Running step: isoform_identification @ Fri Oct 17 22:40:33 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri Oct 17 22:40:34 2025 -------------------
Checking for fastq file(s) /tmp/RtmpjNKYVT/file129c3a6e9cb7/fastq, /tmp/RtmpjNKYVT/file129c3a6e9cb7/fastq/sample1.fq.gz, /tmp/RtmpjNKYVT/file129c3a6e9cb7/fastq/sample2.fq.gz, /tmp/RtmpjNKYVT/file129c3a6e9cb7/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpjNKYVT/file129c3a6e9cb7/sampleA_matched_reads.fastq.gz, /tmp/RtmpjNKYVT/file129c3a6e9cb7/sample1_matched_reads.fastq.gz, /tmp/RtmpjNKYVT/file129c3a6e9cb7/sample2_matched_reads.fastq.gz, /tmp/RtmpjNKYVT/file129c3a6e9cb7/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpjNKYVT/file129c3a6e9cb7/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpjNKYVT/file129c3a6e9cb7/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpjNKYVT/file129c3a6e9cb7/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpjNKYVT/file129c3a6e9cb7/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpjNKYVT/file129c3a6e9cb7/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpjNKYVT/file129c3a6e9cb7/sampleA_realign2transcript.bam
/tmp/RtmpjNKYVT/file129c3a6e9cb7/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpjNKYVT/file129c3a6e9cb7/sample1_realign2transcript.bam
/tmp/RtmpjNKYVT/file129c3a6e9cb7/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpjNKYVT/file129c3a6e9cb7/sample2_realign2transcript.bam
/tmp/RtmpjNKYVT/file129c3a6e9cb7/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpjNKYVT/file129c3a6e9cb7/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Fri Oct 17 22:40:58 2025 ----------
2025-10-18T02:40:58.461911Z  INFO oarfish: setting user-provided filter parameters.
2025-10-18T02:40:58.467721Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpjNKYVT/file129c3a6e9cb7/sampleA_realign2transcript.bam, contains 14 reference sequences.
2025-10-18T02:40:58.467995Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-18T02:40:58.468048Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-18T02:40:58.469875Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-18T02:40:58.470007Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-10-18T02:40:58.505462Z  INFO oarfish::single_cell: Processed 100 cells.
2025-10-18T02:40:59.534455Z  INFO oarfish: setting user-provided filter parameters.
2025-10-18T02:40:59.535501Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpjNKYVT/file129c3a6e9cb7/sample1_realign2transcript.bam, contains 14 reference sequences.
2025-10-18T02:40:59.535588Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-18T02:40:59.535626Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-18T02:40:59.535842Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-18T02:40:59.535876Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-10-18T02:41:00.446902Z  INFO oarfish: setting user-provided filter parameters.
2025-10-18T02:41:00.447864Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpjNKYVT/file129c3a6e9cb7/sample2_realign2transcript.bam, contains 14 reference sequences.
2025-10-18T02:41:00.447932Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-18T02:41:00.447962Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-18T02:41:00.448137Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-18T02:41:00.448160Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-10-18T02:41:01.247801Z  INFO oarfish: setting user-provided filter parameters.
2025-10-18T02:41:01.248488Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpjNKYVT/file129c3a6e9cb7/sample3_realign2transcript.bam, contains 14 reference sequences.
2025-10-18T02:41:01.248535Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-18T02:41:01.248552Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-18T02:41:01.248757Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-18T02:41:01.248784Z  INFO oarfish: parsed reference information for 14 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpjNKYVT/file129c25ecaa5b/config_file_4764.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Fri Oct 17 22:41:03 2025 ----------------
Using flexiplex for barcode demultiplexing.
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/RtmpjNKYVT/file129c25ecaa5b/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpjNKYVT/file129c25ecaa5b/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpjNKYVT/file129c25ecaa5b/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpjNKYVT/file129c25ecaa5b/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!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
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/RtmpjNKYVT/file129c25ecaa5b/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpjNKYVT/file129c25ecaa5b/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!
Reads	Barcodes
4	1
3	9
2	9
1	44
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/RtmpjNKYVT/file129c25ecaa5b/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpjNKYVT/file129c25ecaa5b/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!
Reads	Barcodes
4	2
3	3
2	16
1	47
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/RtmpjNKYVT/file129c25ecaa5b/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpjNKYVT/file129c25ecaa5b/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!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Fri Oct 17 22:41:05 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpjNKYVT/file129c25ecaa5b/sampleA_matched_reads.fastq.gz -> /tmp/RtmpjNKYVT/file129c25ecaa5b/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.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpjNKYVT/file129c25ecaa5b/sample1_matched_reads.fastq.gz -> /tmp/RtmpjNKYVT/file129c25ecaa5b/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.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpjNKYVT/file129c25ecaa5b/sample2_matched_reads.fastq.gz -> /tmp/RtmpjNKYVT/file129c25ecaa5b/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.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpjNKYVT/file129c25ecaa5b/sample3_matched_reads.fastq.gz -> /tmp/RtmpjNKYVT/file129c25ecaa5b/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.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Fri Oct 17 22:41:07 2025 ----------------
22:41:07 Fri Oct 17 2025 quantify genes 
Using BAM(s): '/tmp/RtmpjNKYVT/file129c25ecaa5b/sampleA_align2genome.bam',
'/tmp/RtmpjNKYVT/file129c25ecaa5b/sample1_align2genome.bam',
'/tmp/RtmpjNKYVT/file129c25ecaa5b/sample2_align2genome.bam', and
'/tmp/RtmpjNKYVT/file129c25ecaa5b/sample3_align2genome.bam'
parsing /tmp/RtmpjNKYVT/file129c25ecaa5b/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 18.93gene_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, 272300.82Read/s]
parsing /tmp/RtmpjNKYVT/file129c25ecaa5b/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 36.04gene_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, 1006021.30Read/s]
parsing /tmp/RtmpjNKYVT/file129c25ecaa5b/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 37.13gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1090789.56Read/s]
parsing /tmp/RtmpjNKYVT/file129c25ecaa5b/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  5.18gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  5.17gene_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, 634577.58Read/s]
-- Running step: isoform_identification @ Fri Oct 17 22:41:09 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri Oct 17 22:41:09 2025 -------------------
Checking for fastq file(s) /tmp/RtmpjNKYVT/file129c25ecaa5b/fastq, /tmp/RtmpjNKYVT/file129c25ecaa5b/fastq/sample1.fq.gz, /tmp/RtmpjNKYVT/file129c25ecaa5b/fastq/sample2.fq.gz, /tmp/RtmpjNKYVT/file129c25ecaa5b/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpjNKYVT/file129c25ecaa5b/sampleA_matched_reads.fastq.gz, /tmp/RtmpjNKYVT/file129c25ecaa5b/sample1_matched_reads.fastq.gz, /tmp/RtmpjNKYVT/file129c25ecaa5b/sample2_matched_reads.fastq.gz, /tmp/RtmpjNKYVT/file129c25ecaa5b/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpjNKYVT/file129c25ecaa5b/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpjNKYVT/file129c25ecaa5b/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpjNKYVT/file129c25ecaa5b/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpjNKYVT/file129c25ecaa5b/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpjNKYVT/file129c25ecaa5b/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpjNKYVT/file129c25ecaa5b/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.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample /tmp/RtmpjNKYVT/file129c25ecaa5b/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpjNKYVT/file129c25ecaa5b/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.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample /tmp/RtmpjNKYVT/file129c25ecaa5b/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpjNKYVT/file129c25ecaa5b/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.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample /tmp/RtmpjNKYVT/file129c25ecaa5b/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpjNKYVT/file129c25ecaa5b/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.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Fri Oct 17 22:41:10 2025 ----------
22:41:10 Fri Oct 17 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpjNKYVT/file129c25ecaa5b/sample3_realign2transcript.bam...
parsing /tmp/RtmpjNKYVT/file129c25ecaa5b/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpjNKYVT/file129c25ecaa5b/sample3_realign2transcript.bamdone
parsing /tmp/RtmpjNKYVT/file129c25ecaa5b/sampleA_realign2transcript.bam...
parsing /tmp/RtmpjNKYVT/file129c25ecaa5b/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpjNKYVT/file129c25ecaa5b/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpjNKYVT/file129c25ecaa5b/sample2_realign2transcript.bam...
parsing /tmp/RtmpjNKYVT/file129c25ecaa5b/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpjNKYVT/file129c25ecaa5b/sample2_realign2transcript.bamdone
parsing /tmp/RtmpjNKYVT/file129c25ecaa5b/sample1_realign2transcript.bam...
parsing /tmp/RtmpjNKYVT/file129c25ecaa5b/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpjNKYVT/file129c25ecaa5b/sample1_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpjNKYVT/file129c35f18e11/config_file_4764.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Fri Oct 17 22:41:15 2025 ----------------
Using flexiplex for barcode demultiplexing.
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/RtmpjNKYVT/file129c35f18e11/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpjNKYVT/file129c35f18e11/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpjNKYVT/file129c35f18e11/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpjNKYVT/file129c35f18e11/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!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
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/RtmpjNKYVT/file129c35f18e11/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpjNKYVT/file129c35f18e11/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!
Reads	Barcodes
4	1
3	9
2	9
1	44
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/RtmpjNKYVT/file129c35f18e11/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpjNKYVT/file129c35f18e11/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!
Reads	Barcodes
4	2
3	3
2	16
1	47
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/RtmpjNKYVT/file129c35f18e11/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpjNKYVT/file129c35f18e11/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!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Fri Oct 17 22:41:17 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpjNKYVT/file129c35f18e11/sampleA_matched_reads.fastq.gz ->/tmp/RtmpjNKYVT/file129c35f18e11/sampleA_align2genome.bam
/tmp/RtmpjNKYVT/file129c35f18e11/sample1_matched_reads.fastq.gz ->/tmp/RtmpjNKYVT/file129c35f18e11/sample1_align2genome.bam
/tmp/RtmpjNKYVT/file129c35f18e11/sample2_matched_reads.fastq.gz ->/tmp/RtmpjNKYVT/file129c35f18e11/sample2_align2genome.bam
/tmp/RtmpjNKYVT/file129c35f18e11/sample3_matched_reads.fastq.gz ->/tmp/RtmpjNKYVT/file129c35f18e11/sample3_align2genome.bam
-- Running step: gene_quantification @ Fri Oct 17 22:41:40 2025 ----------------
22:41:40 Fri Oct 17 2025 quantify genes 
Using BAM(s): '/tmp/RtmpjNKYVT/file129c35f18e11/sampleA_align2genome.bam',
'/tmp/RtmpjNKYVT/file129c35f18e11/sample1_align2genome.bam',
'/tmp/RtmpjNKYVT/file129c35f18e11/sample2_align2genome.bam', and
'/tmp/RtmpjNKYVT/file129c35f18e11/sample3_align2genome.bam'
	Counter({'counted_reads': 176})
	Counter({'counted_reads': 358})
	Counter({'counted_reads': 95})
	Counter({'counted_reads': 91})
parsing /tmp/RtmpjNKYVT/file129c35f18e11/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 15.53gene_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, 300761.82Read/s]
parsing /tmp/RtmpjNKYVT/file129c35f18e11/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 37.02gene_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, 1525423.33Read/s]
parsing /tmp/RtmpjNKYVT/file129c35f18e11/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 32.81gene_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, 946282.83Read/s]
parsing /tmp/RtmpjNKYVT/file129c35f18e11/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 24.27gene_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, 617245.11Read/s]
-- Running step: isoform_identification @ Fri Oct 17 22:41:42 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri Oct 17 22:41:42 2025 -------------------
Checking for fastq file(s) /tmp/RtmpjNKYVT/file129c35f18e11/fastq, /tmp/RtmpjNKYVT/file129c35f18e11/fastq/sample1.fq.gz, /tmp/RtmpjNKYVT/file129c35f18e11/fastq/sample2.fq.gz, /tmp/RtmpjNKYVT/file129c35f18e11/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpjNKYVT/file129c35f18e11/sampleA_matched_reads.fastq.gz, /tmp/RtmpjNKYVT/file129c35f18e11/sample1_matched_reads.fastq.gz, /tmp/RtmpjNKYVT/file129c35f18e11/sample2_matched_reads.fastq.gz, /tmp/RtmpjNKYVT/file129c35f18e11/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpjNKYVT/file129c35f18e11/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpjNKYVT/file129c35f18e11/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpjNKYVT/file129c35f18e11/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpjNKYVT/file129c35f18e11/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpjNKYVT/file129c35f18e11/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpjNKYVT/file129c35f18e11/sampleA_realign2transcript.bam
/tmp/RtmpjNKYVT/file129c35f18e11/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpjNKYVT/file129c35f18e11/sample1_realign2transcript.bam
/tmp/RtmpjNKYVT/file129c35f18e11/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpjNKYVT/file129c35f18e11/sample2_realign2transcript.bam
/tmp/RtmpjNKYVT/file129c35f18e11/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpjNKYVT/file129c35f18e11/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Fri Oct 17 22:42:05 2025 ----------
22:42:05 Fri Oct 17 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpjNKYVT/file129c35f18e11/sample3_realign2transcript.bam...
parsing /tmp/RtmpjNKYVT/file129c35f18e11/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpjNKYVT/file129c35f18e11/sample3_realign2transcript.bamdone
parsing /tmp/RtmpjNKYVT/file129c35f18e11/sampleA_realign2transcript.bam...
parsing /tmp/RtmpjNKYVT/file129c35f18e11/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpjNKYVT/file129c35f18e11/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpjNKYVT/file129c35f18e11/sample2_realign2transcript.bam...
parsing /tmp/RtmpjNKYVT/file129c35f18e11/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpjNKYVT/file129c35f18e11/sample2_realign2transcript.bamdone
parsing /tmp/RtmpjNKYVT/file129c35f18e11/sample1_realign2transcript.bam...
parsing /tmp/RtmpjNKYVT/file129c35f18e11/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpjNKYVT/file129c35f18e11/sample1_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
[ FAIL 0 | WARN 183 | SKIP 0 | PASS 59 ]

[ FAIL 0 | WARN 183 | SKIP 0 | PASS 59 ]
	Counter({'counted_reads': 176})
	Counter({'counted_reads': 358})
	Counter({'counted_reads': 95})
	Counter({'counted_reads': 91})
> 
> proc.time()
    user   system  elapsed 
 834.252   80.875 1078.332 
/Users/biocbuild/.pyenv/versions/3.11.9/lib/python3.11/multiprocessing/resource_tracker.py:254: UserWarning: resource_tracker: There appear to be 1 leaked semaphore objects to clean up at shutdown
  warnings.warn('resource_tracker: There appear to be %d '

Example timings

FLAMES.Rcheck/FLAMES-Ex.timings

nameusersystemelapsed
BulkPipeline4.5250.7345.915
MultiSampleSCPipeline13.128 2.69830.323
SingleCellPipeline3.4780.3674.393
add_gene_counts0.2140.0050.284
annotation_to_fasta0.1440.0050.182
blaze 7.659 1.40415.498
bulk_long_pipeline3.8711.0875.833
combine_sce0.9700.0961.096
config-set0.3070.2420.614
config0.3950.4050.857
controllers-set0.4870.0970.672
controllers0.3260.2591.212
convolution_filter0.0000.0010.003
create_config0.0090.0030.015
create_sce_from_dir 7.759 2.20310.216
create_se_from_dir3.7000.8905.056
cutadapt0.1800.0870.384
example_pipeline0.4390.0500.538
experiment2.8650.5286.396
filter_annotation0.0610.0060.068
filter_coverage1.4440.3141.962
find_barcode1.8390.3002.349
find_bin0.0070.0090.028
find_variants25.456 0.50525.852
get_coverage1.5180.5112.205
index_genome0.3140.2060.581
mutation_positions1.6420.0501.713
plot_coverage3.6220.4014.220
plot_demultiplex3.1490.5284.435
plot_demultiplex_raw1.7630.0932.036
plot_durations2.9270.5113.851
plot_isoform_heatmap 9.688 0.60710.505
plot_isoform_reduced_dim27.442 0.60129.832
plot_isoforms2.8430.0473.499
resume_FLAMES2.8630.8899.762
run_FLAMES2.5770.4833.929
run_step1.1680.2922.734
sc_DTU_analysis 9.664 1.75012.639
sc_gene_entropy1.3890.0681.796
sc_genotype4.0641.2114.339
sc_impute_transcript0.8490.0190.882
sc_long_multisample_pipeline17.205 3.99825.200
sc_long_pipeline5.4831.0185.687
sc_mutations3.1930.4923.451
sc_plot_genotype11.301 0.45511.752
show-FLAMESPipeline0.2720.0661.622
steps-set0.4090.0690.930
steps0.2010.1042.758
weight_transcripts0.0320.0550.105