##############################################################################
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###
### 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_2.5.5.tar.gz
###
##############################################################################
##############################################################################


* using log directory ‘/Users/biocbuild/bbs-3.23-bioc/meat/FLAMES.Rcheck’
* using R version 4.6.0 alpha (2026-04-08 r89818)
* using platform: aarch64-apple-darwin23
* R was compiled by
    Apple clang version 17.0.0 (clang-1700.3.19.1)
    GNU Fortran (GCC) 14.2.0
* running under: macOS Tahoe 26.3.1
* using session charset: UTF-8
* current time: 2026-04-24 23:47:39 UTC
* using option ‘--no-vignettes’
* checking for file ‘FLAMES/DESCRIPTION’ ... OK
* this is package ‘FLAMES’ version ‘2.5.5’
* 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 ... OK
* checking for portable file names ... OK
* checking for sufficient/correct file permissions ... OK
* checking whether package ‘FLAMES’ can be installed ... NOTE
Found the following notes/warnings:
  Non-staged installation was used
See ‘/Users/biocbuild/bbs-3.23-bioc/meat/FLAMES.Rcheck/00install.out’ for details.
* used C++ compiler: ‘Apple clang version 17.0.0 (clang-1700.6.4.2)’
* used SDK: ‘MacOSX26.2.sdk’
* checking C++ specification ... INFO
  specified C++17
* checking installed package size ... INFO
  installed size is 11.0Mb
  sub-directories of 1Mb or more:
    bin    5.9Mb
    data   1.8Mb
    libs   1.6Mb
* 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
Namespace in Imports field not imported from: ‘scuttle’
  All declared Imports should be used.
There are ::: calls to the package's namespace in its code. A package
  almost never needs to use ::: for its own objects:
  ‘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’
barcode_group: no visible global function definition for ‘new’
barcode_segment: no visible global function definition for ‘new’
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_diversity: no visible global function definition for ‘as’
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 ‘CB’
plot_demultiplex_raw: no visible binding for global variable ‘UB’
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 ‘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_isoforms: no visible binding for global variable ‘xmin’
plot_isoforms: no visible binding for global variable ‘xmax’
plot_isoforms: no visible binding for global variable ‘ymin’
plot_isoforms: no visible binding for global variable ‘ymax’
plot_isoforms: no visible binding for global variable ‘x’
plot_isoforms: no visible binding for global variable ‘xend’
plot_isoforms: no visible binding for global variable ‘y’
plot_isoforms: no visible binding for global variable ‘yend’
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_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 ‘outdir’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘demultiplexed_fastq’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘barcodes_file’
barcode_demultiplex,FLAMES.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 ‘outdir’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘demultiplexed_fastq’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘barcodes_files’
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:
  CB Count FSM_match FlankEditDist Freq Reads Sample Type UB UMI_count
  adj.p.value allele allele_count as bam_index barcode barcode_rank
  barcodes_file barcodes_files 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 xend xmax xmin y yend ymax ymin
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/4.6/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     8.997  0.129   9.231
sc_long_multisample_pipeline 7.189  1.727   6.929
find_variants                6.377  0.314   6.276
sc_plot_genotype             5.484  0.803   5.616
MultiSampleSCPipeline        4.905  0.850   6.423
* 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: 4 NOTEs
See
  ‘/Users/biocbuild/bbs-3.23-bioc/meat/FLAMES.Rcheck/00check.log’
for details.

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


* installing to library ‘/Library/Frameworks/R.framework/Versions/4.6/Resources/library’
* installing *source* package ‘FLAMES’ ...
** this is package ‘FLAMES’ version ‘2.5.5’
** using non-staged installation via StagedInstall field
** libs
specified C++17
using C++ compiler: ‘Apple clang version 17.0.0 (clang-1700.6.4.2)’
using C++17
using SDK: ‘MacOSX26.2.sdk’
clang++ -arch arm64 -std=gnu++17 -I"/Library/Frameworks/R.framework/Resources/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/Rcpp/include' -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/Rhtslib/include' -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/testthat/include' -I/opt/R/arm64/include    -fPIC  -falign-functions=64 -Wall -g -O2   -c RcppExports.cpp -o RcppExports.o
clang++ -arch arm64 -std=gnu++17 -I"/Library/Frameworks/R.framework/Resources/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/Rcpp/include' -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/Rhtslib/include' -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/testthat/include' -I/opt/R/arm64/include    -fPIC  -falign-functions=64 -Wall -g -O2   -c RcppFunctions.cpp -o RcppFunctions.o
clang++ -arch arm64 -std=gnu++17 -I"/Library/Frameworks/R.framework/Resources/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/Rcpp/include' -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/Rhtslib/include' -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/testthat/include' -I/opt/R/arm64/include    -fPIC  -falign-functions=64 -Wall -g -O2   -c classes/BamRecord.cpp -o classes/BamRecord.o
clang++ -arch arm64 -std=gnu++17 -I"/Library/Frameworks/R.framework/Resources/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/Rcpp/include' -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/Rhtslib/include' -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/testthat/include' -I/opt/R/arm64/include    -fPIC  -falign-functions=64 -Wall -g -O2   -c classes/GFFRecord.cpp -o classes/GFFRecord.o
clang++ -arch arm64 -std=gnu++17 -I"/Library/Frameworks/R.framework/Resources/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/Rcpp/include' -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/Rhtslib/include' -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/testthat/include' -I/opt/R/arm64/include    -fPIC  -falign-functions=64 -Wall -g -O2   -c classes/GeneAnnotationParser.cpp -o classes/GeneAnnotationParser.o
clang++ -arch arm64 -std=gnu++17 -I"/Library/Frameworks/R.framework/Resources/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/Rcpp/include' -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/Rhtslib/include' -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/testthat/include' -I/opt/R/arm64/include    -fPIC  -falign-functions=64 -Wall -g -O2   -c classes/Isoforms.cpp -o classes/Isoforms.o
classes/Isoforms.cpp:725:22: warning: comparisons like 'X<=Y<=Z' don't have their mathematical meaning [-Wparentheses]
  725 |                                 } else if (0 < i < raw_iso_key.size() - 1) { // a site from the middle somewhere
      |                                                  ^
1 warning generated.
clang++ -arch arm64 -std=gnu++17 -I"/Library/Frameworks/R.framework/Resources/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/Rcpp/include' -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/Rhtslib/include' -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/testthat/include' -I/opt/R/arm64/include    -fPIC  -falign-functions=64 -Wall -g -O2   -c classes/junctions.cpp -o classes/junctions.o
clang++ -arch arm64 -std=gnu++17 -I"/Library/Frameworks/R.framework/Resources/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/Rcpp/include' -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/Rhtslib/include' -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/testthat/include' -I/opt/R/arm64/include    -fPIC  -falign-functions=64 -Wall -g -O2   -c main-functions/find_isoform.cpp -o main-functions/find_isoform.o
clang++ -arch arm64 -std=gnu++17 -I"/Library/Frameworks/R.framework/Resources/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/Rcpp/include' -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/Rhtslib/include' -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/testthat/include' -I/opt/R/arm64/include    -fPIC  -falign-functions=64 -Wall -g -O2   -c main-functions/flexiplex.cpp -o main-functions/flexiplex.o
clang++ -arch arm64 -std=gnu++17 -I"/Library/Frameworks/R.framework/Resources/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/Rcpp/include' -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/Rhtslib/include' -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/testthat/include' -I/opt/R/arm64/include    -fPIC  -falign-functions=64 -Wall -g -O2   -c main-functions/get_transcript_seq.cpp -o main-functions/get_transcript_seq.o
clang++ -arch arm64 -std=gnu++17 -I"/Library/Frameworks/R.framework/Resources/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/Rcpp/include' -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/Rhtslib/include' -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/testthat/include' -I/opt/R/arm64/include    -fPIC  -falign-functions=64 -Wall -g -O2   -c main-functions/group_bam2isoform.cpp -o main-functions/group_bam2isoform.o
clang++ -arch arm64 -std=gnu++17 -I"/Library/Frameworks/R.framework/Resources/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/Rcpp/include' -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/Rhtslib/include' -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/testthat/include' -I/opt/R/arm64/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]
   86 |   unsigned int end;
      |                ^~~
1 warning generated.
clang++ -arch arm64 -std=gnu++17 -I"/Library/Frameworks/R.framework/Resources/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/Rcpp/include' -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/Rhtslib/include' -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/testthat/include' -I/opt/R/arm64/include    -fPIC  -falign-functions=64 -Wall -g -O2   -c tests/test-junctions.cpp -o tests/test-junctions.o
clang++ -arch arm64 -std=gnu++17 -I"/Library/Frameworks/R.framework/Resources/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/Rcpp/include' -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/Rhtslib/include' -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/testthat/include' -I/opt/R/arm64/include    -fPIC  -falign-functions=64 -Wall -g -O2   -c tests/test-parsing.cpp -o tests/test-parsing.o
clang++ -arch arm64 -std=gnu++17 -I"/Library/Frameworks/R.framework/Resources/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/Rcpp/include' -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/Rhtslib/include' -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/testthat/include' -I/opt/R/arm64/include    -fPIC  -falign-functions=64 -Wall -g -O2   -c utility/cigars.cpp -o utility/cigars.o
clang++ -arch arm64 -std=gnu++17 -I"/Library/Frameworks/R.framework/Resources/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/Rcpp/include' -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/Rhtslib/include' -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/testthat/include' -I/opt/R/arm64/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 arm64 -I"/Library/Frameworks/R.framework/Resources/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/Rcpp/include' -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/Rhtslib/include' -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/testthat/include' -I/opt/R/arm64/include    -fPIC  -falign-functions=64 -Wall -g -O2  -c utility/bam.c -o utility/bam.o
clang++ -arch arm64 -std=gnu++17 -dynamiclib -Wl,-headerpad_max_install_names -undefined dynamic_lookup -L/Library/Frameworks/R.framework/Resources/lib -L/opt/R/arm64/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/4.6/Resources/library/Rhtslib/usrlib/libhts.a -lcurl -lbz2 -llzma -lz -F/Library/Frameworks/R.framework/.. -framework R
ld: warning: ignoring duplicate libraries: '-lz'
if test -e "/usr/bin/strip" & test -e "/bin/uname" & [[ `uname` == "Linux" ]] ; then /usr/bin/strip --strip-debug FLAMES.so; fi
Building for ARM64
(cd submodule/minimap2 && make -f Makefile CFLAGS="-falign-functions=64 -Wall -g -O2  -Wno-unused-result" arm_neon=1 aarch64=1 minimap2)
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  -Isse2neon main.c -o main.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  -Isse2neon kthread.c -o kthread.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  -Isse2neon kalloc.c -o kalloc.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  -Isse2neon misc.c -o misc.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  -Isse2neon bseq.c -o bseq.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  -Isse2neon sketch.c -o sketch.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  -Isse2neon sdust.c -o sdust.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  -Isse2neon options.c -o options.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  -Isse2neon index.c -o index.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  -Isse2neon lchain.c -o lchain.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  -Isse2neon align.c -o align.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  -Isse2neon hit.c -o hit.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  -Isse2neon seed.c -o seed.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  -Isse2neon jump.c -o jump.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  -Isse2neon map.c -o map.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  -Isse2neon format.c -o format.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  -Isse2neon pe.c -o pe.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  -Isse2neon esterr.c -o esterr.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  -Isse2neon splitidx.c -o splitidx.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  -Isse2neon ksw2_ll_sse.c -o ksw2_ll_sse.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC -DKSW_SSE2_ONLY -D__SSE2__  -Isse2neon ksw2_extz2_sse.c -o ksw2_extz2_neon.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC -DKSW_SSE2_ONLY -D__SSE2__  -Isse2neon ksw2_extd2_sse.c -o ksw2_extd2_neon.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC -DKSW_SSE2_ONLY -D__SSE2__  -Isse2neon ksw2_exts2_sse.c -o ksw2_exts2_neon.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_neon.o ksw2_extd2_neon.o ksw2_exts2_neon.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-3.23-bioc/meat/FLAMES/src/../inst/bin"
Installing binary to /Users/biocbuild/bbs-3.23-bioc/meat/FLAMES/src/../inst/bin
mkdir -p ../inst/bin
cp submodule/minimap2/minimap2 ../inst/bin/
Building oarfish with cargo
mkdir -p ../inst/bin
mkdir cargo_temp
(cargo install oarfish --root cargo_temp --bin oarfish --vers 0.8.0)
    Updating crates.io index
  Installing oarfish v0.8.0
    Updating crates.io index
     Locking 279 packages to latest compatible versions
      Adding generic-array v0.14.7 (available: v0.14.9)
      Adding indicatif v0.17.11 (available: v0.18.4)
      Adding needletail v0.6.3 (available: v0.7.3)
      Adding noodles-bam v0.78.0 (available: v0.85.0)
      Adding noodles-bgzf v0.38.0 (available: v0.45.0)
      Adding noodles-sam v0.74.0 (available: v0.81.0)
      Adding rand v0.9.4 (available: v0.10.1)
      Adding tabled v0.18.0 (available: v0.20.0)
      Adding typed-builder v0.21.2 (available: v0.23.2)
      Adding wasip2 v1.0.3+wasi-0.2.9 (requires Rust 1.87.0)
      Adding wasip3 v0.4.0+wasi-0.3.0-rc-2026-01-06 (requires Rust 1.87.0)
      Adding wit-bindgen v0.51.0 (requires Rust 1.87.0)
      Adding wit-bindgen-core v0.51.0 (requires Rust 1.87.0)
      Adding wit-bindgen-rust v0.51.0 (requires Rust 1.87.0)
 Downloading crates ...
  Downloaded cc v1.2.61
   Compiling libc v0.2.186
   Compiling proc-macro2 v1.0.106
   Compiling unicode-ident v1.0.24
   Compiling quote v1.0.45
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   Compiling adler2 v2.0.1
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   Compiling seqcol_rs v0.4.1
   Compiling statrs v0.18.0
   Compiling oarfish v0.8.0
    Finished `release` profile [optimized] target(s) in 32.68s
  Installing cargo_temp/bin/oarfish
   Installed package `oarfish v0.8.0` (executable `oarfish`)
warning: be sure to add `cargo_temp/bin` to your PATH to be able to run the installed binaries
cp cargo_temp/bin/oarfish ../inst/bin/
cargo uninstall oarfish --root cargo_temp
    Removing cargo_temp/bin/oarfish
rm -rf cargo_temp
installing to /Library/Frameworks/R.framework/Versions/4.6/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)

R version 4.6.0 alpha (2026-04-08 r89818)
Copyright (C) 2026 The R Foundation for Statistical Computing
Platform: aarch64-apple-darwin23

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")
i Writing configuration to: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7e6640c6/config_file_84812.json
i Writing configuration to: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7e6640c6/config_file_84812.json
i Writing configuration to: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7e6640c6/config_file_84812.json
i Writing configuration to: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7b1daab/config_file_84812.json
Converting legacy `pattern` argument to `segments`...
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c3414cefb/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /Library/Frameworks/R.framework/Versions/4.6/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 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...
Converting legacy `pattern` argument to `segments`...
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c3c64f156/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /Library/Frameworks/R.framework/Versions/4.6/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 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...
Converting legacy `pattern` argument to `segments`...
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c3c64f156/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c5f8a60b3/musc_rps24_1.fastq
Searching for barcodes...
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c5f8a60b3/musc_rps24_2.fastq
Searching for barcodes...
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c5f8a60b3/musc_rps24_3.fastq
Searching for barcodes...
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c5f8a60b3/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 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...
Converting legacy `pattern` argument to `segments`...
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c53b412ec/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /Library/Frameworks/R.framework/Versions/4.6/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 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...
2 transcripts found in the BAM file.
1(50%) transcripts failed the filter.
Failed transcripts account for 100 reads, out of 200(50%) reads in total.
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
i Writing configuration to: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c3a16a0d6/config_file_84812.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 Apr 24 19:51:35 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c3a16a0d6/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c3a16a0d6/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c3a16a0d6/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Fri Apr 24 19:51:36 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 6 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Fri Apr 24 19:51:44 2026 -------------------
Realigning sample sample1 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c3a16a0d6/sample1_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample2 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c3a16a0d6/sample2_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample3 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c3a16a0d6/sample3_realign2transcript.bam
Skipped sorting BAM files.

-- Running step: transcript_quantification @ Fri Apr 24 19:51:45 2026 ----------
2026-04-24T23:51:45.529708Z  INFO oarfish: setting user-provided filter parameters.
2026-04-24T23:51:45.530037Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c3a16a0d6/sample1_realign2transcript.bam, contains 5 reference sequences.
2026-04-24T23:51:45.530060Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-24T23:51:45.530073Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-24T23:51:45.530122Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-24T23:51:45.530133Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-04-24T23:51:45.533680Z  INFO oarfish::alignment_parser: the alignment file contained 1 unmapped read records.
2026-04-24T23:51:45.533808Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 686   │
│ aligned fraction too low        │ 16    │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 4     │
│ reads with valid best alignment │ 283   │
╰─────────────────────────────────┴───────╯

2026-04-24T23:51:45.533833Z  INFO oarfish::bulk: Total number of alignment records : 364
2026-04-24T23:51:45.533839Z  INFO oarfish::bulk: number of aligned reads : 283
2026-04-24T23:51:45.533841Z  INFO oarfish::bulk: number of unique alignments : 235
2026-04-24T23:51:45.535524Z  INFO oarfish: oarfish completed successfully.
2026-04-24T23:51:45.553372Z  INFO oarfish: setting user-provided filter parameters.
2026-04-24T23:51:45.553648Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c3a16a0d6/sample2_realign2transcript.bam, contains 5 reference sequences.
2026-04-24T23:51:45.553664Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-24T23:51:45.553681Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-24T23:51:45.553725Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-24T23:51:45.553732Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-04-24T23:51:45.557061Z  INFO oarfish::alignment_parser: the alignment file contained 1 unmapped read records.
2026-04-24T23:51:45.557137Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 695   │
│ aligned fraction too low        │ 14    │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 7     │
│ reads with valid best alignment │ 285   │
╰─────────────────────────────────┴───────╯

2026-04-24T23:51:45.557183Z  INFO oarfish::bulk: Total number of alignment records : 369
2026-04-24T23:51:45.557190Z  INFO oarfish::bulk: number of aligned reads : 285
2026-04-24T23:51:45.557194Z  INFO oarfish::bulk: number of unique alignments : 235
2026-04-24T23:51:45.558625Z  INFO oarfish: oarfish completed successfully.
2026-04-24T23:51:45.576886Z  INFO oarfish: setting user-provided filter parameters.
2026-04-24T23:51:45.577134Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c3a16a0d6/sample3_realign2transcript.bam, contains 5 reference sequences.
2026-04-24T23:51:45.577170Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-24T23:51:45.577185Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-24T23:51:45.577227Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-24T23:51:45.577235Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-04-24T23:51:45.580665Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2026-04-24T23:51:45.580789Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 696   │
│ aligned fraction too low        │ 14    │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 5     │
│ reads with valid best alignment │ 284   │
╰─────────────────────────────────┴───────╯

2026-04-24T23:51:45.580815Z  INFO oarfish::bulk: Total number of alignment records : 362
2026-04-24T23:51:45.580821Z  INFO oarfish::bulk: number of aligned reads : 284
2026-04-24T23:51:45.580824Z  INFO oarfish::bulk: number of unique alignments : 237
2026-04-24T23:51:45.582239Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
i Writing configuration to: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c32099461/config_file_84812.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 Apr 24 19:51:45 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c32099461/sample1_align2genome.bam
sample2 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c32099461/sample2_align2genome.bam
sample3 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c32099461/sample3_align2genome.bam
-- Running step: isoform_identification @ Fri Apr 24 19:51:55 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

  |                                                                            
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Detected 6 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Fri Apr 24 19:52:02 2026 -------------------
Realignment complete for the following samples:
sample1 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c32099461/sample1_realign2transcript.bam
sample2 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c32099461/sample2_realign2transcript.bam
sample3 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c32099461/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Fri Apr 24 19:52:12 2026 ----------
2026-04-24T23:52:12.318481Z  INFO oarfish: setting user-provided filter parameters.
2026-04-24T23:52:12.318764Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c32099461/sample1_realign2transcript.bam, contains 5 reference sequences.
2026-04-24T23:52:12.318826Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-24T23:52:12.318874Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-24T23:52:12.318989Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-24T23:52:12.319007Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-04-24T23:52:12.324145Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2026-04-24T23:52:12.324249Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 702   │
│ aligned fraction too low        │ 13    │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 5     │
│ reads with valid best alignment │ 285   │
╰─────────────────────────────────┴───────╯

2026-04-24T23:52:12.324294Z  INFO oarfish::bulk: Total number of alignment records : 364
2026-04-24T23:52:12.324301Z  INFO oarfish::bulk: number of aligned reads : 285
2026-04-24T23:52:12.324304Z  INFO oarfish::bulk: number of unique alignments : 238
2026-04-24T23:52:12.326394Z  INFO oarfish: oarfish completed successfully.
2026-04-24T23:52:12.347808Z  INFO oarfish: setting user-provided filter parameters.
2026-04-24T23:52:12.348115Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c32099461/sample2_realign2transcript.bam, contains 5 reference sequences.
2026-04-24T23:52:12.348185Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-24T23:52:12.348243Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-24T23:52:12.348351Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-24T23:52:12.348375Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-04-24T23:52:12.352969Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2026-04-24T23:52:12.353139Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 687   │
│ aligned fraction too low        │ 16    │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 6     │
│ reads with valid best alignment │ 282   │
╰─────────────────────────────────┴───────╯

2026-04-24T23:52:12.353184Z  INFO oarfish::bulk: Total number of alignment records : 355
2026-04-24T23:52:12.353195Z  INFO oarfish::bulk: number of aligned reads : 282
2026-04-24T23:52:12.353202Z  INFO oarfish::bulk: number of unique alignments : 240
2026-04-24T23:52:12.355045Z  INFO oarfish: oarfish completed successfully.
2026-04-24T23:52:12.382996Z  INFO oarfish: setting user-provided filter parameters.
2026-04-24T23:52:12.383437Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c32099461/sample3_realign2transcript.bam, contains 5 reference sequences.
2026-04-24T23:52:12.383468Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-24T23:52:12.383492Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-24T23:52:12.383560Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-24T23:52:12.383574Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-04-24T23:52:12.387476Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2026-04-24T23:52:12.387650Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 688   │
│ aligned fraction too low        │ 15    │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 7     │
│ reads with valid best alignment │ 283   │
╰─────────────────────────────────┴───────╯

2026-04-24T23:52:12.387692Z  INFO oarfish::bulk: Total number of alignment records : 354
2026-04-24T23:52:12.387702Z  INFO oarfish::bulk: number of aligned reads : 283
2026-04-24T23:52:12.387709Z  INFO oarfish::bulk: number of unique alignments : 240
2026-04-24T23:52:12.390786Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
i Writing configuration to: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c447d4822/config_file_84812.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 Apr 24 19:52:12 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c447d4822/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c447d4822/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c447d4822/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Fri Apr 24 19:52:13 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 6 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Fri Apr 24 19:52:20 2026 -------------------
Realigning sample sample1 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c447d4822/sample1_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample2 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c447d4822/sample2_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample3 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c447d4822/sample3_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Fri Apr 24 19:52:20 2026 ----------
19:52:20 Fri Apr 24 2026 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
i Writing configuration to: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c629ff11b/config_file_84812.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 Apr 24 19:52:22 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c629ff11b/sample1_align2genome.bam
sample2 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c629ff11b/sample2_align2genome.bam
sample3 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c629ff11b/sample3_align2genome.bam
-- Running step: isoform_identification @ Fri Apr 24 19:52:31 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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Detected 5 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 Apr 24 19:52:37 2026 -------------------
Realignment complete for the following samples:
sample1 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c629ff11b/sample1_realign2transcript.bam
sample2 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c629ff11b/sample2_realign2transcript.bam
sample3 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c629ff11b/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Fri Apr 24 19:52:46 2026 ----------
19:52:46 Fri Apr 24 2026 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Inputs:  ['/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c447d4822/sample3_realign2transcript.bam', '/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c447d4822/sample2_realign2transcript.bam', '/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c447d4822/sample1_realign2transcript.bam'] /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c447d4822/transcript_assembly.fa.fai 5 0.4 0.4
	Counter({'counted_reads': 866, 'not_enough_coverage': 30, 'unmapped': 4})
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
i Writing configuration to: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c452eeaee/config_file_84812.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 Apr 24 19:52:46 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c452eeaee/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c452eeaee/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c452eeaee/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Fri Apr 24 19:52:47 2026 -------------
Inputs:  ['/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c629ff11b/sample3_realign2transcript.bam', '/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c629ff11b/sample2_realign2transcript.bam', '/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c629ff11b/sample1_realign2transcript.bam'] /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c629ff11b/transcript_assembly.fa.fai 5 0.4 0.4
	Counter({'counted_reads': 863, 'not_enough_coverage': 32, 'unmapped': 5})
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri Apr 24 19:52:47 2026 -------------------
Realigning sample sample1 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c452eeaee/sample1_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample2 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c452eeaee/sample2_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample3 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c452eeaee/sample3_realign2transcript.bam
Skipped sorting BAM files.

-- Running step: transcript_quantification @ Fri Apr 24 19:52:49 2026 ----------
2026-04-24T23:52:49.295279Z  INFO oarfish: setting user-provided filter parameters.
2026-04-24T23:52:49.295624Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c452eeaee/sample1_realign2transcript.bam, contains 17 reference sequences.
2026-04-24T23:52:49.295653Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-24T23:52:49.295670Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-24T23:52:49.295745Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-24T23:52:49.295766Z  INFO oarfish: parsed reference information for 17 transcripts.
2026-04-24T23:52:49.303161Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-04-24T23:52:49.303308Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 3436  │
│ aligned fraction too low        │ 7     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 293   │
╰─────────────────────────────────┴───────╯

2026-04-24T23:52:49.303352Z  INFO oarfish::bulk: Total number of alignment records : 513
2026-04-24T23:52:49.303363Z  INFO oarfish::bulk: number of aligned reads : 293
2026-04-24T23:52:49.303368Z  INFO oarfish::bulk: number of unique alignments : 198
2026-04-24T23:52:49.305026Z  INFO oarfish: oarfish completed successfully.
2026-04-24T23:52:49.323719Z  INFO oarfish: setting user-provided filter parameters.
2026-04-24T23:52:49.324071Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c452eeaee/sample2_realign2transcript.bam, contains 17 reference sequences.
2026-04-24T23:52:49.324125Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-24T23:52:49.324172Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-24T23:52:49.324287Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-24T23:52:49.324303Z  INFO oarfish: parsed reference information for 17 transcripts.
2026-04-24T23:52:49.331683Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-04-24T23:52:49.331774Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 3402  │
│ aligned fraction too low        │ 8     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 292   │
╰─────────────────────────────────┴───────╯

2026-04-24T23:52:49.331800Z  INFO oarfish::bulk: Total number of alignment records : 525
2026-04-24T23:52:49.331810Z  INFO oarfish::bulk: number of aligned reads : 292
2026-04-24T23:52:49.331815Z  INFO oarfish::bulk: number of unique alignments : 190
2026-04-24T23:52:49.333327Z  INFO oarfish: oarfish completed successfully.
2026-04-24T23:52:49.351292Z  INFO oarfish: setting user-provided filter parameters.
2026-04-24T23:52:49.351611Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c452eeaee/sample3_realign2transcript.bam, contains 17 reference sequences.
2026-04-24T23:52:49.351629Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-24T23:52:49.351643Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-24T23:52:49.351714Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-24T23:52:49.351724Z  INFO oarfish: parsed reference information for 17 transcripts.
2026-04-24T23:52:49.358477Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-04-24T23:52:49.358564Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 3342  │
│ aligned fraction too low        │ 10    │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 290   │
╰─────────────────────────────────┴───────╯

2026-04-24T23:52:49.358588Z  INFO oarfish::bulk: Total number of alignment records : 517
2026-04-24T23:52:49.358594Z  INFO oarfish::bulk: number of aligned reads : 290
2026-04-24T23:52:49.358597Z  INFO oarfish::bulk: number of unique alignments : 192
2026-04-24T23:52:49.360216Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
i Writing configuration to: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4b5f1d1b/config_file_84812.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 Apr 24 19:52:49 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4b5f1d1b/sample1_align2genome.bam
sample2 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4b5f1d1b/sample2_align2genome.bam
sample3 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4b5f1d1b/sample3_align2genome.bam
-- Running step: isoform_identification @ Fri Apr 24 19:52:58 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri Apr 24 19:52:58 2026 -------------------
Realignment complete for the following samples:
sample1 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4b5f1d1b/sample1_realign2transcript.bam
sample2 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4b5f1d1b/sample2_realign2transcript.bam
sample3 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4b5f1d1b/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Fri Apr 24 19:53:07 2026 ----------
2026-04-24T23:53:07.736751Z  INFO oarfish: setting user-provided filter parameters.
2026-04-24T23:53:07.737050Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4b5f1d1b/sample1_realign2transcript.bam, contains 15 reference sequences.
2026-04-24T23:53:07.737070Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-24T23:53:07.737082Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-24T23:53:07.737145Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-24T23:53:07.737157Z  INFO oarfish: parsed reference information for 15 transcripts.
2026-04-24T23:53:07.743459Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-04-24T23:53:07.743543Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 3045  │
│ aligned fraction too low        │ 7     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 293   │
╰─────────────────────────────────┴───────╯

2026-04-24T23:53:07.743564Z  INFO oarfish::bulk: Total number of alignment records : 495
2026-04-24T23:53:07.743569Z  INFO oarfish::bulk: number of aligned reads : 293
2026-04-24T23:53:07.743573Z  INFO oarfish::bulk: number of unique alignments : 208
2026-04-24T23:53:07.745321Z  INFO oarfish: oarfish completed successfully.
2026-04-24T23:53:07.762574Z  INFO oarfish: setting user-provided filter parameters.
2026-04-24T23:53:07.762806Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4b5f1d1b/sample2_realign2transcript.bam, contains 15 reference sequences.
2026-04-24T23:53:07.762832Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-24T23:53:07.762854Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-24T23:53:07.762923Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-24T23:53:07.762936Z  INFO oarfish: parsed reference information for 15 transcripts.
2026-04-24T23:53:07.769328Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-04-24T23:53:07.769417Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 2896  │
│ aligned fraction too low        │ 10    │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 290   │
╰─────────────────────────────────┴───────╯

2026-04-24T23:53:07.769471Z  INFO oarfish::bulk: Total number of alignment records : 511
2026-04-24T23:53:07.769479Z  INFO oarfish::bulk: number of aligned reads : 290
2026-04-24T23:53:07.769483Z  INFO oarfish::bulk: number of unique alignments : 198
2026-04-24T23:53:07.771416Z  INFO oarfish: oarfish completed successfully.
2026-04-24T23:53:07.792194Z  INFO oarfish: setting user-provided filter parameters.
2026-04-24T23:53:07.792448Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4b5f1d1b/sample3_realign2transcript.bam, contains 15 reference sequences.
2026-04-24T23:53:07.792523Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-24T23:53:07.792575Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-24T23:53:07.792704Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-24T23:53:07.792717Z  INFO oarfish: parsed reference information for 15 transcripts.
2026-04-24T23:53:07.799795Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-04-24T23:53:07.799883Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 2880  │
│ aligned fraction too low        │ 9     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 291   │
╰─────────────────────────────────┴───────╯

2026-04-24T23:53:07.799904Z  INFO oarfish::bulk: Total number of alignment records : 496
2026-04-24T23:53:07.799910Z  INFO oarfish::bulk: number of aligned reads : 291
2026-04-24T23:53:07.799913Z  INFO oarfish::bulk: number of unique alignments : 206
2026-04-24T23:53:07.801356Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
i Writing configuration to: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c20cf2e94/config_file_84812.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 Apr 24 19:53:08 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c20cf2e94/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c20cf2e94/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c20cf2e94/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Fri Apr 24 19:53:08 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri Apr 24 19:53:09 2026 -------------------
Realigning sample sample1 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c20cf2e94/sample1_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample2 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c20cf2e94/sample2_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample3 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c20cf2e94/sample3_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Fri Apr 24 19:53:09 2026 ----------
19:53:09 Fri Apr 24 2026 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)
i Writing configuration to: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c6f3c7774/config_file_84812.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 Apr 24 19:53:10 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c6f3c7774/sample1_align2genome.bam
sample2 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c6f3c7774/sample2_align2genome.bam
sample3 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c6f3c7774/sample3_align2genome.bam
-- Running step: isoform_identification @ Fri Apr 24 19:53:19 2026 -------------
Inputs:  ['/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c20cf2e94/sample3_realign2transcript.bam', '/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c20cf2e94/sample2_realign2transcript.bam', '/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c20cf2e94/sample1_realign2transcript.bam'] /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c20cf2e94/transcript_assembly.fa.fai 5 0.4 0.4
	Counter({'counted_reads': 895, 'not_enough_coverage': 5})
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri Apr 24 19:53:19 2026 -------------------
Realignment complete for the following samples:
sample1 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c6f3c7774/sample1_realign2transcript.bam
sample2 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c6f3c7774/sample2_realign2transcript.bam
sample3 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c6f3c7774/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Fri Apr 24 19:53:27 2026 ----------
19:53:27 Fri Apr 24 2026 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
i Writing configuration to: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c41567e28/config_file_84812.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 Apr 24 19:53:28 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c41567e28/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 8
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /Library/Frameworks/R.framework/Versions/4.6/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 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 Apr 24 19:53:28 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c41567e28/matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c41567e28/align2genome.bam
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: gene_quantification @ Fri Apr 24 19:53:28 2026 ----------------
19:53:28 Fri Apr 24 2026 quantify genes 
Using BAM(s):
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c41567e28/align2genome.bam'
Inputs:  ['/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c6f3c7774/sample3_realign2transcript.bam', '/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c6f3c7774/sample2_realign2transcript.bam', '/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c6f3c7774/sample1_realign2transcript.bam'] /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c6f3c7774/transcript_assembly.fa.fai 5 0.4 0.4
	Counter({'counted_reads': 895, 'not_enough_coverage': 5})
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  1.43gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  1.43gene_group/s]
2026-04-24 19:53:30.216 R[84812:319504159] +[NSXPCSharedListener endpointForReply:withListenerName:replyErrorCode:]: an error occurred while attempting to obtain endpoint for listener 'ClientCallsAuxiliary': Connection interrupted
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1084696.39Read/s]
-- Running step: isoform_identification @ Fri Apr 24 19:53:30 2026 -------------
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 Apr 24 19:53:33 2026 -------------------
Checking for fastq file(s) /Library/Frameworks/R.framework/Versions/4.6/Resources/library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c41567e28/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c41567e28/matched_reads_dedup.fastq.gz
	files found
Realigning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c41567e28/matched_reads_dedup.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c41567e28/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 Apr 24 19:53:33 2026 ----------
2026-04-24T23:53:33.447799Z  INFO oarfish: setting user-provided filter parameters.
2026-04-24T23:53:33.448018Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c41567e28/realign2transcript.bam, contains 5 reference sequences.
2026-04-24T23:53:33.448048Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-24T23:53:33.448066Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-24T23:53:33.448118Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-24T23:53:33.448128Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-04-24T23:53:33.452734Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
i Writing configuration to: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c52132081/config_file_84812.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 Apr 24 19:53:33 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c52132081/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 8
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /Library/Frameworks/R.framework/Versions/4.6/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 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 Apr 24 19:53:33 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c52132081/matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c52132081/align2genome.bam
-- Running step: gene_quantification @ Fri Apr 24 19:53:41 2026 ----------------
19:53:41 Fri Apr 24 2026 quantify genes 
Using BAM(s):
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c52132081/align2genome.bam'
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  1.21gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  1.20gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 917469.59Read/s]
-- Running step: isoform_identification @ Fri Apr 24 19:53:42 2026 -------------
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 Apr 24 19:53:45 2026 -------------------
Checking for fastq file(s) /Library/Frameworks/R.framework/Versions/4.6/Resources/library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c52132081/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c52132081/matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c52132081/matched_reads_dedup.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c52132081/realign2transcript.bam
-- Running step: transcript_quantification @ Fri Apr 24 19:53:53 2026 ----------
2026-04-24T23:53:53.867111Z  INFO oarfish: setting user-provided filter parameters.
2026-04-24T23:53:53.867350Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c52132081/realign2transcript.bam, contains 5 reference sequences.
2026-04-24T23:53:53.867367Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-24T23:53:53.867379Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-24T23:53:53.867432Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-24T23:53:53.867439Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-04-24T23:53:53.871764Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
i Writing configuration to: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4e23a8f1/config_file_84812.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 Apr 24 19:53:54 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4e23a8f1/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 8
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /Library/Frameworks/R.framework/Versions/4.6/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 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 Apr 24 19:53:54 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4e23a8f1/matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4e23a8f1/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: gene_quantification @ Fri Apr 24 19:53:54 2026 ----------------
19:53:54 Fri Apr 24 2026 quantify genes 
Using BAM(s):
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4e23a8f1/align2genome.bam'
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  1.39gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  1.39gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 895759.44Read/s]
-- Running step: isoform_identification @ Fri Apr 24 19:53:55 2026 -------------
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 Apr 24 19:53:58 2026 -------------------
Checking for fastq file(s) /Library/Frameworks/R.framework/Versions/4.6/Resources/library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4e23a8f1/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4e23a8f1/matched_reads_dedup.fastq.gz
	files found
Realigning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4e23a8f1/matched_reads_dedup.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4e23a8f1/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 Apr 24 19:53:58 2026 ----------
19:53:58 Fri Apr 24 2026 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
i Writing configuration to: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c64d0aac1/config_file_84812.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 Apr 24 19:53:58 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c64d0aac1/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 8
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /Library/Frameworks/R.framework/Versions/4.6/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 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 Apr 24 19:53:59 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c64d0aac1/matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c64d0aac1/align2genome.bam
-- Running step: gene_quantification @ Fri Apr 24 19:54:07 2026 ----------------
19:54:07 Fri Apr 24 2026 quantify genes 
Using BAM(s):
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c64d0aac1/align2genome.bam'
	Counter({'counted_reads': 347, 'not_enough_coverage': 9, 'unmapped': 2})
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  1.45gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  1.45gene_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, 949195.26Read/s]
-- Running step: isoform_identification @ Fri Apr 24 19:54:08 2026 -------------
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 Apr 24 19:54:11 2026 -------------------
Checking for fastq file(s) /Library/Frameworks/R.framework/Versions/4.6/Resources/library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c64d0aac1/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c64d0aac1/matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c64d0aac1/matched_reads_dedup.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c64d0aac1/realign2transcript.bam
-- Running step: transcript_quantification @ Fri Apr 24 19:54:19 2026 ----------
19:54:19 Fri Apr 24 2026 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
i Writing configuration to: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c43f905e0/config_file_84812.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 Apr 24 19:54:19 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c43f905e0/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 8
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /Library/Frameworks/R.framework/Versions/4.6/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 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 Apr 24 19:54:19 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c43f905e0/matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c43f905e0/align2genome.bam
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: gene_quantification @ Fri Apr 24 19:54:19 2026 ----------------
19:54:19 Fri Apr 24 2026 quantify genes 
Using BAM(s):
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c43f905e0/align2genome.bam'
	Counter({'counted_reads': 347, 'not_enough_coverage': 9, 'unmapped': 2})
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  1.36gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  1.35gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 556450.86Read/s]
-- Running step: isoform_identification @ Fri Apr 24 19:54:20 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri Apr 24 19:54:20 2026 -------------------
Checking for fastq file(s) /Library/Frameworks/R.framework/Versions/4.6/Resources/library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c43f905e0/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c43f905e0/matched_reads_dedup.fastq.gz
	files found
Realigning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c43f905e0/matched_reads_dedup.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c43f905e0/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 Apr 24 19:54:21 2026 ----------
2026-04-24T23:54:21.175796Z  INFO oarfish: setting user-provided filter parameters.
2026-04-24T23:54:21.176163Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c43f905e0/realign2transcript.bam, contains 10 reference sequences.
2026-04-24T23:54:21.176231Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-24T23:54:21.176269Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-24T23:54:21.176405Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-24T23:54:21.176428Z  INFO oarfish: parsed reference information for 10 transcripts.
2026-04-24T23:54:21.182262Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
i Writing configuration to: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7504f014/config_file_84812.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 Apr 24 19:54:21 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7504f014/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 8
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /Library/Frameworks/R.framework/Versions/4.6/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 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 Apr 24 19:54:21 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7504f014/matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7504f014/align2genome.bam
-- Running step: gene_quantification @ Fri Apr 24 19:54:29 2026 ----------------
19:54:29 Fri Apr 24 2026 quantify genes 
Using BAM(s):
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7504f014/align2genome.bam'
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  1.35gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  1.35gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1024200.04Read/s]
-- Running step: isoform_identification @ Fri Apr 24 19:54:30 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri Apr 24 19:54:30 2026 -------------------
Checking for fastq file(s) /Library/Frameworks/R.framework/Versions/4.6/Resources/library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7504f014/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7504f014/matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7504f014/matched_reads_dedup.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7504f014/realign2transcript.bam
-- Running step: transcript_quantification @ Fri Apr 24 19:54:38 2026 ----------
2026-04-24T23:54:38.790995Z  INFO oarfish: setting user-provided filter parameters.
2026-04-24T23:54:38.791351Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7504f014/realign2transcript.bam, contains 10 reference sequences.
2026-04-24T23:54:38.791371Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-24T23:54:38.791385Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-24T23:54:38.791444Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-24T23:54:38.791454Z  INFO oarfish: parsed reference information for 10 transcripts.
2026-04-24T23:54:38.798940Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
i Writing configuration to: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c851f9fc/config_file_84812.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 Apr 24 19:54:39 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c851f9fc/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 8
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /Library/Frameworks/R.framework/Versions/4.6/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 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 Apr 24 19:54:39 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c851f9fc/matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c851f9fc/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: gene_quantification @ Fri Apr 24 19:54:39 2026 ----------------
19:54:39 Fri Apr 24 2026 quantify genes 
Using BAM(s):
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c851f9fc/align2genome.bam'
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  1.39gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  1.39gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1054375.06Read/s]
-- Running step: isoform_identification @ Fri Apr 24 19:54:40 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri Apr 24 19:54:40 2026 -------------------
Checking for fastq file(s) /Library/Frameworks/R.framework/Versions/4.6/Resources/library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c851f9fc/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c851f9fc/matched_reads_dedup.fastq.gz
	files found
Realigning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c851f9fc/matched_reads_dedup.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c851f9fc/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 Apr 24 19:54:40 2026 ----------
19:54:40 Fri Apr 24 2026 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
i Writing configuration to: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c2efc0c34/config_file_84812.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 Apr 24 19:54:41 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c2efc0c34/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 8
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /Library/Frameworks/R.framework/Versions/4.6/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 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 Apr 24 19:54:41 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c2efc0c34/matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c2efc0c34/align2genome.bam
-- Running step: gene_quantification @ Fri Apr 24 19:54:49 2026 ----------------
19:54:49 Fri Apr 24 2026 quantify genes 
Using BAM(s):
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c2efc0c34/align2genome.bam'
	Counter({'counted_reads': 358})
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  1.50gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  1.50gene_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, 775229.93Read/s]
-- Running step: isoform_identification @ Fri Apr 24 19:54:49 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri Apr 24 19:54:50 2026 -------------------
Checking for fastq file(s) /Library/Frameworks/R.framework/Versions/4.6/Resources/library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c2efc0c34/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c2efc0c34/matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c2efc0c34/matched_reads_dedup.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c2efc0c34/realign2transcript.bam
-- Running step: transcript_quantification @ Fri Apr 24 19:54:58 2026 ----------
19:54:58 Fri Apr 24 2026 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
i Writing configuration to: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4ce86a25e/config_file_84812.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 Apr 24 19:54:58 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4ce86a25e/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4ce86a25e/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4ce86a25e/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4ce86a25e/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 993
Number of reads where at least one barcode was found: 927
Number of chimera reads: 3
All done!
Reads	Barcodes
26	1
23	3
22	1
21	3
20	1
19	1
18	1
16	2
15	1
14	2
13	3
12	5
11	2
10	6
9	9
8	3
7	6
6	11
5	8
4	8
3	39
2	19
1	2
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4ce86a25e/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4ce86a25e/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 300
Number of reads where at least one barcode was found: 280
Number of chimera reads: 1
All done!
Reads	Barcodes
9	1
8	1
7	5
6	2
5	7
4	5
3	19
2	27
1	53
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4ce86a25e/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4ce86a25e/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 300
Number of reads where at least one barcode was found: 279
Number of chimera reads: 1
All done!
Reads	Barcodes
9	1
7	5
6	4
5	1
4	15
3	12
2	23
1	65
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4ce86a25e/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4ce86a25e/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 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 Apr 24 19:54:59 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4ce86a25e/sampleA_matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4ce86a25e/sampleA_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4ce86a25e/sample1_matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4ce86a25e/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4ce86a25e/sample2_matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4ce86a25e/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4ce86a25e/sample3_matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4ce86a25e/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Fri Apr 24 19:55:01 2026 ----------------
19:55:01 Fri Apr 24 2026 quantify genes 
Using BAM(s):
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4ce86a25e/sampleA_align2genome.bam',
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4ce86a25e/sample1_align2genome.bam',
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4ce86a25e/sample2_align2genome.bam',
and
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4ce86a25e/sample3_align2genome.bam'
	Counter({'counted_reads': 358})
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4ce86a25e/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 40.03gene_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, 330073.03Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4ce86a25e/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 74.12gene_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, 1062817.76Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4ce86a25e/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 88.55gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1377530.22Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4ce86a25e/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 39.82gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1135191.08Read/s]
-- Running step: isoform_identification @ Fri Apr 24 19:55:01 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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  |                                                                            
  |==================                                                    |  25%
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Detected 7 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)
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Fri Apr 24 19:55:09 2026 -------------------
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4ce86a25e/fastq, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4ce86a25e/fastq/sample1.fq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4ce86a25e/fastq/sample2.fq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4ce86a25e/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4ce86a25e/sampleA_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4ce86a25e/sample1_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4ce86a25e/sample2_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4ce86a25e/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4ce86a25e/sampleA_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4ce86a25e/sample1_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4ce86a25e/sample2_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4ce86a25e/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4ce86a25e/sampleA_matched_reads_dedup.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4ce86a25e/sampleA_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4ce86a25e/sample1_matched_reads_dedup.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4ce86a25e/sample1_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4ce86a25e/sample2_matched_reads_dedup.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4ce86a25e/sample2_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4ce86a25e/sample3_matched_reads_dedup.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4ce86a25e/sample3_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

-- Running step: transcript_quantification @ Fri Apr 24 19:55:10 2026 ----------
2026-04-24T23:55:10.779365Z  INFO oarfish: setting user-provided filter parameters.
2026-04-24T23:55:10.779721Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4ce86a25e/sampleA_realign2transcript.bam, contains 6 reference sequences.
2026-04-24T23:55:10.779762Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-24T23:55:10.779789Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-24T23:55:10.779878Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-24T23:55:10.779894Z  INFO oarfish: parsed reference information for 6 transcripts.
2026-04-24T23:55:10.789089Z  INFO oarfish::single_cell: Processed 100 cells.
2026-04-24T23:55:10.911324Z  INFO oarfish: setting user-provided filter parameters.
2026-04-24T23:55:10.911582Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4ce86a25e/sample1_realign2transcript.bam, contains 6 reference sequences.
2026-04-24T23:55:10.911593Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-24T23:55:10.911627Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-24T23:55:10.911716Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-24T23:55:10.911728Z  INFO oarfish: parsed reference information for 6 transcripts.
2026-04-24T23:55:10.916125Z  INFO oarfish::single_cell: Processed 100 cells.
2026-04-24T23:55:11.033367Z  INFO oarfish: setting user-provided filter parameters.
2026-04-24T23:55:11.033603Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4ce86a25e/sample2_realign2transcript.bam, contains 6 reference sequences.
2026-04-24T23:55:11.033621Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-24T23:55:11.033636Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-24T23:55:11.033699Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-24T23:55:11.033708Z  INFO oarfish: parsed reference information for 6 transcripts.
2026-04-24T23:55:11.039109Z  INFO oarfish::single_cell: Processed 100 cells.
2026-04-24T23:55:11.146841Z  INFO oarfish: setting user-provided filter parameters.
2026-04-24T23:55:11.147095Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4ce86a25e/sample3_realign2transcript.bam, contains 6 reference sequences.
2026-04-24T23:55:11.147114Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-24T23:55:11.147128Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-24T23:55:11.147176Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-24T23:55:11.147185Z  INFO oarfish: parsed reference information for 6 transcripts.
2026-04-24T23:55:11.151526Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
i Writing configuration to: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c6b79b3d9/config_file_84812.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 Apr 24 19:55:11 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c6b79b3d9/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c6b79b3d9/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c6b79b3d9/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c6b79b3d9/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 993
Number of reads where at least one barcode was found: 930
Number of chimera reads: 3
All done!
Reads	Barcodes
28	1
25	1
23	3
22	1
21	1
19	2
18	1
17	1
16	2
15	2
14	2
13	4
12	2
11	5
10	4
9	7
8	7
7	2
6	11
5	12
4	7
3	37
2	20
1	2
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c6b79b3d9/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c6b79b3d9/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 300
Number of reads where at least one barcode was found: 284
Number of chimera reads: 1
All done!
Reads	Barcodes
10	1
9	1
8	3
7	2
6	4
5	7
4	6
3	15
2	21
1	60
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c6b79b3d9/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c6b79b3d9/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 300
Number of reads where at least one barcode was found: 278
Number of chimera reads: 1
All done!
Reads	Barcodes
8	2
7	3
6	1
5	6
4	9
3	19
2	29
1	56
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c6b79b3d9/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c6b79b3d9/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 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 Apr 24 19:55:12 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c6b79b3d9/sampleA_matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c6b79b3d9/sampleA_align2genome.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c6b79b3d9/sample1_matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c6b79b3d9/sample1_align2genome.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c6b79b3d9/sample2_matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c6b79b3d9/sample2_align2genome.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c6b79b3d9/sample3_matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c6b79b3d9/sample3_align2genome.bam
-- Running step: gene_quantification @ Fri Apr 24 19:55:21 2026 ----------------
19:55:21 Fri Apr 24 2026 quantify genes 
Using BAM(s):
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c6b79b3d9/sampleA_align2genome.bam',
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c6b79b3d9/sample1_align2genome.bam',
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c6b79b3d9/sample2_align2genome.bam',
and
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c6b79b3d9/sample3_align2genome.bam'
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c6b79b3d9/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 48.34gene_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, 357302.62Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c6b79b3d9/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 85.41gene_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, 1379705.26Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c6b79b3d9/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 45.02gene_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, 1553676.10Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c6b79b3d9/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 75.43gene_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, 1032266.19Read/s]
-- Running step: isoform_identification @ Fri Apr 24 19:55:22 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

  |                                                                            
  |                                                                      |   0%
  |                                                                            
  |==================                                                    |  25%
  |                                                                            
  |===================================                                   |  50%
  |                                                                            
  |====================================================                  |  75%
  |                                                                            
  |======================================================================| 100%

Detected 8 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)
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Fri Apr 24 19:55:30 2026 -------------------
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c6b79b3d9/fastq, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c6b79b3d9/fastq/sample1.fq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c6b79b3d9/fastq/sample2.fq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c6b79b3d9/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c6b79b3d9/sampleA_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c6b79b3d9/sample1_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c6b79b3d9/sample2_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c6b79b3d9/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c6b79b3d9/sampleA_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c6b79b3d9/sample1_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c6b79b3d9/sample2_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c6b79b3d9/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c6b79b3d9/sampleA_matched_reads_dedup.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c6b79b3d9/sampleA_realign2transcript.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c6b79b3d9/sample1_matched_reads_dedup.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c6b79b3d9/sample1_realign2transcript.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c6b79b3d9/sample2_matched_reads_dedup.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c6b79b3d9/sample2_realign2transcript.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c6b79b3d9/sample3_matched_reads_dedup.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c6b79b3d9/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Fri Apr 24 19:55:39 2026 ----------
2026-04-24T23:55:39.461374Z  INFO oarfish: setting user-provided filter parameters.
2026-04-24T23:55:39.461643Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c6b79b3d9/sampleA_realign2transcript.bam, contains 6 reference sequences.
2026-04-24T23:55:39.461664Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-24T23:55:39.461679Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-24T23:55:39.461735Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-24T23:55:39.461743Z  INFO oarfish: parsed reference information for 6 transcripts.
2026-04-24T23:55:39.470754Z  INFO oarfish::single_cell: Processed 100 cells.
2026-04-24T23:55:39.618079Z  INFO oarfish: setting user-provided filter parameters.
2026-04-24T23:55:39.618288Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c6b79b3d9/sample1_realign2transcript.bam, contains 6 reference sequences.
2026-04-24T23:55:39.618303Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-24T23:55:39.618321Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-24T23:55:39.618372Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-24T23:55:39.618380Z  INFO oarfish: parsed reference information for 6 transcripts.
2026-04-24T23:55:39.622874Z  INFO oarfish::single_cell: Processed 100 cells.
2026-04-24T23:55:39.785019Z  INFO oarfish: setting user-provided filter parameters.
2026-04-24T23:55:39.785256Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c6b79b3d9/sample2_realign2transcript.bam, contains 6 reference sequences.
2026-04-24T23:55:39.785274Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-24T23:55:39.785287Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-24T23:55:39.785348Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-24T23:55:39.785365Z  INFO oarfish: parsed reference information for 6 transcripts.
2026-04-24T23:55:39.789014Z  INFO oarfish::single_cell: Processed 100 cells.
2026-04-24T23:55:39.935202Z  INFO oarfish: setting user-provided filter parameters.
2026-04-24T23:55:39.935481Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c6b79b3d9/sample3_realign2transcript.bam, contains 6 reference sequences.
2026-04-24T23:55:39.935582Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-24T23:55:39.935608Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-24T23:55:39.935665Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-24T23:55:39.935675Z  INFO oarfish: parsed reference information for 6 transcripts.
2026-04-24T23:55:39.940134Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
i Writing configuration to: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4d5f16fd/config_file_84812.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 Apr 24 19:55:40 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4d5f16fd/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4d5f16fd/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4d5f16fd/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4d5f16fd/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 993
Number of reads where at least one barcode was found: 929
Number of chimera reads: 2
All done!
Reads	Barcodes
27	1
26	1
24	1
22	2
21	1
20	3
19	1
18	1
17	2
16	2
15	2
14	1
12	1
11	7
10	5
9	5
8	9
7	5
6	13
5	11
4	5
3	33
2	22
1	3
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4d5f16fd/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4d5f16fd/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 300
Number of reads where at least one barcode was found: 281
Number of chimera reads: 1
All done!
Reads	Barcodes
9	1
8	3
7	2
6	5
5	3
4	9
3	13
2	31
1	55
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4d5f16fd/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4d5f16fd/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 300
Number of reads where at least one barcode was found: 280
Number of chimera reads: 0
All done!
Reads	Barcodes
9	1
8	3
7	1
6	5
5	5
4	6
3	18
2	30
1	50
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4d5f16fd/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4d5f16fd/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 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 Apr 24 19:55:41 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4d5f16fd/sampleA_matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4d5f16fd/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 /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4d5f16fd/sample1_matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4d5f16fd/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 /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4d5f16fd/sample2_matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4d5f16fd/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 /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4d5f16fd/sample3_matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4d5f16fd/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 Apr 24 19:55:42 2026 ----------------
19:55:42 Fri Apr 24 2026 quantify genes 
Using BAM(s):
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4d5f16fd/sampleA_align2genome.bam',
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4d5f16fd/sample1_align2genome.bam',
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4d5f16fd/sample2_align2genome.bam',
and
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4d5f16fd/sample3_align2genome.bam'
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4d5f16fd/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 46.40gene_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, 395987.92Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4d5f16fd/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 87.68gene_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, 1335595.47Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4d5f16fd/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 92.09gene_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, 1306799.60Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4d5f16fd/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 75.88gene_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, 1030035.36Read/s]
-- Running step: isoform_identification @ Fri Apr 24 19:55:43 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

  |                                                                            
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  |                                                                            
  |==================                                                    |  25%
  |                                                                            
  |===================================                                   |  50%
  |                                                                            
  |====================================================                  |  75%
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  |======================================================================| 100%

Detected 8 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)
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Fri Apr 24 19:55:50 2026 -------------------
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4d5f16fd/fastq, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4d5f16fd/fastq/sample1.fq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4d5f16fd/fastq/sample2.fq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4d5f16fd/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4d5f16fd/sampleA_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4d5f16fd/sample1_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4d5f16fd/sample2_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4d5f16fd/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4d5f16fd/sampleA_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4d5f16fd/sample1_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4d5f16fd/sample2_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4d5f16fd/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4d5f16fd/sampleA_matched_reads_dedup.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4d5f16fd/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 /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4d5f16fd/sample1_matched_reads_dedup.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4d5f16fd/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 /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4d5f16fd/sample2_matched_reads_dedup.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4d5f16fd/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 /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4d5f16fd/sample3_matched_reads_dedup.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4d5f16fd/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 Apr 24 19:55:51 2026 ----------
19:55:51 Fri Apr 24 2026 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4d5f16fd/sample3_realign2transcript.bam...
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4d5f16fd/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4d5f16fd/sample3_realign2transcript.bamdone
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4d5f16fd/sampleA_realign2transcript.bam...
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4d5f16fd/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4d5f16fd/sampleA_realign2transcript.bamdone
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4d5f16fd/sample2_realign2transcript.bam...
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4d5f16fd/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4d5f16fd/sample2_realign2transcript.bamdone
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4d5f16fd/sample1_realign2transcript.bam...
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4d5f16fd/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c4d5f16fd/sample1_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
i Writing configuration to: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4cb9e1f56/config_file_84812.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 Apr 24 19:55:52 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4cb9e1f56/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4cb9e1f56/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4cb9e1f56/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4cb9e1f56/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 993
Number of reads where at least one barcode was found: 924
Number of chimera reads: 2
All done!
Reads	Barcodes
26	1
25	1
22	1
21	3
20	2
19	2
18	1
17	4
16	1
15	1
14	1
13	1
12	1
11	7
10	5
9	3
8	11
7	3
6	5
5	19
4	7
3	35
2	19
1	3
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4cb9e1f56/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4cb9e1f56/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 300
Number of reads where at least one barcode was found: 279
Number of chimera reads: 1
All done!
Reads	Barcodes
9	1
8	1
7	3
6	5
5	8
4	7
3	11
2	23
1	66
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4cb9e1f56/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4cb9e1f56/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 300
Number of reads where at least one barcode was found: 277
Number of chimera reads: 0
All done!
Reads	Barcodes
8	1
7	3
6	8
5	5
4	4
3	19
2	22
1	60
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4cb9e1f56/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4cb9e1f56/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 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 Apr 24 19:55:53 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4cb9e1f56/sampleA_matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4cb9e1f56/sampleA_align2genome.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4cb9e1f56/sample1_matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4cb9e1f56/sample1_align2genome.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4cb9e1f56/sample2_matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4cb9e1f56/sample2_align2genome.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4cb9e1f56/sample3_matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4cb9e1f56/sample3_align2genome.bam
-- Running step: gene_quantification @ Fri Apr 24 19:56:02 2026 ----------------
19:56:02 Fri Apr 24 2026 quantify genes 
Using BAM(s):
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4cb9e1f56/sampleA_align2genome.bam',
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4cb9e1f56/sample1_align2genome.bam',
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4cb9e1f56/sample2_align2genome.bam',
and
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4cb9e1f56/sample3_align2genome.bam'
	Counter({'counted_reads': 347, 'not_enough_coverage': 9, 'unmapped': 2})
	Counter({'counted_reads': 347, 'not_enough_coverage': 9, 'unmapped': 5})
	Counter({'counted_reads': 264, 'not_enough_coverage': 8, 'unmapped': 1})
	Counter({'counted_reads': 262, 'not_enough_coverage': 8, 'unmapped': 2})
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4cb9e1f56/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 44.76gene_group/s]
/Library/Frameworks/R.framework/Versions/4.6/Resources/library/FLAMES/python/count_gene.py:704: RuntimeWarning: More than 20 figures have been opened. Figures created through the pyplot interface (`matplotlib.pyplot.figure`) are retained until explicitly closed and may consume too much memory. (To control this warning, see the rcParam `figure.max_open_warning`). Consider using `matplotlib.pyplot.close()`.
  plt.figure(figsize=(8, 6))
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 359396.76Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4cb9e1f56/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 95.47gene_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, 1341747.92Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4cb9e1f56/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 16.70gene_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, 1445115.77Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4cb9e1f56/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 77.54gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1069211.79Read/s]
-- Running step: isoform_identification @ Fri Apr 24 19:56:02 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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Detected 8 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 Apr 24 19:56:10 2026 -------------------
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4cb9e1f56/fastq, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4cb9e1f56/fastq/sample1.fq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4cb9e1f56/fastq/sample2.fq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4cb9e1f56/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4cb9e1f56/sampleA_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4cb9e1f56/sample1_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4cb9e1f56/sample2_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4cb9e1f56/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4cb9e1f56/sampleA_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4cb9e1f56/sample1_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4cb9e1f56/sample2_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4cb9e1f56/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4cb9e1f56/sampleA_matched_reads_dedup.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4cb9e1f56/sampleA_realign2transcript.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4cb9e1f56/sample1_matched_reads_dedup.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4cb9e1f56/sample1_realign2transcript.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4cb9e1f56/sample2_matched_reads_dedup.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4cb9e1f56/sample2_realign2transcript.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4cb9e1f56/sample3_matched_reads_dedup.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4cb9e1f56/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Fri Apr 24 19:56:18 2026 ----------
19:56:18 Fri Apr 24 2026 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4cb9e1f56/sample3_realign2transcript.bam...
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4cb9e1f56/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4cb9e1f56/sample3_realign2transcript.bamdone
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4cb9e1f56/sampleA_realign2transcript.bam...
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4cb9e1f56/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4cb9e1f56/sampleA_realign2transcript.bamdone
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4cb9e1f56/sample2_realign2transcript.bam...
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4cb9e1f56/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4cb9e1f56/sample2_realign2transcript.bamdone
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4cb9e1f56/sample1_realign2transcript.bam...
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4cb9e1f56/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4cb9e1f56/sample1_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
i Writing configuration to: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c9f98a14/config_file_84812.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 Apr 24 19:56:19 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c9f98a14/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c9f98a14/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c9f98a14/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c9f98a14/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 993
Number of reads where at least one barcode was found: 928
Number of chimera reads: 2
All done!
Reads	Barcodes
28	1
26	1
21	2
20	3
19	4
17	2
14	4
13	2
12	5
11	7
10	1
9	6
8	4
7	7
6	12
5	15
4	4
3	35
2	19
1	3
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c9f98a14/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c9f98a14/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 300
Number of reads where at least one barcode was found: 278
Number of chimera reads: 1
All done!
Reads	Barcodes
9	1
8	1
7	4
6	3
5	5
4	8
3	12
2	36
1	54
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c9f98a14/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c9f98a14/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 300
Number of reads where at least one barcode was found: 282
Number of chimera reads: 0
All done!
Reads	Barcodes
9	2
7	1
6	4
5	8
4	14
3	9
2	27
1	59
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c9f98a14/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c9f98a14/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 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 Apr 24 19:56:20 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c9f98a14/sampleA_matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c9f98a14/sampleA_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c9f98a14/sample1_matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c9f98a14/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c9f98a14/sample2_matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c9f98a14/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c9f98a14/sample3_matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c9f98a14/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Fri Apr 24 19:56:21 2026 ----------------
19:56:21 Fri Apr 24 2026 quantify genes 
Using BAM(s):
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c9f98a14/sampleA_align2genome.bam',
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c9f98a14/sample1_align2genome.bam',
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c9f98a14/sample2_align2genome.bam',
and
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c9f98a14/sample3_align2genome.bam'
	Counter({'counted_reads': 347, 'not_enough_coverage': 9, 'unmapped': 2})
	Counter({'counted_reads': 347, 'not_enough_coverage': 9, 'unmapped': 5})
	Counter({'counted_reads': 261, 'not_enough_coverage': 9, 'unmapped': 1})
	Counter({'counted_reads': 265, 'not_enough_coverage': 8, 'unmapped': 2})
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c9f98a14/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 47.90gene_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, 385265.09Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c9f98a14/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 86.80gene_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, 1285965.17Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c9f98a14/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 89.71gene_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, 1329667.77Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c9f98a14/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 74.16gene_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, 999310.02Read/s]
-- Running step: isoform_identification @ Fri Apr 24 19:56:22 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri Apr 24 19:56:22 2026 -------------------
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c9f98a14/fastq, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c9f98a14/fastq/sample1.fq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c9f98a14/fastq/sample2.fq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c9f98a14/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c9f98a14/sampleA_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c9f98a14/sample1_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c9f98a14/sample2_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c9f98a14/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c9f98a14/sampleA_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c9f98a14/sample1_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c9f98a14/sample2_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c9f98a14/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c9f98a14/sampleA_matched_reads_dedup.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c9f98a14/sampleA_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c9f98a14/sample1_matched_reads_dedup.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c9f98a14/sample1_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c9f98a14/sample2_matched_reads_dedup.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c9f98a14/sample2_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c9f98a14/sample3_matched_reads_dedup.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c9f98a14/sample3_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

-- Running step: transcript_quantification @ Fri Apr 24 19:56:25 2026 ----------
2026-04-24T23:56:25.693453Z  INFO oarfish: setting user-provided filter parameters.
2026-04-24T23:56:25.693792Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c9f98a14/sampleA_realign2transcript.bam, contains 27 reference sequences.
2026-04-24T23:56:25.693825Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-24T23:56:25.693840Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-24T23:56:25.693931Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-24T23:56:25.693953Z  INFO oarfish: parsed reference information for 27 transcripts.
2026-04-24T23:56:25.711985Z  INFO oarfish::single_cell: Processed 100 cells.
2026-04-24T23:56:25.903989Z  INFO oarfish: setting user-provided filter parameters.
2026-04-24T23:56:25.904311Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c9f98a14/sample1_realign2transcript.bam, contains 27 reference sequences.
2026-04-24T23:56:25.904343Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-24T23:56:25.904359Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-24T23:56:25.904457Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-24T23:56:25.904478Z  INFO oarfish: parsed reference information for 27 transcripts.
2026-04-24T23:56:25.910709Z  INFO oarfish::single_cell: Processed 100 cells.
2026-04-24T23:56:26.100074Z  INFO oarfish: setting user-provided filter parameters.
2026-04-24T23:56:26.100298Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c9f98a14/sample2_realign2transcript.bam, contains 27 reference sequences.
2026-04-24T23:56:26.100315Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-24T23:56:26.100328Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-24T23:56:26.100402Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-24T23:56:26.100414Z  INFO oarfish: parsed reference information for 27 transcripts.
2026-04-24T23:56:26.108399Z  INFO oarfish::single_cell: Processed 100 cells.
2026-04-24T23:56:26.296794Z  INFO oarfish: setting user-provided filter parameters.
2026-04-24T23:56:26.297129Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c9f98a14/sample3_realign2transcript.bam, contains 27 reference sequences.
2026-04-24T23:56:26.297151Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-24T23:56:26.297167Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-24T23:56:26.297248Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-24T23:56:26.297266Z  INFO oarfish: parsed reference information for 27 transcripts.
2026-04-24T23:56:26.305619Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
i Writing configuration to: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c57e8a851/config_file_84812.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 Apr 24 19:56:26 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c57e8a851/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c57e8a851/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c57e8a851/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c57e8a851/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 993
Number of reads where at least one barcode was found: 932
Number of chimera reads: 3
All done!
Reads	Barcodes
26	1
25	1
24	1
22	2
21	1
20	4
19	1
18	1
15	3
14	1
13	1
12	3
11	9
10	5
9	5
8	6
7	3
6	15
5	10
4	6
3	37
2	20
1	1
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c57e8a851/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c57e8a851/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 300
Number of reads where at least one barcode was found: 280
Number of chimera reads: 1
All done!
Reads	Barcodes
8	2
7	4
6	5
5	2
4	9
3	18
2	27
1	56
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c57e8a851/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c57e8a851/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 300
Number of reads where at least one barcode was found: 284
Number of chimera reads: 1
All done!
Reads	Barcodes
8	3
7	4
6	4
5	3
4	9
3	17
2	26
1	58
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c57e8a851/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c57e8a851/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 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 Apr 24 19:56:27 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c57e8a851/sampleA_matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c57e8a851/sampleA_align2genome.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c57e8a851/sample1_matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c57e8a851/sample1_align2genome.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c57e8a851/sample2_matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c57e8a851/sample2_align2genome.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c57e8a851/sample3_matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c57e8a851/sample3_align2genome.bam
-- Running step: gene_quantification @ Fri Apr 24 19:56:36 2026 ----------------
19:56:36 Fri Apr 24 2026 quantify genes 
Using BAM(s):
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c57e8a851/sampleA_align2genome.bam',
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c57e8a851/sample1_align2genome.bam',
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c57e8a851/sample2_align2genome.bam',
and
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c57e8a851/sample3_align2genome.bam'
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c57e8a851/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 46.12gene_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, 404902.50Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c57e8a851/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 89.70gene_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, 1385905.37Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c57e8a851/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 88.54gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1299350.68Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c57e8a851/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 73.08gene_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, 1027512.00Read/s]
-- Running step: isoform_identification @ Fri Apr 24 19:56:37 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri Apr 24 19:56:37 2026 -------------------
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c57e8a851/fastq, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c57e8a851/fastq/sample1.fq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c57e8a851/fastq/sample2.fq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c57e8a851/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c57e8a851/sampleA_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c57e8a851/sample1_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c57e8a851/sample2_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c57e8a851/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c57e8a851/sampleA_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c57e8a851/sample1_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c57e8a851/sample2_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c57e8a851/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c57e8a851/sampleA_matched_reads_dedup.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c57e8a851/sampleA_realign2transcript.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c57e8a851/sample1_matched_reads_dedup.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c57e8a851/sample1_realign2transcript.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c57e8a851/sample2_matched_reads_dedup.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c57e8a851/sample2_realign2transcript.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c57e8a851/sample3_matched_reads_dedup.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c57e8a851/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Fri Apr 24 19:56:49 2026 ----------
2026-04-24T23:56:49.083501Z  INFO oarfish: setting user-provided filter parameters.
2026-04-24T23:56:49.083739Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c57e8a851/sampleA_realign2transcript.bam, contains 28 reference sequences.
2026-04-24T23:56:49.083770Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-24T23:56:49.083787Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-24T23:56:49.083881Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-24T23:56:49.083895Z  INFO oarfish: parsed reference information for 28 transcripts.
2026-04-24T23:56:49.108233Z  INFO oarfish::single_cell: Processed 100 cells.
2026-04-24T23:56:49.344778Z  INFO oarfish: setting user-provided filter parameters.
2026-04-24T23:56:49.345051Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c57e8a851/sample1_realign2transcript.bam, contains 28 reference sequences.
2026-04-24T23:56:49.345073Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-24T23:56:49.345089Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-24T23:56:49.345177Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-24T23:56:49.345191Z  INFO oarfish: parsed reference information for 28 transcripts.
2026-04-24T23:56:49.352378Z  INFO oarfish::single_cell: Processed 100 cells.
2026-04-24T23:56:49.575919Z  INFO oarfish: setting user-provided filter parameters.
2026-04-24T23:56:49.576196Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c57e8a851/sample2_realign2transcript.bam, contains 28 reference sequences.
2026-04-24T23:56:49.576229Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-24T23:56:49.576266Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-24T23:56:49.576380Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-24T23:56:49.576397Z  INFO oarfish: parsed reference information for 28 transcripts.
2026-04-24T23:56:49.582987Z  INFO oarfish::single_cell: Processed 100 cells.
2026-04-24T23:56:49.799495Z  INFO oarfish: setting user-provided filter parameters.
2026-04-24T23:56:49.799708Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c57e8a851/sample3_realign2transcript.bam, contains 28 reference sequences.
2026-04-24T23:56:49.799730Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-24T23:56:49.799745Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-24T23:56:49.799831Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-24T23:56:49.799845Z  INFO oarfish: parsed reference information for 28 transcripts.
2026-04-24T23:56:49.808138Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
i Writing configuration to: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f18d5db/config_file_84812.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 Apr 24 19:56:50 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f18d5db/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f18d5db/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f18d5db/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f18d5db/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 993
Number of reads where at least one barcode was found: 928
Number of chimera reads: 2
All done!
Reads	Barcodes
26	1
23	2
22	1
21	1
20	2
19	2
18	4
16	2
15	1
14	1
13	1
12	5
11	6
10	5
9	4
8	7
7	3
6	15
5	11
4	2
3	35
2	23
1	3
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f18d5db/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f18d5db/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 300
Number of reads where at least one barcode was found: 278
Number of chimera reads: 0
All done!
Reads	Barcodes
7	4
6	3
5	9
4	11
3	13
2	23
1	59
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f18d5db/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f18d5db/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 300
Number of reads where at least one barcode was found: 282
Number of chimera reads: 1
All done!
Reads	Barcodes
9	1
8	1
7	5
6	2
5	8
4	7
3	14
2	32
1	46
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f18d5db/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f18d5db/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 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 Apr 24 19:56:50 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f18d5db/sampleA_matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f18d5db/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 /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f18d5db/sample1_matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f18d5db/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 /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f18d5db/sample2_matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f18d5db/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 /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f18d5db/sample3_matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f18d5db/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 Apr 24 19:56:52 2026 ----------------
19:56:52 Fri Apr 24 2026 quantify genes 
Using BAM(s):
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f18d5db/sampleA_align2genome.bam',
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f18d5db/sample1_align2genome.bam',
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f18d5db/sample2_align2genome.bam',
and
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f18d5db/sample3_align2genome.bam'
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f18d5db/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 48.04gene_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, 461846.37Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f18d5db/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 12.47gene_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, 1232024.44Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f18d5db/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 93.02gene_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, 1317968.83Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f18d5db/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 73.77gene_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, 1097410.78Read/s]
-- Running step: isoform_identification @ Fri Apr 24 19:56:53 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri Apr 24 19:56:53 2026 -------------------
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f18d5db/fastq, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f18d5db/fastq/sample1.fq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f18d5db/fastq/sample2.fq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f18d5db/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f18d5db/sampleA_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f18d5db/sample1_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f18d5db/sample2_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f18d5db/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f18d5db/sampleA_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f18d5db/sample1_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f18d5db/sample2_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f18d5db/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f18d5db/sampleA_matched_reads_dedup.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f18d5db/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 /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f18d5db/sample1_matched_reads_dedup.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f18d5db/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 /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f18d5db/sample2_matched_reads_dedup.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f18d5db/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 /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f18d5db/sample3_matched_reads_dedup.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f18d5db/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 Apr 24 19:56:54 2026 ----------
19:56:54 Fri Apr 24 2026 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f18d5db/sample3_realign2transcript.bam...
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f18d5db/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f18d5db/sample3_realign2transcript.bamdone
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f18d5db/sampleA_realign2transcript.bam...
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f18d5db/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f18d5db/sampleA_realign2transcript.bamdone
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f18d5db/sample2_realign2transcript.bam...
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f18d5db/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f18d5db/sample2_realign2transcript.bamdone
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f18d5db/sample1_realign2transcript.bam...
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f18d5db/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f18d5db/sample1_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
i Writing configuration to: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f92dd3f/config_file_84812.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 Apr 24 19:56:56 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f92dd3f/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f92dd3f/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f92dd3f/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f92dd3f/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 993
Number of reads where at least one barcode was found: 929
Number of chimera reads: 2
All done!
Reads	Barcodes
29	1
24	1
22	1
21	2
20	3
19	1
18	2
16	4
15	1
14	1
13	5
11	4
10	7
9	5
8	9
7	3
6	13
5	14
4	2
3	32
2	20
1	6
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f92dd3f/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f92dd3f/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 300
Number of reads where at least one barcode was found: 281
Number of chimera reads: 1
All done!
Reads	Barcodes
9	1
8	3
7	1
6	4
5	5
4	8
3	16
2	33
1	50
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f92dd3f/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f92dd3f/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 300
Number of reads where at least one barcode was found: 280
Number of chimera reads: 0
All done!
Reads	Barcodes
10	1
9	1
7	1
6	4
5	10
4	7
3	16
2	28
1	51
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f92dd3f/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f92dd3f/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 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 Apr 24 19:56:56 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f92dd3f/sampleA_matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f92dd3f/sampleA_align2genome.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f92dd3f/sample1_matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f92dd3f/sample1_align2genome.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f92dd3f/sample2_matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f92dd3f/sample2_align2genome.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f92dd3f/sample3_matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f92dd3f/sample3_align2genome.bam
-- Running step: gene_quantification @ Fri Apr 24 19:57:06 2026 ----------------
19:57:06 Fri Apr 24 2026 quantify genes 
Using BAM(s):
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f92dd3f/sampleA_align2genome.bam',
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f92dd3f/sample1_align2genome.bam',
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f92dd3f/sample2_align2genome.bam',
and
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f92dd3f/sample3_align2genome.bam'
	Counter({'counted_reads': 357, 'not_enough_coverage': 1})
	Counter({'counted_reads': 357, 'not_enough_coverage': 1})
	Counter({'counted_reads': 275, 'not_enough_coverage': 1})
	Counter({'counted_reads': 275})
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f92dd3f/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 48.61gene_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, 474748.04Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f92dd3f/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 87.68gene_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, 1381159.11Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f92dd3f/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 92.01gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1324963.36Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f92dd3f/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 79.60gene_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, 1110074.11Read/s]
-- Running step: isoform_identification @ Fri Apr 24 19:57:06 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri Apr 24 19:57:06 2026 -------------------
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f92dd3f/fastq, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f92dd3f/fastq/sample1.fq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f92dd3f/fastq/sample2.fq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f92dd3f/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f92dd3f/sampleA_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f92dd3f/sample1_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f92dd3f/sample2_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f92dd3f/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f92dd3f/sampleA_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f92dd3f/sample1_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f92dd3f/sample2_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f92dd3f/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f92dd3f/sampleA_matched_reads_dedup.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f92dd3f/sampleA_realign2transcript.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f92dd3f/sample1_matched_reads_dedup.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f92dd3f/sample1_realign2transcript.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f92dd3f/sample2_matched_reads_dedup.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f92dd3f/sample2_realign2transcript.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f92dd3f/sample3_matched_reads_dedup.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f92dd3f/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Fri Apr 24 19:57:16 2026 ----------
19:57:16 Fri Apr 24 2026 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f92dd3f/sample3_realign2transcript.bam...
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f92dd3f/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f92dd3f/sample3_realign2transcript.bamdone
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f92dd3f/sampleA_realign2transcript.bam...
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f92dd3f/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f92dd3f/sampleA_realign2transcript.bamdone
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f92dd3f/sample2_realign2transcript.bam...
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f92dd3f/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f92dd3f/sample2_realign2transcript.bamdone
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f92dd3f/sample1_realign2transcript.bam...
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f92dd3f/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp5IExr9/file14b4c7f92dd3f/sample1_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Filtering for genes with at least 2 detected isoforms ...
	6 isoform(s) left.

Aggregating counts by cluster labels ...

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

Filtering for genes with at least 2 isoforms expressing more than 1 counts ...
	3 gene(s), 6 transcript(s) left.

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

[ FAIL 0 | WARN 182 | SKIP 0 | PASS 98 ]

[ FAIL 0 | WARN 182 | SKIP 0 | PASS 98 ]
	Counter({'counted_reads': 358})
	Counter({'counted_reads': 358})
	Counter({'counted_reads': 272})
	Counter({'counted_reads': 275})
> 
> proc.time()
   user  system elapsed 
326.102  23.101 354.659 
/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 '