##############################################################################
##############################################################################
###
### 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_2.7.0.tar.gz
###
##############################################################################
##############################################################################


* using log directory ‘/Users/biocbuild/bbs-3.24-bioc/meat/FLAMES.Rcheck’
* using R version 4.6.0 Patched (2026-05-01 r89994)
* 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: 2026-05-21 23:31:57 UTC
* using option ‘--no-vignettes’
* checking for file ‘FLAMES/DESCRIPTION’ ... OK
* this is package ‘FLAMES’ version ‘2.7.0’
* 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-3.24-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 11.1Mb
  sub-directories of 1Mb or more:
    bin    6.0Mb
    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     8.599  0.083   8.710
sc_long_multisample_pipeline 6.642  1.527   6.354
find_variants                6.233  0.318   6.056
sc_plot_genotype             5.344  0.595   5.425
MultiSampleSCPipeline        4.581  0.796   6.036
* 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-3.24-bioc/meat/FLAMES.Rcheck/00check.log’
for details.

##############################################################################
##############################################################################
###
### 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 ‘2.7.0’
** 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-3.24-bioc/meat/FLAMES/src/../inst/bin"
Installing binary to /Users/biocbuild/bbs-3.24-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)
   Compiling libc v0.2.186
   Compiling proc-macro2 v1.0.106
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   Compiling minimap2 v0.1.31+minimap2.2.30
   Compiling statrs v0.18.0
   Compiling oarfish v0.8.0
    Finished `release` profile [optimized] target(s) in 29.93s
  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 Patched (2026-05-01 r89994) -- "Because it was There"
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//RtmpMunpLR/file2ca5293995e6/config_file_11429.json
i Writing configuration to: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5293995e6/config_file_11429.json
i Writing configuration to: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5293995e6/config_file_11429.json
i Writing configuration to: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5986a4df/config_file_11429.json
Converting legacy `pattern` argument to `segments`...
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca577a78604/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//RtmpMunpLR/file2ca571eb4e39/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//RtmpMunpLR/file2ca571eb4e39/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//RtmpMunpLR/file2ca510dfa14c/musc_rps24_1.fastq
Searching for barcodes...
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca510dfa14c/musc_rps24_2.fastq
Searching for barcodes...
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca510dfa14c/musc_rps24_3.fastq
Searching for barcodes...
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca510dfa14c/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//RtmpMunpLR/file2ca5710dbfce/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//RtmpMunpLR/file2ca51fe39825/config_file_11429.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 @ Thu May 21 19:35:40 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca51fe39825/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca51fe39825/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca51fe39825/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Thu May 21 19:35:41 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 @ Thu May 21 19:35:50 2026 -------------------
Realigning sample sample1 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca51fe39825/sample1_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample2 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca51fe39825/sample2_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample3 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca51fe39825/sample3_realign2transcript.bam
Skipped sorting BAM files.

-- Running step: transcript_quantification @ Thu May 21 19:35:50 2026 ----------
2026-05-21T23:35:50.847582Z  INFO oarfish: setting user-provided filter parameters.
2026-05-21T23:35:50.847817Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca51fe39825/sample1_realign2transcript.bam, contains 5 reference sequences.
2026-05-21T23:35:50.847832Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-21T23:35:50.847847Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-21T23:35:50.847891Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-21T23:35:50.847898Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-05-21T23:35:50.850081Z  INFO oarfish::alignment_parser: the alignment file contained 1 unmapped read records.
2026-05-21T23:35:50.850151Z  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   │
╰─────────────────────────────────┴───────╯

2026-05-21T23:35:50.850174Z  INFO oarfish::bulk: Total number of alignment records : 364
2026-05-21T23:35:50.850180Z  INFO oarfish::bulk: number of aligned reads : 283
2026-05-21T23:35:50.850183Z  INFO oarfish::bulk: number of unique alignments : 235
2026-05-21T23:35:50.851563Z  INFO oarfish: oarfish completed successfully.
2026-05-21T23:35:50.867754Z  INFO oarfish: setting user-provided filter parameters.
2026-05-21T23:35:50.867966Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca51fe39825/sample2_realign2transcript.bam, contains 5 reference sequences.
2026-05-21T23:35:50.867978Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-21T23:35:50.867988Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-21T23:35:50.868027Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-21T23:35:50.868034Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-05-21T23:35:50.870119Z  INFO oarfish::alignment_parser: the alignment file contained 1 unmapped read records.
2026-05-21T23:35:50.870178Z  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   │
╰─────────────────────────────────┴───────╯

2026-05-21T23:35:50.870210Z  INFO oarfish::bulk: Total number of alignment records : 369
2026-05-21T23:35:50.870215Z  INFO oarfish::bulk: number of aligned reads : 285
2026-05-21T23:35:50.870218Z  INFO oarfish::bulk: number of unique alignments : 235
2026-05-21T23:35:50.871543Z  INFO oarfish: oarfish completed successfully.
2026-05-21T23:35:50.889199Z  INFO oarfish: setting user-provided filter parameters.
2026-05-21T23:35:50.889395Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca51fe39825/sample3_realign2transcript.bam, contains 5 reference sequences.
2026-05-21T23:35:50.889424Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-21T23:35:50.889442Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-21T23:35:50.889496Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-21T23:35:50.889509Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-05-21T23:35:50.891712Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2026-05-21T23:35:50.891791Z  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   │
╰─────────────────────────────────┴───────╯

2026-05-21T23:35:50.891814Z  INFO oarfish::bulk: Total number of alignment records : 362
2026-05-21T23:35:50.891819Z  INFO oarfish::bulk: number of aligned reads : 284
2026-05-21T23:35:50.891822Z  INFO oarfish::bulk: number of unique alignments : 237
2026-05-21T23:35:50.893198Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
i Writing configuration to: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca57bd7f84/config_file_11429.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 @ Thu May 21 19:35:51 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca57bd7f84/sample1_align2genome.bam
sample2 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca57bd7f84/sample2_align2genome.bam
sample3 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca57bd7f84/sample3_align2genome.bam
-- Running step: isoform_identification @ Thu May 21 19:35:59 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 @ Thu May 21 19:36:06 2026 -------------------
Realignment complete for the following samples:
sample1 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca57bd7f84/sample1_realign2transcript.bam
sample2 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca57bd7f84/sample2_realign2transcript.bam
sample3 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca57bd7f84/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Thu May 21 19:36:15 2026 ----------
2026-05-21T23:36:15.393383Z  INFO oarfish: setting user-provided filter parameters.
2026-05-21T23:36:15.393720Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca57bd7f84/sample1_realign2transcript.bam, contains 5 reference sequences.
2026-05-21T23:36:15.393776Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-21T23:36:15.393807Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-21T23:36:15.393893Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-21T23:36:15.393910Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-05-21T23:36:15.398294Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2026-05-21T23:36:15.398426Z  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   │
╰─────────────────────────────────┴───────╯

2026-05-21T23:36:15.398482Z  INFO oarfish::bulk: Total number of alignment records : 364
2026-05-21T23:36:15.398491Z  INFO oarfish::bulk: number of aligned reads : 285
2026-05-21T23:36:15.398495Z  INFO oarfish::bulk: number of unique alignments : 238
2026-05-21T23:36:15.399919Z  INFO oarfish: oarfish completed successfully.
2026-05-21T23:36:15.421921Z  INFO oarfish: setting user-provided filter parameters.
2026-05-21T23:36:15.422240Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca57bd7f84/sample2_realign2transcript.bam, contains 5 reference sequences.
2026-05-21T23:36:15.422265Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-21T23:36:15.422288Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-21T23:36:15.422359Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-21T23:36:15.422373Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-05-21T23:36:15.426128Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2026-05-21T23:36:15.426261Z  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   │
╰─────────────────────────────────┴───────╯

2026-05-21T23:36:15.426296Z  INFO oarfish::bulk: Total number of alignment records : 355
2026-05-21T23:36:15.426305Z  INFO oarfish::bulk: number of aligned reads : 282
2026-05-21T23:36:15.426311Z  INFO oarfish::bulk: number of unique alignments : 240
2026-05-21T23:36:15.429555Z  INFO oarfish: oarfish completed successfully.
2026-05-21T23:36:15.456127Z  INFO oarfish: setting user-provided filter parameters.
2026-05-21T23:36:15.456478Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca57bd7f84/sample3_realign2transcript.bam, contains 5 reference sequences.
2026-05-21T23:36:15.456509Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-21T23:36:15.456533Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-21T23:36:15.456609Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-21T23:36:15.456623Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-05-21T23:36:15.460704Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2026-05-21T23:36:15.460845Z  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   │
╰─────────────────────────────────┴───────╯

2026-05-21T23:36:15.460887Z  INFO oarfish::bulk: Total number of alignment records : 354
2026-05-21T23:36:15.460897Z  INFO oarfish::bulk: number of aligned reads : 283
2026-05-21T23:36:15.460903Z  INFO oarfish::bulk: number of unique alignments : 240
2026-05-21T23:36:15.463990Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
i Writing configuration to: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5ffb9297/config_file_11429.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 @ Thu May 21 19:36:15 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5ffb9297/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5ffb9297/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5ffb9297/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Thu May 21 19:36:16 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

  |                                                                            
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  |                                                                            
  |=======================                                               |  33%
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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 @ Thu May 21 19:36:22 2026 -------------------
Realigning sample sample1 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5ffb9297/sample1_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample2 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5ffb9297/sample2_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample3 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5ffb9297/sample3_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Thu May 21 19:36:23 2026 ----------
19:36:23 Thu May 21 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//RtmpMunpLR/file2ca54131ebe9/config_file_11429.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 @ Thu May 21 19:36:25 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca54131ebe9/sample1_align2genome.bam
sample2 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca54131ebe9/sample2_align2genome.bam
sample3 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca54131ebe9/sample3_align2genome.bam
-- Running step: isoform_identification @ Thu May 21 19:36:33 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 @ Thu May 21 19:36:39 2026 -------------------
Realignment complete for the following samples:
sample1 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca54131ebe9/sample1_realign2transcript.bam
sample2 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca54131ebe9/sample2_realign2transcript.bam
sample3 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca54131ebe9/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Thu May 21 19:36:47 2026 ----------
19:36:47 Thu May 21 2026 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Inputs:  ['/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5ffb9297/sample3_realign2transcript.bam', '/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5ffb9297/sample2_realign2transcript.bam', '/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5ffb9297/sample1_realign2transcript.bam'] /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5ffb9297/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//RtmpMunpLR/file2ca5db52481/config_file_11429.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 @ Thu May 21 19:36:48 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5db52481/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5db52481/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5db52481/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Thu May 21 19:36:49 2026 -------------
Inputs:  ['/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca54131ebe9/sample3_realign2transcript.bam', '/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca54131ebe9/sample2_realign2transcript.bam', '/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca54131ebe9/sample1_realign2transcript.bam'] /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca54131ebe9/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 @ Thu May 21 19:36:49 2026 -------------------
Realigning sample sample1 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5db52481/sample1_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample2 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5db52481/sample2_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample3 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5db52481/sample3_realign2transcript.bam
Skipped sorting BAM files.

-- Running step: transcript_quantification @ Thu May 21 19:36:50 2026 ----------
2026-05-21T23:36:50.535200Z  INFO oarfish: setting user-provided filter parameters.
2026-05-21T23:36:50.535509Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5db52481/sample1_realign2transcript.bam, contains 17 reference sequences.
2026-05-21T23:36:50.535524Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-21T23:36:50.535536Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-21T23:36:50.535602Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-21T23:36:50.535611Z  INFO oarfish: parsed reference information for 17 transcripts.
2026-05-21T23:36:50.541260Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-05-21T23:36:50.541344Z  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   │
╰─────────────────────────────────┴───────╯

2026-05-21T23:36:50.541368Z  INFO oarfish::bulk: Total number of alignment records : 513
2026-05-21T23:36:50.541374Z  INFO oarfish::bulk: number of aligned reads : 293
2026-05-21T23:36:50.541377Z  INFO oarfish::bulk: number of unique alignments : 198
2026-05-21T23:36:50.542854Z  INFO oarfish: oarfish completed successfully.
2026-05-21T23:36:50.559082Z  INFO oarfish: setting user-provided filter parameters.
2026-05-21T23:36:50.559326Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5db52481/sample2_realign2transcript.bam, contains 17 reference sequences.
2026-05-21T23:36:50.559339Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-21T23:36:50.559354Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-21T23:36:50.559419Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-21T23:36:50.559429Z  INFO oarfish: parsed reference information for 17 transcripts.
2026-05-21T23:36:50.565190Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-05-21T23:36:50.565271Z  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   │
╰─────────────────────────────────┴───────╯

2026-05-21T23:36:50.565295Z  INFO oarfish::bulk: Total number of alignment records : 525
2026-05-21T23:36:50.565301Z  INFO oarfish::bulk: number of aligned reads : 292
2026-05-21T23:36:50.565304Z  INFO oarfish::bulk: number of unique alignments : 190
2026-05-21T23:36:50.566911Z  INFO oarfish: oarfish completed successfully.
2026-05-21T23:36:50.583010Z  INFO oarfish: setting user-provided filter parameters.
2026-05-21T23:36:50.583307Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5db52481/sample3_realign2transcript.bam, contains 17 reference sequences.
2026-05-21T23:36:50.583324Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-21T23:36:50.583341Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-21T23:36:50.583410Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-21T23:36:50.583422Z  INFO oarfish: parsed reference information for 17 transcripts.
2026-05-21T23:36:50.588982Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-05-21T23:36:50.589063Z  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   │
╰─────────────────────────────────┴───────╯

2026-05-21T23:36:50.589087Z  INFO oarfish::bulk: Total number of alignment records : 517
2026-05-21T23:36:50.589092Z  INFO oarfish::bulk: number of aligned reads : 290
2026-05-21T23:36:50.589094Z  INFO oarfish::bulk: number of unique alignments : 192
2026-05-21T23:36:50.590838Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
i Writing configuration to: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca53ab3b070/config_file_11429.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 @ Thu May 21 19:36:50 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca53ab3b070/sample1_align2genome.bam
sample2 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca53ab3b070/sample2_align2genome.bam
sample3 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca53ab3b070/sample3_align2genome.bam
-- Running step: isoform_identification @ Thu May 21 19:36:59 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Thu May 21 19:36:59 2026 -------------------
Realignment complete for the following samples:
sample1 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca53ab3b070/sample1_realign2transcript.bam
sample2 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca53ab3b070/sample2_realign2transcript.bam
sample3 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca53ab3b070/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Thu May 21 19:37:08 2026 ----------
2026-05-21T23:37:08.167591Z  INFO oarfish: setting user-provided filter parameters.
2026-05-21T23:37:08.167993Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca53ab3b070/sample1_realign2transcript.bam, contains 15 reference sequences.
2026-05-21T23:37:08.168007Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-21T23:37:08.168022Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-21T23:37:08.168084Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-21T23:37:08.168095Z  INFO oarfish: parsed reference information for 15 transcripts.
2026-05-21T23:37:08.173812Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-05-21T23:37:08.173893Z  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   │
╰─────────────────────────────────┴───────╯

2026-05-21T23:37:08.173918Z  INFO oarfish::bulk: Total number of alignment records : 495
2026-05-21T23:37:08.173924Z  INFO oarfish::bulk: number of aligned reads : 293
2026-05-21T23:37:08.173927Z  INFO oarfish::bulk: number of unique alignments : 208
2026-05-21T23:37:08.175883Z  INFO oarfish: oarfish completed successfully.
2026-05-21T23:37:08.191086Z  INFO oarfish: setting user-provided filter parameters.
2026-05-21T23:37:08.191307Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca53ab3b070/sample2_realign2transcript.bam, contains 15 reference sequences.
2026-05-21T23:37:08.191318Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-21T23:37:08.191330Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-21T23:37:08.191391Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-21T23:37:08.191398Z  INFO oarfish: parsed reference information for 15 transcripts.
2026-05-21T23:37:08.197186Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-05-21T23:37:08.197292Z  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   │
╰─────────────────────────────────┴───────╯

2026-05-21T23:37:08.197351Z  INFO oarfish::bulk: Total number of alignment records : 511
2026-05-21T23:37:08.197363Z  INFO oarfish::bulk: number of aligned reads : 290
2026-05-21T23:37:08.197376Z  INFO oarfish::bulk: number of unique alignments : 198
2026-05-21T23:37:08.199181Z  INFO oarfish: oarfish completed successfully.
2026-05-21T23:37:08.218075Z  INFO oarfish: setting user-provided filter parameters.
2026-05-21T23:37:08.218534Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca53ab3b070/sample3_realign2transcript.bam, contains 15 reference sequences.
2026-05-21T23:37:08.218545Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-21T23:37:08.218563Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-21T23:37:08.218630Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-21T23:37:08.218641Z  INFO oarfish: parsed reference information for 15 transcripts.
2026-05-21T23:37:08.223895Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-05-21T23:37:08.223984Z  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   │
╰─────────────────────────────────┴───────╯

2026-05-21T23:37:08.224012Z  INFO oarfish::bulk: Total number of alignment records : 496
2026-05-21T23:37:08.224020Z  INFO oarfish::bulk: number of aligned reads : 291
2026-05-21T23:37:08.224027Z  INFO oarfish::bulk: number of unique alignments : 206
2026-05-21T23:37:08.856984Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
i Writing configuration to: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5456c17eb/config_file_11429.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 @ Thu May 21 19:37:09 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5456c17eb/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5456c17eb/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5456c17eb/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Thu May 21 19:37:10 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Thu May 21 19:37:10 2026 -------------------
Realigning sample sample1 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5456c17eb/sample1_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample2 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5456c17eb/sample2_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample3 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5456c17eb/sample3_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Thu May 21 19:37:11 2026 ----------
19:37:11 Thu May 21 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//RtmpMunpLR/file2ca548d665d/config_file_11429.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 @ Thu May 21 19:37:11 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca548d665d/sample1_align2genome.bam
sample2 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca548d665d/sample2_align2genome.bam
sample3 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca548d665d/sample3_align2genome.bam
-- Running step: isoform_identification @ Thu May 21 19:37:20 2026 -------------
Inputs:  ['/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5456c17eb/sample3_realign2transcript.bam', '/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5456c17eb/sample2_realign2transcript.bam', '/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5456c17eb/sample1_realign2transcript.bam'] /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5456c17eb/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 @ Thu May 21 19:37:20 2026 -------------------
Realignment complete for the following samples:
sample1 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca548d665d/sample1_realign2transcript.bam
sample2 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca548d665d/sample2_realign2transcript.bam
sample3 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca548d665d/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Thu May 21 19:37:28 2026 ----------
19:37:28 Thu May 21 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//RtmpMunpLR/file2ca566f68875/config_file_11429.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 @ Thu May 21 19:37:28 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca566f68875/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 @ Thu May 21 19:37:28 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca566f68875/matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca566f68875/align2genome.bam
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: gene_quantification @ Thu May 21 19:37:28 2026 ----------------
19:37:28 Thu May 21 2026 quantify genes 
Using BAM(s):
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca566f68875/align2genome.bam'
Inputs:  ['/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca548d665d/sample3_realign2transcript.bam', '/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca548d665d/sample2_realign2transcript.bam', '/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca548d665d/sample1_realign2transcript.bam'] /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca548d665d/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,  1.49gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  1.49gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
2026-05-21 19:37:30.458 R[11429:28231022] +[NSXPCSharedListener endpointForReply:withListenerName:replyErrorCode:]: an error occurred while attempting to obtain endpoint for listener 'ClientCallsAuxiliary': Connection interrupted
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 987824.78Read/s]
-- Running step: isoform_identification @ Thu May 21 19:37:30 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 @ Thu May 21 19:37:33 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//RtmpMunpLR/file2ca566f68875/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca566f68875/matched_reads_dedup.fastq.gz
	files found
Realigning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca566f68875/matched_reads_dedup.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca566f68875/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 @ Thu May 21 19:37:33 2026 ----------
2026-05-21T23:37:33.701558Z  INFO oarfish: setting user-provided filter parameters.
2026-05-21T23:37:33.701839Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca566f68875/realign2transcript.bam, contains 5 reference sequences.
2026-05-21T23:37:33.701854Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-21T23:37:33.701867Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-21T23:37:33.701922Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-21T23:37:33.701930Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-05-21T23:37:33.705212Z  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//RtmpMunpLR/file2ca5562e1a00/config_file_11429.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 @ Thu May 21 19:37:33 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5562e1a00/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 @ Thu May 21 19:37:34 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5562e1a00/matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5562e1a00/align2genome.bam
-- Running step: gene_quantification @ Thu May 21 19:37:41 2026 ----------------
19:37:41 Thu May 21 2026 quantify genes 
Using BAM(s):
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5562e1a00/align2genome.bam'
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  1.53gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  1.53gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 904100.71Read/s]
-- Running step: isoform_identification @ Thu May 21 19:37:42 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 @ Thu May 21 19:37:45 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//RtmpMunpLR/file2ca5562e1a00/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5562e1a00/matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5562e1a00/matched_reads_dedup.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5562e1a00/realign2transcript.bam
-- Running step: transcript_quantification @ Thu May 21 19:37:53 2026 ----------
2026-05-21T23:37:53.397281Z  INFO oarfish: setting user-provided filter parameters.
2026-05-21T23:37:53.397496Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5562e1a00/realign2transcript.bam, contains 5 reference sequences.
2026-05-21T23:37:53.397508Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-21T23:37:53.397520Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-21T23:37:53.397565Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-21T23:37:53.397572Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-05-21T23:37:53.401305Z  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//RtmpMunpLR/file2ca56539092/config_file_11429.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 @ Thu May 21 19:37:53 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca56539092/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 @ Thu May 21 19:37:53 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca56539092/matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca56539092/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 @ Thu May 21 19:37:53 2026 ----------------
19:37:53 Thu May 21 2026 quantify genes 
Using BAM(s):
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca56539092/align2genome.bam'
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  1.41gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  1.41gene_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, 940595.62Read/s]
-- Running step: isoform_identification @ Thu May 21 19:37:54 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 @ Thu May 21 19:37:57 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//RtmpMunpLR/file2ca56539092/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca56539092/matched_reads_dedup.fastq.gz
	files found
Realigning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca56539092/matched_reads_dedup.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca56539092/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 @ Thu May 21 19:37:57 2026 ----------
19:37:57 Thu May 21 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//RtmpMunpLR/file2ca51b7d658c/config_file_11429.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 @ Thu May 21 19:37:58 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca51b7d658c/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 @ Thu May 21 19:37:58 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca51b7d658c/matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca51b7d658c/align2genome.bam
-- Running step: gene_quantification @ Thu May 21 19:38:06 2026 ----------------
19:38:06 Thu May 21 2026 quantify genes 
Using BAM(s):
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca51b7d658c/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,  1.47gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  1.47gene_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, 861819.68Read/s]
-- Running step: isoform_identification @ Thu May 21 19:38:06 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 @ Thu May 21 19:38:09 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//RtmpMunpLR/file2ca51b7d658c/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca51b7d658c/matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca51b7d658c/matched_reads_dedup.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca51b7d658c/realign2transcript.bam
-- Running step: transcript_quantification @ Thu May 21 19:38:17 2026 ----------
19:38:17 Thu May 21 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//RtmpMunpLR/file2ca520227725/config_file_11429.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 @ Thu May 21 19:38:17 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca520227725/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 @ Thu May 21 19:38:18 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca520227725/matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca520227725/align2genome.bam
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: gene_quantification @ Thu May 21 19:38:18 2026 ----------------
19:38:18 Thu May 21 2026 quantify genes 
Using BAM(s):
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca520227725/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,  1.34gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  1.34gene_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, 942709.70Read/s]
-- Running step: isoform_identification @ Thu May 21 19:38:19 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Thu May 21 19:38:19 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//RtmpMunpLR/file2ca520227725/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca520227725/matched_reads_dedup.fastq.gz
	files found
Realigning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca520227725/matched_reads_dedup.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca520227725/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 @ Thu May 21 19:38:19 2026 ----------
2026-05-21T23:38:19.422575Z  INFO oarfish: setting user-provided filter parameters.
2026-05-21T23:38:19.422890Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca520227725/realign2transcript.bam, contains 10 reference sequences.
2026-05-21T23:38:19.422926Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-21T23:38:19.422945Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-21T23:38:19.423007Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-21T23:38:19.423020Z  INFO oarfish: parsed reference information for 10 transcripts.
2026-05-21T23:38:19.428121Z  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//RtmpMunpLR/file2ca54e4914cc/config_file_11429.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 @ Thu May 21 19:38:19 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca54e4914cc/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 @ Thu May 21 19:38:19 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca54e4914cc/matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca54e4914cc/align2genome.bam
-- Running step: gene_quantification @ Thu May 21 19:38:27 2026 ----------------
19:38:27 Thu May 21 2026 quantify genes 
Using BAM(s):
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca54e4914cc/align2genome.bam'
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, 997219.21Read/s]
-- Running step: isoform_identification @ Thu May 21 19:38:28 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Thu May 21 19:38:28 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//RtmpMunpLR/file2ca54e4914cc/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca54e4914cc/matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca54e4914cc/matched_reads_dedup.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca54e4914cc/realign2transcript.bam
-- Running step: transcript_quantification @ Thu May 21 19:38:35 2026 ----------
2026-05-21T23:38:35.974512Z  INFO oarfish: setting user-provided filter parameters.
2026-05-21T23:38:35.974756Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca54e4914cc/realign2transcript.bam, contains 10 reference sequences.
2026-05-21T23:38:35.974776Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-21T23:38:35.974795Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-21T23:38:35.974853Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-21T23:38:35.974864Z  INFO oarfish: parsed reference information for 10 transcripts.
2026-05-21T23:38:35.979137Z  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//RtmpMunpLR/file2ca55d5e875/config_file_11429.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 @ Thu May 21 19:38:36 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca55d5e875/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 @ Thu May 21 19:38:36 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca55d5e875/matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca55d5e875/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 @ Thu May 21 19:38:36 2026 ----------------
19:38:36 Thu May 21 2026 quantify genes 
Using BAM(s):
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca55d5e875/align2genome.bam'
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  1.42gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  1.42gene_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, 1037372.38Read/s]
-- Running step: isoform_identification @ Thu May 21 19:38:37 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Thu May 21 19:38:37 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//RtmpMunpLR/file2ca55d5e875/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca55d5e875/matched_reads_dedup.fastq.gz
	files found
Realigning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca55d5e875/matched_reads_dedup.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca55d5e875/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 @ Thu May 21 19:38:37 2026 ----------
19:38:37 Thu May 21 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//RtmpMunpLR/file2ca5efeee20/config_file_11429.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 @ Thu May 21 19:38:38 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5efeee20/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 @ Thu May 21 19:38:38 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5efeee20/matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5efeee20/align2genome.bam
-- Running step: gene_quantification @ Thu May 21 19:38:45 2026 ----------------
19:38:45 Thu May 21 2026 quantify genes 
Using BAM(s):
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5efeee20/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, 969019.50Read/s]
-- Running step: isoform_identification @ Thu May 21 19:38:46 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Thu May 21 19:38:46 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//RtmpMunpLR/file2ca5efeee20/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5efeee20/matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5efeee20/matched_reads_dedup.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5efeee20/realign2transcript.bam
-- Running step: transcript_quantification @ Thu May 21 19:38:54 2026 ----------
19:38:54 Thu May 21 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//RtmpMunpLR/file2ca557203ca8/config_file_11429.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 @ Thu May 21 19:38:54 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca557203ca8/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//RtmpMunpLR/file2ca557203ca8/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca557203ca8/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca557203ca8/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//RtmpMunpLR/file2ca557203ca8/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//RtmpMunpLR/file2ca557203ca8/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//RtmpMunpLR/file2ca557203ca8/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//RtmpMunpLR/file2ca557203ca8/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//RtmpMunpLR/file2ca557203ca8/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//RtmpMunpLR/file2ca557203ca8/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 @ Thu May 21 19:38:55 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca557203ca8/sampleA_matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca557203ca8/sampleA_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca557203ca8/sample1_matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca557203ca8/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca557203ca8/sample2_matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca557203ca8/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca557203ca8/sample3_matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca557203ca8/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Thu May 21 19:38:57 2026 ----------------
19:38:57 Thu May 21 2026 quantify genes 
Using BAM(s):
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca557203ca8/sampleA_align2genome.bam',
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca557203ca8/sample1_align2genome.bam',
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca557203ca8/sample2_align2genome.bam',
and
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca557203ca8/sample3_align2genome.bam'
	Counter({'counted_reads': 358})
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca557203ca8/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 44.39gene_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, 405246.76Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca557203ca8/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 89.00gene_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, 1454335.64Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca557203ca8/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 90.49gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1420449.74Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca557203ca8/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 73.19gene_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, 1102371.74Read/s]
-- Running step: isoform_identification @ Thu May 21 19:38:57 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

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 @ Thu May 21 19:39:05 2026 -------------------
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca557203ca8/fastq, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca557203ca8/fastq/sample1.fq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca557203ca8/fastq/sample2.fq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca557203ca8/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca557203ca8/sampleA_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca557203ca8/sample1_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca557203ca8/sample2_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca557203ca8/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca557203ca8/sampleA_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca557203ca8/sample1_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca557203ca8/sample2_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca557203ca8/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca557203ca8/sampleA_matched_reads_dedup.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca557203ca8/sampleA_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca557203ca8/sample1_matched_reads_dedup.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca557203ca8/sample1_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca557203ca8/sample2_matched_reads_dedup.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca557203ca8/sample2_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca557203ca8/sample3_matched_reads_dedup.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca557203ca8/sample3_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

-- Running step: transcript_quantification @ Thu May 21 19:39:05 2026 ----------
2026-05-21T23:39:05.995287Z  INFO oarfish: setting user-provided filter parameters.
2026-05-21T23:39:05.995532Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca557203ca8/sampleA_realign2transcript.bam, contains 6 reference sequences.
2026-05-21T23:39:05.995565Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-21T23:39:05.995585Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-21T23:39:05.995640Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-21T23:39:05.995652Z  INFO oarfish: parsed reference information for 6 transcripts.
2026-05-21T23:39:06.001478Z  INFO oarfish::single_cell: Processed 100 cells.
2026-05-21T23:39:06.122290Z  INFO oarfish: setting user-provided filter parameters.
2026-05-21T23:39:06.122548Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca557203ca8/sample1_realign2transcript.bam, contains 6 reference sequences.
2026-05-21T23:39:06.122578Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-21T23:39:06.122596Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-21T23:39:06.122648Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-21T23:39:06.122658Z  INFO oarfish: parsed reference information for 6 transcripts.
2026-05-21T23:39:06.125990Z  INFO oarfish::single_cell: Processed 100 cells.
2026-05-21T23:39:06.229917Z  INFO oarfish: setting user-provided filter parameters.
2026-05-21T23:39:06.230188Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca557203ca8/sample2_realign2transcript.bam, contains 6 reference sequences.
2026-05-21T23:39:06.230222Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-21T23:39:06.230239Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-21T23:39:06.230294Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-21T23:39:06.230304Z  INFO oarfish: parsed reference information for 6 transcripts.
2026-05-21T23:39:06.233197Z  INFO oarfish::single_cell: Processed 100 cells.
2026-05-21T23:39:06.341686Z  INFO oarfish: setting user-provided filter parameters.
2026-05-21T23:39:06.341933Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca557203ca8/sample3_realign2transcript.bam, contains 6 reference sequences.
2026-05-21T23:39:06.341971Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-21T23:39:06.341992Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-21T23:39:06.342056Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-21T23:39:06.342070Z  INFO oarfish: parsed reference information for 6 transcripts.
2026-05-21T23:39:06.345988Z  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//RtmpMunpLR/file2ca52cc281ee/config_file_11429.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 @ Thu May 21 19:39:06 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52cc281ee/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//RtmpMunpLR/file2ca52cc281ee/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52cc281ee/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52cc281ee/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//RtmpMunpLR/file2ca52cc281ee/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//RtmpMunpLR/file2ca52cc281ee/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//RtmpMunpLR/file2ca52cc281ee/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//RtmpMunpLR/file2ca52cc281ee/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//RtmpMunpLR/file2ca52cc281ee/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//RtmpMunpLR/file2ca52cc281ee/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 @ Thu May 21 19:39:07 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52cc281ee/sampleA_matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52cc281ee/sampleA_align2genome.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52cc281ee/sample1_matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52cc281ee/sample1_align2genome.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52cc281ee/sample2_matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52cc281ee/sample2_align2genome.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52cc281ee/sample3_matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52cc281ee/sample3_align2genome.bam
-- Running step: gene_quantification @ Thu May 21 19:39:16 2026 ----------------
19:39:16 Thu May 21 2026 quantify genes 
Using BAM(s):
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52cc281ee/sampleA_align2genome.bam',
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52cc281ee/sample1_align2genome.bam',
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52cc281ee/sample2_align2genome.bam',
and
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52cc281ee/sample3_align2genome.bam'
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52cc281ee/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 47.97gene_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, 457553.78Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52cc281ee/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 85.79gene_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, 1406163.34Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52cc281ee/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 87.39gene_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, 1383345.65Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52cc281ee/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 78.83gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1123634.80Read/s]
-- Running step: isoform_identification @ Thu May 21 19:39:16 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

  |                                                                            
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  |                                                                            
  |==================                                                    |  25%
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  |===================================                                   |  50%
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  |====================================================                  |  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 @ Thu May 21 19:39:24 2026 -------------------
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52cc281ee/fastq, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52cc281ee/fastq/sample1.fq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52cc281ee/fastq/sample2.fq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52cc281ee/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52cc281ee/sampleA_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52cc281ee/sample1_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52cc281ee/sample2_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52cc281ee/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52cc281ee/sampleA_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52cc281ee/sample1_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52cc281ee/sample2_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52cc281ee/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52cc281ee/sampleA_matched_reads_dedup.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52cc281ee/sampleA_realign2transcript.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52cc281ee/sample1_matched_reads_dedup.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52cc281ee/sample1_realign2transcript.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52cc281ee/sample2_matched_reads_dedup.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52cc281ee/sample2_realign2transcript.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52cc281ee/sample3_matched_reads_dedup.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52cc281ee/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Thu May 21 19:39:32 2026 ----------
2026-05-21T23:39:32.825672Z  INFO oarfish: setting user-provided filter parameters.
2026-05-21T23:39:32.825928Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52cc281ee/sampleA_realign2transcript.bam, contains 6 reference sequences.
2026-05-21T23:39:32.825962Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-21T23:39:32.825986Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-21T23:39:32.826057Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-21T23:39:32.826071Z  INFO oarfish: parsed reference information for 6 transcripts.
2026-05-21T23:39:32.832738Z  INFO oarfish::single_cell: Processed 100 cells.
2026-05-21T23:39:32.958712Z  INFO oarfish: setting user-provided filter parameters.
2026-05-21T23:39:32.958975Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52cc281ee/sample1_realign2transcript.bam, contains 6 reference sequences.
2026-05-21T23:39:32.959009Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-21T23:39:32.959026Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-21T23:39:32.959083Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-21T23:39:32.959094Z  INFO oarfish: parsed reference information for 6 transcripts.
2026-05-21T23:39:32.962345Z  INFO oarfish::single_cell: Processed 100 cells.
2026-05-21T23:39:33.078535Z  INFO oarfish: setting user-provided filter parameters.
2026-05-21T23:39:33.078752Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52cc281ee/sample2_realign2transcript.bam, contains 6 reference sequences.
2026-05-21T23:39:33.078784Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-21T23:39:33.078801Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-21T23:39:33.078861Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-21T23:39:33.078872Z  INFO oarfish: parsed reference information for 6 transcripts.
2026-05-21T23:39:33.082322Z  INFO oarfish::single_cell: Processed 100 cells.
2026-05-21T23:39:33.196979Z  INFO oarfish: setting user-provided filter parameters.
2026-05-21T23:39:33.197207Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52cc281ee/sample3_realign2transcript.bam, contains 6 reference sequences.
2026-05-21T23:39:33.197244Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-21T23:39:33.197262Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-21T23:39:33.197316Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-21T23:39:33.197332Z  INFO oarfish: parsed reference information for 6 transcripts.
2026-05-21T23:39:33.201108Z  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//RtmpMunpLR/file2ca5764e2d86/config_file_11429.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 @ Thu May 21 19:39:33 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5764e2d86/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//RtmpMunpLR/file2ca5764e2d86/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5764e2d86/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5764e2d86/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//RtmpMunpLR/file2ca5764e2d86/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//RtmpMunpLR/file2ca5764e2d86/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//RtmpMunpLR/file2ca5764e2d86/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//RtmpMunpLR/file2ca5764e2d86/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//RtmpMunpLR/file2ca5764e2d86/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//RtmpMunpLR/file2ca5764e2d86/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 @ Thu May 21 19:39:34 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5764e2d86/sampleA_matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5764e2d86/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//RtmpMunpLR/file2ca5764e2d86/sample1_matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5764e2d86/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//RtmpMunpLR/file2ca5764e2d86/sample2_matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5764e2d86/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//RtmpMunpLR/file2ca5764e2d86/sample3_matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5764e2d86/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 @ Thu May 21 19:39:35 2026 ----------------
19:39:35 Thu May 21 2026 quantify genes 
Using BAM(s):
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5764e2d86/sampleA_align2genome.bam',
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5764e2d86/sample1_align2genome.bam',
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5764e2d86/sample2_align2genome.bam',
and
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5764e2d86/sample3_align2genome.bam'
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5764e2d86/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 45.22gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 421453.38Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5764e2d86/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 86.04gene_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, 1345535.74Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5764e2d86/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 91.34gene_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, 1302740.71Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5764e2d86/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, 1100405.08Read/s]
-- Running step: isoform_identification @ Thu May 21 19:39:36 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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  |                                                                            
  |==================                                                    |  25%
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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)
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Thu May 21 19:39:43 2026 -------------------
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5764e2d86/fastq, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5764e2d86/fastq/sample1.fq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5764e2d86/fastq/sample2.fq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5764e2d86/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5764e2d86/sampleA_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5764e2d86/sample1_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5764e2d86/sample2_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5764e2d86/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5764e2d86/sampleA_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5764e2d86/sample1_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5764e2d86/sample2_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5764e2d86/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5764e2d86/sampleA_matched_reads_dedup.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5764e2d86/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//RtmpMunpLR/file2ca5764e2d86/sample1_matched_reads_dedup.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5764e2d86/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//RtmpMunpLR/file2ca5764e2d86/sample2_matched_reads_dedup.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5764e2d86/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//RtmpMunpLR/file2ca5764e2d86/sample3_matched_reads_dedup.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5764e2d86/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 @ Thu May 21 19:39:44 2026 ----------
19:39:44 Thu May 21 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//RtmpMunpLR/file2ca5764e2d86/sample3_realign2transcript.bam...
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5764e2d86/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5764e2d86/sample3_realign2transcript.bamdone
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5764e2d86/sampleA_realign2transcript.bam...
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5764e2d86/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5764e2d86/sampleA_realign2transcript.bamdone
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5764e2d86/sample2_realign2transcript.bam...
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5764e2d86/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5764e2d86/sample2_realign2transcript.bamdone
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5764e2d86/sample1_realign2transcript.bam...
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5764e2d86/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca5764e2d86/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//RtmpMunpLR/file2ca574077bd5/config_file_11429.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 @ Thu May 21 19:39:45 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca574077bd5/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//RtmpMunpLR/file2ca574077bd5/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca574077bd5/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca574077bd5/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//RtmpMunpLR/file2ca574077bd5/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//RtmpMunpLR/file2ca574077bd5/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//RtmpMunpLR/file2ca574077bd5/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//RtmpMunpLR/file2ca574077bd5/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//RtmpMunpLR/file2ca574077bd5/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//RtmpMunpLR/file2ca574077bd5/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 @ Thu May 21 19:39:45 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca574077bd5/sampleA_matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca574077bd5/sampleA_align2genome.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca574077bd5/sample1_matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca574077bd5/sample1_align2genome.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca574077bd5/sample2_matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca574077bd5/sample2_align2genome.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca574077bd5/sample3_matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca574077bd5/sample3_align2genome.bam
-- Running step: gene_quantification @ Thu May 21 19:39:54 2026 ----------------
19:39:54 Thu May 21 2026 quantify genes 
Using BAM(s):
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca574077bd5/sampleA_align2genome.bam',
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca574077bd5/sample1_align2genome.bam',
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca574077bd5/sample2_align2genome.bam',
and
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca574077bd5/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//RtmpMunpLR/file2ca574077bd5/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 48.56gene_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, 428287.38Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca574077bd5/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 95.38gene_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, 1417952.67Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca574077bd5/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 21.27gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1355800.36Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca574077bd5/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 79.52gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1161728.34Read/s]
-- Running step: isoform_identification @ Thu May 21 19:39:55 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

  |                                                                            
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  |                                                                            
  |==================                                                    |  25%
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  |===================================                                   |  50%
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  |====================================================                  |  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)
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 @ Thu May 21 19:40:02 2026 -------------------
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca574077bd5/fastq, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca574077bd5/fastq/sample1.fq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca574077bd5/fastq/sample2.fq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca574077bd5/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca574077bd5/sampleA_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca574077bd5/sample1_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca574077bd5/sample2_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca574077bd5/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca574077bd5/sampleA_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca574077bd5/sample1_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca574077bd5/sample2_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca574077bd5/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca574077bd5/sampleA_matched_reads_dedup.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca574077bd5/sampleA_realign2transcript.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca574077bd5/sample1_matched_reads_dedup.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca574077bd5/sample1_realign2transcript.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca574077bd5/sample2_matched_reads_dedup.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca574077bd5/sample2_realign2transcript.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca574077bd5/sample3_matched_reads_dedup.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca574077bd5/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Thu May 21 19:40:10 2026 ----------
19:40:10 Thu May 21 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//RtmpMunpLR/file2ca574077bd5/sample3_realign2transcript.bam...
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca574077bd5/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca574077bd5/sample3_realign2transcript.bamdone
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca574077bd5/sampleA_realign2transcript.bam...
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca574077bd5/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca574077bd5/sampleA_realign2transcript.bamdone
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca574077bd5/sample2_realign2transcript.bam...
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca574077bd5/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca574077bd5/sample2_realign2transcript.bamdone
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca574077bd5/sample1_realign2transcript.bam...
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca574077bd5/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca574077bd5/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//RtmpMunpLR/file2ca55f4f46b5/config_file_11429.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 @ Thu May 21 19:40:11 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca55f4f46b5/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//RtmpMunpLR/file2ca55f4f46b5/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca55f4f46b5/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca55f4f46b5/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//RtmpMunpLR/file2ca55f4f46b5/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//RtmpMunpLR/file2ca55f4f46b5/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//RtmpMunpLR/file2ca55f4f46b5/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//RtmpMunpLR/file2ca55f4f46b5/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//RtmpMunpLR/file2ca55f4f46b5/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//RtmpMunpLR/file2ca55f4f46b5/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 @ Thu May 21 19:40:11 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca55f4f46b5/sampleA_matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca55f4f46b5/sampleA_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca55f4f46b5/sample1_matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca55f4f46b5/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca55f4f46b5/sample2_matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca55f4f46b5/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca55f4f46b5/sample3_matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca55f4f46b5/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Thu May 21 19:40:13 2026 ----------------
19:40:13 Thu May 21 2026 quantify genes 
Using BAM(s):
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca55f4f46b5/sampleA_align2genome.bam',
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca55f4f46b5/sample1_align2genome.bam',
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca55f4f46b5/sample2_align2genome.bam',
and
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca55f4f46b5/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//RtmpMunpLR/file2ca55f4f46b5/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 49.78gene_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, 425472.10Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca55f4f46b5/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 97.75gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1363912.59Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca55f4f46b5/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 96.36gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1382616.03Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca55f4f46b5/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 74.01gene_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, 1197414.64Read/s]
-- Running step: isoform_identification @ Thu May 21 19:40:13 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Thu May 21 19:40:13 2026 -------------------
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca55f4f46b5/fastq, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca55f4f46b5/fastq/sample1.fq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca55f4f46b5/fastq/sample2.fq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca55f4f46b5/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca55f4f46b5/sampleA_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca55f4f46b5/sample1_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca55f4f46b5/sample2_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca55f4f46b5/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca55f4f46b5/sampleA_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca55f4f46b5/sample1_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca55f4f46b5/sample2_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca55f4f46b5/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca55f4f46b5/sampleA_matched_reads_dedup.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca55f4f46b5/sampleA_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca55f4f46b5/sample1_matched_reads_dedup.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca55f4f46b5/sample1_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca55f4f46b5/sample2_matched_reads_dedup.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca55f4f46b5/sample2_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca55f4f46b5/sample3_matched_reads_dedup.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca55f4f46b5/sample3_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

-- Running step: transcript_quantification @ Thu May 21 19:40:17 2026 ----------
2026-05-21T23:40:17.404528Z  INFO oarfish: setting user-provided filter parameters.
2026-05-21T23:40:17.404799Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca55f4f46b5/sampleA_realign2transcript.bam, contains 27 reference sequences.
2026-05-21T23:40:17.404824Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-21T23:40:17.404842Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-21T23:40:17.404916Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-21T23:40:17.404931Z  INFO oarfish: parsed reference information for 27 transcripts.
2026-05-21T23:40:17.421921Z  INFO oarfish::single_cell: Processed 100 cells.
2026-05-21T23:40:17.604219Z  INFO oarfish: setting user-provided filter parameters.
2026-05-21T23:40:17.604493Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca55f4f46b5/sample1_realign2transcript.bam, contains 27 reference sequences.
2026-05-21T23:40:17.604531Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-21T23:40:17.604551Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-21T23:40:17.604650Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-21T23:40:17.604668Z  INFO oarfish: parsed reference information for 27 transcripts.
2026-05-21T23:40:17.613655Z  INFO oarfish::single_cell: Processed 100 cells.
2026-05-21T23:40:17.808816Z  INFO oarfish: setting user-provided filter parameters.
2026-05-21T23:40:17.809099Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca55f4f46b5/sample2_realign2transcript.bam, contains 27 reference sequences.
2026-05-21T23:40:17.809124Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-21T23:40:17.809145Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-21T23:40:17.809237Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-21T23:40:17.809254Z  INFO oarfish: parsed reference information for 27 transcripts.
2026-05-21T23:40:17.817981Z  INFO oarfish::single_cell: Processed 100 cells.
2026-05-21T23:40:17.996827Z  INFO oarfish: setting user-provided filter parameters.
2026-05-21T23:40:17.997089Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca55f4f46b5/sample3_realign2transcript.bam, contains 27 reference sequences.
2026-05-21T23:40:17.997104Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-21T23:40:17.997117Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-21T23:40:17.997188Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-21T23:40:17.997200Z  INFO oarfish: parsed reference information for 27 transcripts.
2026-05-21T23:40:18.002755Z  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//RtmpMunpLR/file2ca528f7776b/config_file_11429.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 @ Thu May 21 19:40:18 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca528f7776b/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//RtmpMunpLR/file2ca528f7776b/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca528f7776b/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca528f7776b/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//RtmpMunpLR/file2ca528f7776b/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//RtmpMunpLR/file2ca528f7776b/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//RtmpMunpLR/file2ca528f7776b/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//RtmpMunpLR/file2ca528f7776b/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//RtmpMunpLR/file2ca528f7776b/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//RtmpMunpLR/file2ca528f7776b/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 @ Thu May 21 19:40:19 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca528f7776b/sampleA_matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca528f7776b/sampleA_align2genome.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca528f7776b/sample1_matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca528f7776b/sample1_align2genome.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca528f7776b/sample2_matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca528f7776b/sample2_align2genome.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca528f7776b/sample3_matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca528f7776b/sample3_align2genome.bam
-- Running step: gene_quantification @ Thu May 21 19:40:28 2026 ----------------
19:40:28 Thu May 21 2026 quantify genes 
Using BAM(s):
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca528f7776b/sampleA_align2genome.bam',
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca528f7776b/sample1_align2genome.bam',
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca528f7776b/sample2_align2genome.bam',
and
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca528f7776b/sample3_align2genome.bam'
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca528f7776b/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 46.28gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 382789.76Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca528f7776b/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 86.10gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1280000.00Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca528f7776b/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 89.44gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1416420.37Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca528f7776b/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 72.83gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1043774.64Read/s]
-- Running step: isoform_identification @ Thu May 21 19:40:28 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Thu May 21 19:40:28 2026 -------------------
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca528f7776b/fastq, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca528f7776b/fastq/sample1.fq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca528f7776b/fastq/sample2.fq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca528f7776b/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca528f7776b/sampleA_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca528f7776b/sample1_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca528f7776b/sample2_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca528f7776b/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca528f7776b/sampleA_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca528f7776b/sample1_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca528f7776b/sample2_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca528f7776b/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca528f7776b/sampleA_matched_reads_dedup.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca528f7776b/sampleA_realign2transcript.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca528f7776b/sample1_matched_reads_dedup.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca528f7776b/sample1_realign2transcript.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca528f7776b/sample2_matched_reads_dedup.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca528f7776b/sample2_realign2transcript.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca528f7776b/sample3_matched_reads_dedup.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca528f7776b/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Thu May 21 19:40:39 2026 ----------
2026-05-21T23:40:39.747499Z  INFO oarfish: setting user-provided filter parameters.
2026-05-21T23:40:39.747751Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca528f7776b/sampleA_realign2transcript.bam, contains 28 reference sequences.
2026-05-21T23:40:39.747786Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-21T23:40:39.747805Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-21T23:40:39.747882Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-21T23:40:39.747900Z  INFO oarfish: parsed reference information for 28 transcripts.
2026-05-21T23:40:39.766142Z  INFO oarfish::single_cell: Processed 100 cells.
2026-05-21T23:40:40.024419Z  INFO oarfish: setting user-provided filter parameters.
2026-05-21T23:40:40.024676Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca528f7776b/sample1_realign2transcript.bam, contains 28 reference sequences.
2026-05-21T23:40:40.024700Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-21T23:40:40.024718Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-21T23:40:40.024798Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-21T23:40:40.024812Z  INFO oarfish: parsed reference information for 28 transcripts.
2026-05-21T23:40:40.033418Z  INFO oarfish::single_cell: Processed 100 cells.
2026-05-21T23:40:40.222777Z  INFO oarfish: setting user-provided filter parameters.
2026-05-21T23:40:40.223036Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca528f7776b/sample2_realign2transcript.bam, contains 28 reference sequences.
2026-05-21T23:40:40.223058Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-21T23:40:40.223077Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-21T23:40:40.223166Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-21T23:40:40.223183Z  INFO oarfish: parsed reference information for 28 transcripts.
2026-05-21T23:40:40.229177Z  INFO oarfish::single_cell: Processed 100 cells.
2026-05-21T23:40:40.416789Z  INFO oarfish: setting user-provided filter parameters.
2026-05-21T23:40:40.417037Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca528f7776b/sample3_realign2transcript.bam, contains 28 reference sequences.
2026-05-21T23:40:40.417059Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-21T23:40:40.417076Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-21T23:40:40.417155Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-21T23:40:40.417168Z  INFO oarfish: parsed reference information for 28 transcripts.
2026-05-21T23:40:40.427376Z  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//RtmpMunpLR/file2ca573ce8567/config_file_11429.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 @ Thu May 21 19:40:40 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca573ce8567/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//RtmpMunpLR/file2ca573ce8567/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca573ce8567/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca573ce8567/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//RtmpMunpLR/file2ca573ce8567/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//RtmpMunpLR/file2ca573ce8567/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//RtmpMunpLR/file2ca573ce8567/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//RtmpMunpLR/file2ca573ce8567/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//RtmpMunpLR/file2ca573ce8567/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//RtmpMunpLR/file2ca573ce8567/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 @ Thu May 21 19:40:41 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca573ce8567/sampleA_matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca573ce8567/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//RtmpMunpLR/file2ca573ce8567/sample1_matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca573ce8567/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//RtmpMunpLR/file2ca573ce8567/sample2_matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca573ce8567/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//RtmpMunpLR/file2ca573ce8567/sample3_matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca573ce8567/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 @ Thu May 21 19:40:43 2026 ----------------
19:40:43 Thu May 21 2026 quantify genes 
Using BAM(s):
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca573ce8567/sampleA_align2genome.bam',
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca573ce8567/sample1_align2genome.bam',
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca573ce8567/sample2_align2genome.bam',
and
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca573ce8567/sample3_align2genome.bam'
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca573ce8567/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 46.60gene_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, 437289.29Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca573ce8567/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 15.63gene_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, 1311047.76Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca573ce8567/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 95.57gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1249494.76Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca573ce8567/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 75.93gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1055223.91Read/s]
-- Running step: isoform_identification @ Thu May 21 19:40:43 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Thu May 21 19:40:43 2026 -------------------
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca573ce8567/fastq, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca573ce8567/fastq/sample1.fq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca573ce8567/fastq/sample2.fq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca573ce8567/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca573ce8567/sampleA_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca573ce8567/sample1_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca573ce8567/sample2_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca573ce8567/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca573ce8567/sampleA_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca573ce8567/sample1_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca573ce8567/sample2_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca573ce8567/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca573ce8567/sampleA_matched_reads_dedup.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca573ce8567/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//RtmpMunpLR/file2ca573ce8567/sample1_matched_reads_dedup.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca573ce8567/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//RtmpMunpLR/file2ca573ce8567/sample2_matched_reads_dedup.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca573ce8567/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//RtmpMunpLR/file2ca573ce8567/sample3_matched_reads_dedup.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca573ce8567/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 @ Thu May 21 19:40:45 2026 ----------
19:40:45 Thu May 21 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//RtmpMunpLR/file2ca573ce8567/sample3_realign2transcript.bam...
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca573ce8567/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca573ce8567/sample3_realign2transcript.bamdone
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca573ce8567/sampleA_realign2transcript.bam...
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca573ce8567/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca573ce8567/sampleA_realign2transcript.bamdone
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca573ce8567/sample2_realign2transcript.bam...
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca573ce8567/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca573ce8567/sample2_realign2transcript.bamdone
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca573ce8567/sample1_realign2transcript.bam...
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca573ce8567/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca573ce8567/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//RtmpMunpLR/file2ca52de770a9/config_file_11429.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 @ Thu May 21 19:40:46 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52de770a9/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//RtmpMunpLR/file2ca52de770a9/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52de770a9/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52de770a9/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//RtmpMunpLR/file2ca52de770a9/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//RtmpMunpLR/file2ca52de770a9/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//RtmpMunpLR/file2ca52de770a9/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//RtmpMunpLR/file2ca52de770a9/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//RtmpMunpLR/file2ca52de770a9/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//RtmpMunpLR/file2ca52de770a9/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 @ Thu May 21 19:40:47 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52de770a9/sampleA_matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52de770a9/sampleA_align2genome.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52de770a9/sample1_matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52de770a9/sample1_align2genome.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52de770a9/sample2_matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52de770a9/sample2_align2genome.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52de770a9/sample3_matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52de770a9/sample3_align2genome.bam
-- Running step: gene_quantification @ Thu May 21 19:40:56 2026 ----------------
19:40:56 Thu May 21 2026 quantify genes 
Using BAM(s):
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52de770a9/sampleA_align2genome.bam',
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52de770a9/sample1_align2genome.bam',
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52de770a9/sample2_align2genome.bam',
and
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52de770a9/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//RtmpMunpLR/file2ca52de770a9/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 48.30gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 426025.27Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52de770a9/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 94.92gene_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, 1351954.62Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52de770a9/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 92.24gene_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, 1397542.32Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52de770a9/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 74.91gene_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, 1089429.61Read/s]
-- Running step: isoform_identification @ Thu May 21 19:40:56 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Thu May 21 19:40:56 2026 -------------------
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52de770a9/fastq, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52de770a9/fastq/sample1.fq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52de770a9/fastq/sample2.fq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52de770a9/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52de770a9/sampleA_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52de770a9/sample1_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52de770a9/sample2_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52de770a9/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52de770a9/sampleA_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52de770a9/sample1_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52de770a9/sample2_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52de770a9/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52de770a9/sampleA_matched_reads_dedup.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52de770a9/sampleA_realign2transcript.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52de770a9/sample1_matched_reads_dedup.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52de770a9/sample1_realign2transcript.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52de770a9/sample2_matched_reads_dedup.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52de770a9/sample2_realign2transcript.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52de770a9/sample3_matched_reads_dedup.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52de770a9/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Thu May 21 19:41:05 2026 ----------
19:41:05 Thu May 21 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//RtmpMunpLR/file2ca52de770a9/sample3_realign2transcript.bam...
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52de770a9/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52de770a9/sample3_realign2transcript.bamdone
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52de770a9/sampleA_realign2transcript.bam...
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52de770a9/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52de770a9/sampleA_realign2transcript.bamdone
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52de770a9/sample2_realign2transcript.bam...
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52de770a9/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52de770a9/sample2_realign2transcript.bamdone
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52de770a9/sample1_realign2transcript.bam...
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52de770a9/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpMunpLR/file2ca52de770a9/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%
  |                                                                            
  |===============================================                       |  67%
  |                                                                            
  |======================================================================| 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 
315.096  19.632 336.936 
/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 '