##############################################################################
##############################################################################
###
### Running command:
###
###   /Library/Frameworks/R.framework/Resources/bin/R CMD check --install=check:FLAMES.install-out.txt --library=/Library/Frameworks/R.framework/Resources/library --no-vignettes --timings FLAMES_2.5.5.tar.gz
###
##############################################################################
##############################################################################


* using log directory ‘/Users/biocbuild/bbs-3.23-bioc/meat/FLAMES.Rcheck’
* using R version 4.6.0 alpha (2026-04-08 r89818)
* using platform: aarch64-apple-darwin23
* R was compiled by
    Apple clang version 17.0.0 (clang-1700.3.19.1)
    GNU Fortran (GCC) 14.2.0
* running under: macOS Tahoe 26.3.1
* using session charset: UTF-8
* current time: 2026-04-20 23:47:20 UTC
* using option ‘--no-vignettes’
* checking for file ‘FLAMES/DESCRIPTION’ ... OK
* this is package ‘FLAMES’ version ‘2.5.5’
* package encoding: UTF-8
* checking package namespace information ... OK
* checking package dependencies ... INFO
Imports includes 47 non-default packages.
Importing from so many packages makes the package vulnerable to any of
them becoming unavailable.  Move as many as possible to Suggests and
use conditionally.
* checking if this is a source package ... OK
* checking if there is a namespace ... OK
* checking for hidden files and directories ... OK
* checking for portable file names ... OK
* checking for sufficient/correct file permissions ... OK
* checking whether package ‘FLAMES’ can be installed ... NOTE
Found the following notes/warnings:
  Non-staged installation was used
See ‘/Users/biocbuild/bbs-3.23-bioc/meat/FLAMES.Rcheck/00install.out’ for details.
* used C++ compiler: ‘Apple clang version 17.0.0 (clang-1700.6.4.2)’
* used SDK: ‘MacOSX26.2.sdk’
* checking C++ specification ... INFO
  specified C++17
* checking installed package size ... INFO
  installed size is 11.0Mb
  sub-directories of 1Mb or more:
    bin    5.9Mb
    data   1.8Mb
    libs   1.6Mb
* checking package directory ... OK
* checking ‘build’ directory ... OK
* checking DESCRIPTION meta-information ... OK
* checking top-level files ... OK
* checking for left-over files ... OK
* checking index information ... OK
* checking package subdirectories ... OK
* checking code files for non-ASCII characters ... OK
* checking R files for syntax errors ... OK
* checking whether the package can be loaded ... OK
* checking whether the package can be loaded with stated dependencies ... OK
* checking whether the package can be unloaded cleanly ... OK
* checking whether the namespace can be loaded with stated dependencies ... OK
* checking whether the namespace can be unloaded cleanly ... OK
* checking dependencies in R code ... NOTE
Namespace in Imports field not imported from: ‘scuttle’
  All declared Imports should be used.
There are ::: calls to the package's namespace in its code. A package
  almost never needs to use ::: for its own objects:
  ‘gene_quantification’ ‘isoform_identification’ ‘minimap2_align’
  ‘transcript_quantification’
* checking S3 generic/method consistency ... OK
* checking replacement functions ... OK
* checking foreign function calls ... OK
* checking R code for possible problems ... NOTE
BulkPipeline: no visible global function definition for ‘new’
BulkPipeline: no visible global function definition for ‘setNames’
MultiSampleSCPipeline: no visible global function definition for ‘new’
MultiSampleSCPipeline: no visible global function definition for
  ‘setNames’
SingleCellPipeline: no visible global function definition for ‘new’
SingleCellPipeline: no visible global function definition for
  ‘setNames’
addRowRanges: no visible global function definition for ‘head’
addRowRanges: no visible global function definition for ‘as’
add_gene_counts: no visible global function definition for ‘as’
barcode_group: no visible global function definition for ‘new’
barcode_segment: no visible global function definition for ‘new’
cache_dir: no visible global function definition for ‘packageVersion’
chisq_test_by_gene: no visible global function definition for
  ‘chisq.test’
create_sce_from_dir: no visible global function definition for
  ‘setNames’
create_spe: no visible binding for global variable ‘barcode’
create_spe: no visible binding for global variable ‘in_tissue’
download_oarfish: no visible global function definition for
  ‘download.file’
download_oarfish: no visible global function definition for ‘unzip’
filter_coverage: no visible global function definition for
  ‘starts_with’
filter_coverage: no visible binding for global variable ‘filter_res’
find_diversity: no visible global function definition for ‘as’
find_variants: no visible global function definition for ‘setNames’
find_variants_grange: no visible binding for global variable
  ‘which_label’
find_variants_grange: no visible binding for global variable
  ‘nucleotide’
find_variants_grange: no visible binding for global variable ‘pos’
find_variants_grange: no visible binding for global variable ‘count’
find_variants_grange: no visible binding for global variable
  ‘counts_no_ins’
find_variants_grange: no visible binding for global variable ‘ref’
generate_sc_sce: no visible binding for global variable ‘FSM_match’
get_coverage: no visible binding for global variable ‘Freq’
homopolymer_pct : <anonymous>: no visible binding for global variable
  ‘Freq’
homopolymer_pct : <anonymous>: no visible binding for global variable
  ‘pct’
plot_coverage: no visible binding for global variable ‘tr_length’
plot_coverage: no visible binding for global variable ‘read_counts’
plot_coverage: no visible binding for global variable ‘total_counts’
plot_coverage: no visible binding for global variable ‘cumpct’
plot_coverage: no visible binding for global variable ‘length_bin’
plot_coverage: no visible binding for global variable ‘min_length’
plot_coverage: no visible binding for global variable ‘max_length’
plot_coverage: no visible global function definition for ‘head’
plot_coverage: no visible binding for global variable ‘transcript’
plot_demultiplex_raw: no visible binding for global variable ‘Sample’
plot_demultiplex_raw: no visible binding for global variable ‘CB’
plot_demultiplex_raw: no visible binding for global variable ‘UB’
plot_demultiplex_raw: no visible binding for global variable
  ‘UMI_count’
plot_demultiplex_raw: no visible binding for global variable
  ‘barcode_rank’
plot_demultiplex_raw: no visible binding for global variable
  ‘FlankEditDist’
plot_demultiplex_raw: no visible binding for global variable ‘n_reads’
plot_demultiplex_raw: no visible binding for global variable ‘total
  reads’
plot_demultiplex_raw: no visible binding for global variable
  ‘demultiplexed reads’
plot_demultiplex_raw: no visible binding for global variable ‘single
  match reads’
plot_demultiplex_raw: no visible binding for global variable
  ‘undemultiplexted reads’
plot_demultiplex_raw: no visible binding for global variable
  ‘multi-matching reads’
plot_demultiplex_raw: no visible binding for global variable ‘Type’
plot_demultiplex_raw: no visible binding for global variable ‘Reads’
plot_demultiplex_raw: no visible binding for global variable ‘input’
plot_demultiplex_raw: no visible binding for global variable ‘output’
plot_demultiplex_raw: no visible binding for global variable
  ‘read1_with_adapter’
plot_demultiplex_raw: no visible binding for global variable ‘Count’
plot_flagstat: no visible global function definition for ‘everything’
plot_flagstat: no visible binding for global variable ‘name’
plot_flagstat: no visible binding for global variable ‘value’
plot_isoform_reduced_dim: no visible binding for global variable ‘x’
plot_isoform_reduced_dim: no visible binding for global variable ‘y’
plot_isoform_reduced_dim: no visible binding for global variable ‘expr’
plot_isoforms: no visible binding for global variable ‘xmin’
plot_isoforms: no visible binding for global variable ‘xmax’
plot_isoforms: no visible binding for global variable ‘ymin’
plot_isoforms: no visible binding for global variable ‘ymax’
plot_isoforms: no visible binding for global variable ‘x’
plot_isoforms: no visible binding for global variable ‘xend’
plot_isoforms: no visible binding for global variable ‘y’
plot_isoforms: no visible binding for global variable ‘yend’
plot_spatial: no visible binding for global variable ‘imageX’
plot_spatial: no visible binding for global variable ‘imageY’
plot_spatial_feature: no visible binding for global variable ‘imageX’
plot_spatial_feature: no visible binding for global variable ‘imageY’
plot_spatial_feature: no visible binding for global variable ‘x’
plot_spatial_feature: no visible binding for global variable ‘y’
plot_spatial_feature: no visible global function definition for
  ‘scale_alpha_continuous’
plot_spatial_feature: no visible global function definition for
  ‘scale_colour_gradient’
plot_spatial_isoform: no visible global function definition for ‘head’
plot_spatial_pie: no visible global function definition for ‘setNames’
plot_spatial_pie: no visible global function definition for ‘head’
plot_spatial_pie: no visible binding for global variable ‘imageX’
plot_spatial_pie: no visible binding for global variable ‘imageY’
sc_genotype: no visible binding for global variable ‘allele’
sc_genotype: no visible binding for global variable ‘allele_count’
sc_genotype: no visible binding for global variable ‘barcode’
sc_genotype: no visible binding for global variable ‘pct’
sc_mutations: no visible binding for global variable ‘mutation_index’
sc_mutations: no visible binding for global variable ‘bam_index’
sc_plot_genotype: no visible global function definition for ‘setNames’
sc_plot_genotype: no visible binding for global variable ‘barcode’
sc_plot_genotype: no visible binding for global variable ‘genotype’
sc_plot_genotype: no visible binding for global variable ‘x’
sc_plot_genotype: no visible binding for global variable ‘y’
sc_transcript_usage_chisq: no visible global function definition for
  ‘as’
sc_transcript_usage_chisq: no visible binding for global variable
  ‘p.value’
sc_transcript_usage_chisq: no visible binding for global variable
  ‘adj.p.value’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘total’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘test’
sc_transcript_usage_permutation: no visible global function definition
  for ‘as’
sc_transcript_usage_permutation : <anonymous>: no visible global
  function definition for ‘as’
sc_transcript_usage_permutation : <anonymous> : <anonymous>: no visible
  global function definition for ‘na.omit’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘transcript’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘p.value’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘adj.p.value’
variant_count_tb: no visible binding for global variable ‘barcode’
variant_count_tb: no visible binding for global variable ‘allele_count’
variant_count_tb: no visible binding for global variable
  ‘cell_total_reads’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘expect_cell_number’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘fastq’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘outdir’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘demultiplexed_fastq’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘barcodes_file’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible global
  function definition for ‘setNames’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline : <anonymous>: no
  visible global function definition for ‘setNames’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘expect_cell_number’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘fastq’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘outdir’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘demultiplexed_fastq’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘barcodes_files’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible global
  function definition for ‘setNames’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘j’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘genome_bam’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘minimap2’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘samtools’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘threads’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘outdir’
plot_durations,FLAMES.Pipeline: no visible binding for global variable
  ‘step’
plot_durations,FLAMES.Pipeline: no visible binding for global variable
  ‘duration’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘j’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘transcriptome_assembly’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘transcriptome_bam’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘minimap2’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘samtools’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘outdir’
resume_FLAMES,FLAMES.Pipeline : <anonymous>: no visible global function
  definition for ‘capture.output’
run_FLAMES,FLAMES.Pipeline : <anonymous>: no visible global function
  definition for ‘capture.output’
Undefined global functions or variables:
  CB Count FSM_match FlankEditDist Freq Reads Sample Type UB UMI_count
  adj.p.value allele allele_count as bam_index barcode barcode_rank
  barcodes_file barcodes_files capture.output cell_total_reads
  chisq.test count counts_no_ins cumpct demultiplexed reads
  demultiplexed_fastq download.file duration everything
  expect_cell_number expr fastq filter_res genome_bam genotype head
  imageX imageY in_tissue input j length_bin max_length min_length
  minimap2 multi-matching reads mutation_index n_reads na.omit name new
  nucleotide outdir output p.value packageVersion pct pos
  read1_with_adapter read_counts ref samtools scale_alpha_continuous
  scale_colour_gradient setNames single match reads starts_with step
  test threads total total reads total_counts tr_length transcript
  transcriptome_assembly transcriptome_bam undemultiplexted reads unzip
  value which_label x xend xmax xmin y yend ymax ymin
Consider adding
  importFrom("base", "match", "single")
  importFrom("methods", "as", "new")
  importFrom("stats", "chisq.test", "na.omit", "setNames", "step")
  importFrom("utils", "capture.output", "download.file", "head",
             "packageVersion", "unzip")
to your NAMESPACE file (and ensure that your DESCRIPTION Imports field
contains 'methods').
* checking Rd files ... OK
* checking Rd metadata ... OK
* checking Rd cross-references ... OK
* checking for missing documentation entries ... OK
* checking for code/documentation mismatches ... OK
* checking Rd \usage sections ... OK
* checking Rd contents ... OK
* checking for unstated dependencies in examples ... OK
* checking contents of ‘data’ directory ... OK
* checking data for non-ASCII characters ... OK
* checking data for ASCII and uncompressed saves ... OK
* checking line endings in shell scripts ... OK
* checking line endings in C/C++/Fortran sources/headers ... OK
* checking line endings in Makefiles ... OK
* checking compilation flags in Makevars ... OK
* checking for GNU extensions in Makefiles ... INFO
GNU make is a SystemRequirements.
* checking for portable use of $(BLAS_LIBS) and $(LAPACK_LIBS) ... OK
* checking use of PKG_*FLAGS in Makefiles ... OK
* checking include directives in Makefiles ... OK
* checking compiled code ... NOTE
Note: information on .o files is not available
File ‘/Library/Frameworks/R.framework/Versions/4.6/Resources/library/FLAMES/libs/FLAMES.so’:
  Found ‘___assert_rtn’, possibly from ‘assert’ (C)
  Found ‘___stderrp’, possibly from ‘stderr’ (C)
  Found ‘___stdoutp’, possibly from ‘stdout’ (C)
  Found ‘_abort’, possibly from ‘abort’ (C)
  Found ‘_exit’, possibly from ‘exit’ (C)

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

See ‘Writing portable packages’ in the ‘Writing R Extensions’ manual.
* checking files in ‘vignettes’ ... OK
* checking examples ... OK
Examples with CPU (user + system) or elapsed time > 5s
                              user system elapsed
plot_isoform_reduced_dim     9.067  0.142   9.346
sc_long_multisample_pipeline 6.129  1.284   5.552
find_variants                6.571  0.405   6.581
MultiSampleSCPipeline        4.438  0.686   5.564
* checking for unstated dependencies in ‘tests’ ... OK
* checking tests ...
  Running ‘testthat.R’
 OK
* checking for unstated dependencies in vignettes ... OK
* checking package vignettes ... OK
* checking running R code from vignettes ... SKIPPED
* checking re-building of vignette outputs ... SKIPPED
* checking PDF version of manual ... OK
* DONE

Status: 4 NOTEs
See
  ‘/Users/biocbuild/bbs-3.23-bioc/meat/FLAMES.Rcheck/00check.log’
for details.

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


* installing to library ‘/Library/Frameworks/R.framework/Versions/4.6/Resources/library’
* installing *source* package ‘FLAMES’ ...
** this is package ‘FLAMES’ version ‘2.5.5’
** using non-staged installation via StagedInstall field
** libs
specified C++17
using C++ compiler: ‘Apple clang version 17.0.0 (clang-1700.6.4.2)’
using C++17
using SDK: ‘MacOSX26.2.sdk’
clang++ -arch arm64 -std=gnu++17 -I"/Library/Frameworks/R.framework/Resources/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/Rcpp/include' -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/Rhtslib/include' -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/testthat/include' -I/opt/R/arm64/include    -fPIC  -falign-functions=64 -Wall -g -O2   -c RcppExports.cpp -o RcppExports.o
clang++ -arch arm64 -std=gnu++17 -I"/Library/Frameworks/R.framework/Resources/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/Rcpp/include' -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/Rhtslib/include' -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/testthat/include' -I/opt/R/arm64/include    -fPIC  -falign-functions=64 -Wall -g -O2   -c RcppFunctions.cpp -o RcppFunctions.o
clang++ -arch arm64 -std=gnu++17 -I"/Library/Frameworks/R.framework/Resources/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/Rcpp/include' -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/Rhtslib/include' -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/testthat/include' -I/opt/R/arm64/include    -fPIC  -falign-functions=64 -Wall -g -O2   -c classes/BamRecord.cpp -o classes/BamRecord.o
clang++ -arch arm64 -std=gnu++17 -I"/Library/Frameworks/R.framework/Resources/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/Rcpp/include' -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/Rhtslib/include' -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/testthat/include' -I/opt/R/arm64/include    -fPIC  -falign-functions=64 -Wall -g -O2   -c classes/GFFRecord.cpp -o classes/GFFRecord.o
clang++ -arch arm64 -std=gnu++17 -I"/Library/Frameworks/R.framework/Resources/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/Rcpp/include' -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/Rhtslib/include' -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/testthat/include' -I/opt/R/arm64/include    -fPIC  -falign-functions=64 -Wall -g -O2   -c classes/GeneAnnotationParser.cpp -o classes/GeneAnnotationParser.o
clang++ -arch arm64 -std=gnu++17 -I"/Library/Frameworks/R.framework/Resources/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/Rcpp/include' -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/Rhtslib/include' -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/testthat/include' -I/opt/R/arm64/include    -fPIC  -falign-functions=64 -Wall -g -O2   -c classes/Isoforms.cpp -o classes/Isoforms.o
classes/Isoforms.cpp:725:22: warning: comparisons like 'X<=Y<=Z' don't have their mathematical meaning [-Wparentheses]
  725 |                                 } else if (0 < i < raw_iso_key.size() - 1) { // a site from the middle somewhere
      |                                                  ^
1 warning generated.
clang++ -arch arm64 -std=gnu++17 -I"/Library/Frameworks/R.framework/Resources/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/Rcpp/include' -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/Rhtslib/include' -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/testthat/include' -I/opt/R/arm64/include    -fPIC  -falign-functions=64 -Wall -g -O2   -c classes/junctions.cpp -o classes/junctions.o
clang++ -arch arm64 -std=gnu++17 -I"/Library/Frameworks/R.framework/Resources/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/Rcpp/include' -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/Rhtslib/include' -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/testthat/include' -I/opt/R/arm64/include    -fPIC  -falign-functions=64 -Wall -g -O2   -c main-functions/find_isoform.cpp -o main-functions/find_isoform.o
clang++ -arch arm64 -std=gnu++17 -I"/Library/Frameworks/R.framework/Resources/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/Rcpp/include' -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/Rhtslib/include' -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/testthat/include' -I/opt/R/arm64/include    -fPIC  -falign-functions=64 -Wall -g -O2   -c main-functions/flexiplex.cpp -o main-functions/flexiplex.o
clang++ -arch arm64 -std=gnu++17 -I"/Library/Frameworks/R.framework/Resources/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/Rcpp/include' -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/Rhtslib/include' -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/testthat/include' -I/opt/R/arm64/include    -fPIC  -falign-functions=64 -Wall -g -O2   -c main-functions/get_transcript_seq.cpp -o main-functions/get_transcript_seq.o
clang++ -arch arm64 -std=gnu++17 -I"/Library/Frameworks/R.framework/Resources/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/Rcpp/include' -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/Rhtslib/include' -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/testthat/include' -I/opt/R/arm64/include    -fPIC  -falign-functions=64 -Wall -g -O2   -c main-functions/group_bam2isoform.cpp -o main-functions/group_bam2isoform.o
clang++ -arch arm64 -std=gnu++17 -I"/Library/Frameworks/R.framework/Resources/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/Rcpp/include' -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/Rhtslib/include' -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/testthat/include' -I/opt/R/arm64/include    -fPIC  -falign-functions=64 -Wall -g -O2   -c main-functions/pileup_readid.cpp -o main-functions/pileup_readid.o
main-functions/pileup_readid.cpp:86:16: warning: unused variable 'end' [-Wunused-variable]
   86 |   unsigned int end;
      |                ^~~
1 warning generated.
clang++ -arch arm64 -std=gnu++17 -I"/Library/Frameworks/R.framework/Resources/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/Rcpp/include' -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/Rhtslib/include' -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/testthat/include' -I/opt/R/arm64/include    -fPIC  -falign-functions=64 -Wall -g -O2   -c tests/test-junctions.cpp -o tests/test-junctions.o
clang++ -arch arm64 -std=gnu++17 -I"/Library/Frameworks/R.framework/Resources/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/Rcpp/include' -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/Rhtslib/include' -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/testthat/include' -I/opt/R/arm64/include    -fPIC  -falign-functions=64 -Wall -g -O2   -c tests/test-parsing.cpp -o tests/test-parsing.o
clang++ -arch arm64 -std=gnu++17 -I"/Library/Frameworks/R.framework/Resources/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/Rcpp/include' -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/Rhtslib/include' -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/testthat/include' -I/opt/R/arm64/include    -fPIC  -falign-functions=64 -Wall -g -O2   -c utility/cigars.cpp -o utility/cigars.o
clang++ -arch arm64 -std=gnu++17 -I"/Library/Frameworks/R.framework/Resources/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/Rcpp/include' -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/Rhtslib/include' -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/testthat/include' -I/opt/R/arm64/include    -fPIC  -falign-functions=64 -Wall -g -O2   -c utility/edlib-1.2.7/edlib.cpp -o utility/edlib-1.2.7/edlib.o
clang -arch arm64 -I"/Library/Frameworks/R.framework/Resources/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/Rcpp/include' -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/Rhtslib/include' -I'/Library/Frameworks/R.framework/Versions/4.6/Resources/library/testthat/include' -I/opt/R/arm64/include    -fPIC  -falign-functions=64 -Wall -g -O2  -c utility/bam.c -o utility/bam.o
clang++ -arch arm64 -std=gnu++17 -dynamiclib -Wl,-headerpad_max_install_names -undefined dynamic_lookup -L/Library/Frameworks/R.framework/Resources/lib -L/opt/R/arm64/lib -o FLAMES.so RcppExports.o RcppFunctions.o classes/BamRecord.o classes/GFFRecord.o classes/GeneAnnotationParser.o classes/Isoforms.o classes/junctions.o main-functions/find_isoform.o main-functions/flexiplex.o main-functions/get_transcript_seq.o main-functions/group_bam2isoform.o main-functions/pileup_readid.o tests/test-junctions.o tests/test-parsing.o utility/cigars.o utility/edlib-1.2.7/edlib.o utility/bam.o -pthread -lz /Library/Frameworks/R.framework/Versions/4.6/Resources/library/Rhtslib/usrlib/libhts.a -lcurl -lbz2 -llzma -lz -F/Library/Frameworks/R.framework/.. -framework R
ld: warning: ignoring duplicate libraries: '-lz'
if test -e "/usr/bin/strip" & test -e "/bin/uname" & [[ `uname` == "Linux" ]] ; then /usr/bin/strip --strip-debug FLAMES.so; fi
Building for ARM64
(cd submodule/minimap2 && make -f Makefile CFLAGS="-falign-functions=64 -Wall -g -O2  -Wno-unused-result" arm_neon=1 aarch64=1 minimap2)
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  -Isse2neon main.c -o main.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  -Isse2neon kthread.c -o kthread.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  -Isse2neon kalloc.c -o kalloc.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  -Isse2neon misc.c -o misc.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  -Isse2neon bseq.c -o bseq.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  -Isse2neon sketch.c -o sketch.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  -Isse2neon sdust.c -o sdust.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  -Isse2neon options.c -o options.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  -Isse2neon index.c -o index.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  -Isse2neon lchain.c -o lchain.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  -Isse2neon align.c -o align.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  -Isse2neon hit.c -o hit.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  -Isse2neon seed.c -o seed.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  -Isse2neon jump.c -o jump.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  -Isse2neon map.c -o map.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  -Isse2neon format.c -o format.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  -Isse2neon pe.c -o pe.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  -Isse2neon esterr.c -o esterr.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  -Isse2neon splitidx.c -o splitidx.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  -Isse2neon ksw2_ll_sse.c -o ksw2_ll_sse.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC -DKSW_SSE2_ONLY -D__SSE2__  -Isse2neon ksw2_extz2_sse.c -o ksw2_extz2_neon.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC -DKSW_SSE2_ONLY -D__SSE2__  -Isse2neon ksw2_extd2_sse.c -o ksw2_extd2_neon.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC -DKSW_SSE2_ONLY -D__SSE2__  -Isse2neon ksw2_exts2_sse.c -o ksw2_exts2_neon.o
ar -csru libminimap2.a kthread.o kalloc.o misc.o bseq.o sketch.o sdust.o options.o index.o lchain.o align.o hit.o seed.o jump.o map.o format.o pe.o esterr.o splitidx.o ksw2_ll_sse.o ksw2_extz2_neon.o ksw2_extd2_neon.o ksw2_exts2_neon.o
cc -falign-functions=64 -Wall -g -O2  -Wno-unused-result main.o -o minimap2 -L. -lminimap2 -lm -lz -lpthread
echo "Installing binary to /Users/biocbuild/bbs-3.23-bioc/meat/FLAMES/src/../inst/bin"
Installing binary to /Users/biocbuild/bbs-3.23-bioc/meat/FLAMES/src/../inst/bin
mkdir -p ../inst/bin
cp submodule/minimap2/minimap2 ../inst/bin/
Building oarfish with cargo
mkdir -p ../inst/bin
mkdir cargo_temp
(cargo install oarfish --root cargo_temp --bin oarfish --vers 0.8.0)
    Updating crates.io index
  Installing oarfish v0.8.0
    Updating crates.io index
     Locking 279 packages to latest compatible versions
      Adding generic-array v0.14.7 (available: v0.14.9)
      Adding indicatif v0.17.11 (available: v0.18.4)
      Adding needletail v0.6.3 (available: v0.7.3)
      Adding noodles-bam v0.78.0 (available: v0.85.0)
      Adding noodles-bgzf v0.38.0 (available: v0.45.0)
      Adding noodles-sam v0.74.0 (available: v0.81.0)
      Adding rand v0.9.4 (available: v0.10.1)
      Adding tabled v0.18.0 (available: v0.20.0)
      Adding typed-builder v0.21.2 (available: v0.23.2)
      Adding wasip2 v1.0.3+wasi-0.2.9 (requires Rust 1.87.0)
      Adding wasip3 v0.4.0+wasi-0.3.0-rc-2026-01-06 (requires Rust 1.87.0)
      Adding wit-bindgen v0.51.0 (requires Rust 1.87.0)
      Adding wit-bindgen-core v0.51.0 (requires Rust 1.87.0)
      Adding wit-bindgen-rust v0.51.0 (requires Rust 1.87.0)
 Downloading crates ...
  Downloaded ethnum v1.5.3
   Compiling libc v0.2.185
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   Compiling unicode-ident v1.0.24
   Compiling quote v1.0.45
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   Compiling minimap2 v0.1.31+minimap2.2.30
   Compiling seqcol_rs v0.4.1
   Compiling statrs v0.18.0
   Compiling oarfish v0.8.0
    Finished `release` profile [optimized] target(s) in 32.06s
  Installing cargo_temp/bin/oarfish
   Installed package `oarfish v0.8.0` (executable `oarfish`)
warning: be sure to add `cargo_temp/bin` to your PATH to be able to run the installed binaries
cp cargo_temp/bin/oarfish ../inst/bin/
cargo uninstall oarfish --root cargo_temp
    Removing cargo_temp/bin/oarfish
rm -rf cargo_temp
installing to /Library/Frameworks/R.framework/Versions/4.6/Resources/library/FLAMES/libs
** R
** data
** inst
** byte-compile and prepare package for lazy loading
** help
*** installing help indices
*** copying figures
** building package indices
** installing vignettes
** testing if installed package can be loaded
* DONE (FLAMES)

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

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

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

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

> # This file is part of the standard setup for testthat.
> # It is recommended that you do not modify it.
> #
> # Where should you do additional test configuration?
> # Learn more about the roles of various files in:
> # * https://r-pkgs.org/tests.html
> # * https://testthat.r-lib.org/reference/test_package.html#special-files
> 
> library(testthat)
> library(FLAMES)
> 
> test_check("FLAMES")
i Writing configuration to: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file14593106e15d/config_file_83347.json
i Writing configuration to: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file14593106e15d/config_file_83347.json
i Writing configuration to: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file14593106e15d/config_file_83347.json
i Writing configuration to: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936c2c2cc8/config_file_83347.json
Converting legacy `pattern` argument to `segments`...
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145933f7d5871/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
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//RtmpdnWSzD/file145932893474f/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
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//RtmpdnWSzD/file145932893474f/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
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file14593222e5933/musc_rps24_1.fastq
Searching for barcodes...
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file14593222e5933/musc_rps24_2.fastq
Searching for barcodes...
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file14593222e5933/musc_rps24_3.fastq
Searching for barcodes...
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file14593222e5933/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//RtmpdnWSzD/file1459341ebe4a2/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
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//RtmpdnWSzD/file145936e2c6dfc/config_file_83347.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 @ Mon Apr 20 19:51:13 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936e2c6dfc/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936e2c6dfc/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936e2c6dfc/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Mon Apr 20 19:51:14 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 6 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Mon Apr 20 19:51:23 2026 -------------------
Realigning sample sample1 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936e2c6dfc/sample1_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample2 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936e2c6dfc/sample2_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample3 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936e2c6dfc/sample3_realign2transcript.bam
Skipped sorting BAM files.

-- Running step: transcript_quantification @ Mon Apr 20 19:51:24 2026 ----------
2026-04-20T23:51:24.100048Z  INFO oarfish: setting user-provided filter parameters.
2026-04-20T23:51:24.100301Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936e2c6dfc/sample1_realign2transcript.bam, contains 5 reference sequences.
2026-04-20T23:51:24.100317Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-20T23:51:24.100330Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-20T23:51:24.100395Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-20T23:51:24.100457Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-04-20T23:51:24.103108Z  INFO oarfish::alignment_parser: the alignment file contained 1 unmapped read records.
2026-04-20T23:51:24.103188Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 686   │
│ aligned fraction too low        │ 16    │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 4     │
│ reads with valid best alignment │ 283   │
╰─────────────────────────────────┴───────╯

2026-04-20T23:51:24.103210Z  INFO oarfish::bulk: Total number of alignment records : 364
2026-04-20T23:51:24.103215Z  INFO oarfish::bulk: number of aligned reads : 283
2026-04-20T23:51:24.103218Z  INFO oarfish::bulk: number of unique alignments : 235
2026-04-20T23:51:24.104970Z  INFO oarfish: oarfish completed successfully.
2026-04-20T23:51:24.120749Z  INFO oarfish: setting user-provided filter parameters.
2026-04-20T23:51:24.121028Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936e2c6dfc/sample2_realign2transcript.bam, contains 5 reference sequences.
2026-04-20T23:51:24.121057Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-20T23:51:24.121078Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-20T23:51:24.121146Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-20T23:51:24.121156Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-04-20T23:51:24.124247Z  INFO oarfish::alignment_parser: the alignment file contained 1 unmapped read records.
2026-04-20T23:51:24.124425Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 695   │
│ aligned fraction too low        │ 14    │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 7     │
│ reads with valid best alignment │ 285   │
╰─────────────────────────────────┴───────╯

2026-04-20T23:51:24.124493Z  INFO oarfish::bulk: Total number of alignment records : 369
2026-04-20T23:51:24.124500Z  INFO oarfish::bulk: number of aligned reads : 285
2026-04-20T23:51:24.124505Z  INFO oarfish::bulk: number of unique alignments : 235
2026-04-20T23:51:24.126048Z  INFO oarfish: oarfish completed successfully.
2026-04-20T23:51:24.144226Z  INFO oarfish: setting user-provided filter parameters.
2026-04-20T23:51:24.144521Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936e2c6dfc/sample3_realign2transcript.bam, contains 5 reference sequences.
2026-04-20T23:51:24.144545Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-20T23:51:24.144562Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-20T23:51:24.144620Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-20T23:51:24.144634Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-04-20T23:51:24.148191Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2026-04-20T23:51:24.148270Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 696   │
│ aligned fraction too low        │ 14    │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 5     │
│ reads with valid best alignment │ 284   │
╰─────────────────────────────────┴───────╯

2026-04-20T23:51:24.148295Z  INFO oarfish::bulk: Total number of alignment records : 362
2026-04-20T23:51:24.148300Z  INFO oarfish::bulk: number of aligned reads : 284
2026-04-20T23:51:24.148303Z  INFO oarfish::bulk: number of unique alignments : 237
2026-04-20T23:51:24.149803Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
i Writing configuration to: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459318955ad8/config_file_83347.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 @ Mon Apr 20 19:51:24 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459318955ad8/sample1_align2genome.bam
sample2 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459318955ad8/sample2_align2genome.bam
sample3 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459318955ad8/sample3_align2genome.bam
-- Running step: isoform_identification @ Mon Apr 20 19:51:33 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 @ Mon Apr 20 19:51:41 2026 -------------------
Realignment complete for the following samples:
sample1 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459318955ad8/sample1_realign2transcript.bam
sample2 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459318955ad8/sample2_realign2transcript.bam
sample3 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459318955ad8/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Mon Apr 20 19:51:50 2026 ----------
2026-04-20T23:51:50.490615Z  INFO oarfish: setting user-provided filter parameters.
2026-04-20T23:51:50.490858Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459318955ad8/sample1_realign2transcript.bam, contains 5 reference sequences.
2026-04-20T23:51:50.490871Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-20T23:51:50.490902Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-20T23:51:50.491074Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-20T23:51:50.491093Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-04-20T23:51:50.495019Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2026-04-20T23:51:50.495137Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 702   │
│ aligned fraction too low        │ 13    │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 5     │
│ reads with valid best alignment │ 285   │
╰─────────────────────────────────┴───────╯

2026-04-20T23:51:50.495181Z  INFO oarfish::bulk: Total number of alignment records : 364
2026-04-20T23:51:50.495187Z  INFO oarfish::bulk: number of aligned reads : 285
2026-04-20T23:51:50.495191Z  INFO oarfish::bulk: number of unique alignments : 238
2026-04-20T23:51:50.496662Z  INFO oarfish: oarfish completed successfully.
2026-04-20T23:51:50.514514Z  INFO oarfish: setting user-provided filter parameters.
2026-04-20T23:51:50.514781Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459318955ad8/sample2_realign2transcript.bam, contains 5 reference sequences.
2026-04-20T23:51:50.514797Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-20T23:51:50.514811Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-20T23:51:50.514861Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-20T23:51:50.514869Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-04-20T23:51:50.518558Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2026-04-20T23:51:50.518651Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 687   │
│ aligned fraction too low        │ 16    │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 6     │
│ reads with valid best alignment │ 282   │
╰─────────────────────────────────┴───────╯

2026-04-20T23:51:50.518678Z  INFO oarfish::bulk: Total number of alignment records : 355
2026-04-20T23:51:50.518685Z  INFO oarfish::bulk: number of aligned reads : 282
2026-04-20T23:51:50.518688Z  INFO oarfish::bulk: number of unique alignments : 240
2026-04-20T23:51:50.520133Z  INFO oarfish: oarfish completed successfully.
2026-04-20T23:51:50.536389Z  INFO oarfish: setting user-provided filter parameters.
2026-04-20T23:51:50.536666Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459318955ad8/sample3_realign2transcript.bam, contains 5 reference sequences.
2026-04-20T23:51:50.536751Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-20T23:51:50.536858Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-20T23:51:50.536933Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-20T23:51:50.536952Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-04-20T23:51:50.541122Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2026-04-20T23:51:50.541221Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 688   │
│ aligned fraction too low        │ 15    │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 7     │
│ reads with valid best alignment │ 283   │
╰─────────────────────────────────┴───────╯

2026-04-20T23:51:50.541251Z  INFO oarfish::bulk: Total number of alignment records : 354
2026-04-20T23:51:50.541257Z  INFO oarfish::bulk: number of aligned reads : 283
2026-04-20T23:51:50.541261Z  INFO oarfish::bulk: number of unique alignments : 240
2026-04-20T23:51:50.542695Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
i Writing configuration to: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459376f66d6e/config_file_83347.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 @ Mon Apr 20 19:51:50 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459376f66d6e/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459376f66d6e/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459376f66d6e/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Mon Apr 20 19:51:52 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

  |                                                                            
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  |                                                                            
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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 @ Mon Apr 20 19:51:58 2026 -------------------
Realigning sample sample1 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459376f66d6e/sample1_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample2 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459376f66d6e/sample2_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample3 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459376f66d6e/sample3_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Mon Apr 20 19:51:59 2026 ----------
19:51:59 Mon Apr 20 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//RtmpdnWSzD/file145932c4113fc/config_file_83347.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 @ Mon Apr 20 19:52:01 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145932c4113fc/sample1_align2genome.bam
sample2 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145932c4113fc/sample2_align2genome.bam
sample3 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145932c4113fc/sample3_align2genome.bam
-- Running step: isoform_identification @ Mon Apr 20 19:52:10 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 @ Mon Apr 20 19:52:16 2026 -------------------
Realignment complete for the following samples:
sample1 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145932c4113fc/sample1_realign2transcript.bam
sample2 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145932c4113fc/sample2_realign2transcript.bam
sample3 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145932c4113fc/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Mon Apr 20 19:52:25 2026 ----------
19:52:25 Mon Apr 20 2026 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Inputs:  ['/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459376f66d6e/sample3_realign2transcript.bam', '/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459376f66d6e/sample2_realign2transcript.bam', '/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459376f66d6e/sample1_realign2transcript.bam'] /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459376f66d6e/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//RtmpdnWSzD/file14593650b80f/config_file_83347.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 @ Mon Apr 20 19:52:26 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file14593650b80f/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file14593650b80f/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file14593650b80f/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Mon Apr 20 19:52:26 2026 -------------
Inputs:  ['/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145932c4113fc/sample3_realign2transcript.bam', '/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145932c4113fc/sample2_realign2transcript.bam', '/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145932c4113fc/sample1_realign2transcript.bam'] /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145932c4113fc/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 @ Mon Apr 20 19:52:27 2026 -------------------
Realigning sample sample1 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file14593650b80f/sample1_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample2 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file14593650b80f/sample2_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample3 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file14593650b80f/sample3_realign2transcript.bam
Skipped sorting BAM files.

-- Running step: transcript_quantification @ Mon Apr 20 19:52:28 2026 ----------
2026-04-20T23:52:28.548252Z  INFO oarfish: setting user-provided filter parameters.
2026-04-20T23:52:28.548600Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file14593650b80f/sample1_realign2transcript.bam, contains 17 reference sequences.
2026-04-20T23:52:28.548615Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-20T23:52:28.548628Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-20T23:52:28.548711Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-20T23:52:28.548722Z  INFO oarfish: parsed reference information for 17 transcripts.
2026-04-20T23:52:28.558079Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-04-20T23:52:28.558248Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 3436  │
│ aligned fraction too low        │ 7     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 293   │
╰─────────────────────────────────┴───────╯

2026-04-20T23:52:28.558290Z  INFO oarfish::bulk: Total number of alignment records : 513
2026-04-20T23:52:28.558301Z  INFO oarfish::bulk: number of aligned reads : 293
2026-04-20T23:52:28.558309Z  INFO oarfish::bulk: number of unique alignments : 198
2026-04-20T23:52:28.560962Z  INFO oarfish: oarfish completed successfully.
2026-04-20T23:52:28.589568Z  INFO oarfish: setting user-provided filter parameters.
2026-04-20T23:52:28.589982Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file14593650b80f/sample2_realign2transcript.bam, contains 17 reference sequences.
2026-04-20T23:52:28.590012Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-20T23:52:28.590036Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-20T23:52:28.590157Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-20T23:52:28.590175Z  INFO oarfish: parsed reference information for 17 transcripts.
2026-04-20T23:52:28.599917Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-04-20T23:52:28.600162Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 3402  │
│ aligned fraction too low        │ 8     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 292   │
╰─────────────────────────────────┴───────╯

2026-04-20T23:52:28.600203Z  INFO oarfish::bulk: Total number of alignment records : 525
2026-04-20T23:52:28.600210Z  INFO oarfish::bulk: number of aligned reads : 292
2026-04-20T23:52:28.600214Z  INFO oarfish::bulk: number of unique alignments : 190
2026-04-20T23:52:28.602241Z  INFO oarfish: oarfish completed successfully.
2026-04-20T23:52:28.621878Z  INFO oarfish: setting user-provided filter parameters.
2026-04-20T23:52:28.622231Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file14593650b80f/sample3_realign2transcript.bam, contains 17 reference sequences.
2026-04-20T23:52:28.622287Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-20T23:52:28.622326Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-20T23:52:28.622465Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-20T23:52:28.622486Z  INFO oarfish: parsed reference information for 17 transcripts.
2026-04-20T23:52:28.632045Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-04-20T23:52:28.632220Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 3342  │
│ aligned fraction too low        │ 10    │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 290   │
╰─────────────────────────────────┴───────╯

2026-04-20T23:52:28.632262Z  INFO oarfish::bulk: Total number of alignment records : 517
2026-04-20T23:52:28.632272Z  INFO oarfish::bulk: number of aligned reads : 290
2026-04-20T23:52:28.632280Z  INFO oarfish::bulk: number of unique alignments : 192
2026-04-20T23:52:28.634681Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
i Writing configuration to: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459373c3dc36/config_file_83347.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 @ Mon Apr 20 19:52:28 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459373c3dc36/sample1_align2genome.bam
sample2 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459373c3dc36/sample2_align2genome.bam
sample3 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459373c3dc36/sample3_align2genome.bam
-- Running step: isoform_identification @ Mon Apr 20 19:52:38 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Mon Apr 20 19:52:38 2026 -------------------
Realignment complete for the following samples:
sample1 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459373c3dc36/sample1_realign2transcript.bam
sample2 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459373c3dc36/sample2_realign2transcript.bam
sample3 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459373c3dc36/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Mon Apr 20 19:52:47 2026 ----------
2026-04-20T23:52:47.506783Z  INFO oarfish: setting user-provided filter parameters.
2026-04-20T23:52:47.507038Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459373c3dc36/sample1_realign2transcript.bam, contains 15 reference sequences.
2026-04-20T23:52:47.507076Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-20T23:52:47.507107Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-20T23:52:47.507210Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-20T23:52:47.507232Z  INFO oarfish: parsed reference information for 15 transcripts.
2026-04-20T23:52:47.513623Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-04-20T23:52:47.513750Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 3045  │
│ aligned fraction too low        │ 7     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 293   │
╰─────────────────────────────────┴───────╯

2026-04-20T23:52:47.513775Z  INFO oarfish::bulk: Total number of alignment records : 495
2026-04-20T23:52:47.513780Z  INFO oarfish::bulk: number of aligned reads : 293
2026-04-20T23:52:47.513783Z  INFO oarfish::bulk: number of unique alignments : 208
2026-04-20T23:52:47.515441Z  INFO oarfish: oarfish completed successfully.
2026-04-20T23:52:47.532641Z  INFO oarfish: setting user-provided filter parameters.
2026-04-20T23:52:47.532862Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459373c3dc36/sample2_realign2transcript.bam, contains 15 reference sequences.
2026-04-20T23:52:47.532873Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-20T23:52:47.532886Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-20T23:52:47.532961Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-20T23:52:47.532972Z  INFO oarfish: parsed reference information for 15 transcripts.
2026-04-20T23:52:47.539203Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-04-20T23:52:47.539284Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 2896  │
│ aligned fraction too low        │ 10    │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 290   │
╰─────────────────────────────────┴───────╯

2026-04-20T23:52:47.539312Z  INFO oarfish::bulk: Total number of alignment records : 511
2026-04-20T23:52:47.539317Z  INFO oarfish::bulk: number of aligned reads : 290
2026-04-20T23:52:47.539331Z  INFO oarfish::bulk: number of unique alignments : 198
2026-04-20T23:52:47.540787Z  INFO oarfish: oarfish completed successfully.
2026-04-20T23:52:47.555816Z  INFO oarfish: setting user-provided filter parameters.
2026-04-20T23:52:47.556077Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459373c3dc36/sample3_realign2transcript.bam, contains 15 reference sequences.
2026-04-20T23:52:47.556090Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-20T23:52:47.556101Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-20T23:52:47.556176Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-20T23:52:47.556184Z  INFO oarfish: parsed reference information for 15 transcripts.
2026-04-20T23:52:47.562453Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-04-20T23:52:47.562532Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 2880  │
│ aligned fraction too low        │ 9     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 291   │
╰─────────────────────────────────┴───────╯

2026-04-20T23:52:47.562554Z  INFO oarfish::bulk: Total number of alignment records : 496
2026-04-20T23:52:47.562559Z  INFO oarfish::bulk: number of aligned reads : 291
2026-04-20T23:52:47.562562Z  INFO oarfish::bulk: number of unique alignments : 206
2026-04-20T23:52:47.564496Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
i Writing configuration to: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145937a2681ce/config_file_83347.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 @ Mon Apr 20 19:52:47 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145937a2681ce/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145937a2681ce/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145937a2681ce/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Mon Apr 20 19:52:48 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Mon Apr 20 19:52:48 2026 -------------------
Realigning sample sample1 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145937a2681ce/sample1_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample2 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145937a2681ce/sample2_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample3 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145937a2681ce/sample3_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Mon Apr 20 19:52:49 2026 ----------
19:52:49 Mon Apr 20 2026 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Inputs:  ['/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145937a2681ce/sample3_realign2transcript.bam', '/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145937a2681ce/sample2_realign2transcript.bam', '/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145937a2681ce/sample1_realign2transcript.bam'] /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145937a2681ce/transcript_assembly.fa.fai 5 0.4 0.4
	Counter({'counted_reads': 895, 'not_enough_coverage': 5})
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
i Writing configuration to: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459342cf7880/config_file_83347.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 @ Mon Apr 20 19:52:50 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459342cf7880/sample1_align2genome.bam
sample2 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459342cf7880/sample2_align2genome.bam
sample3 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459342cf7880/sample3_align2genome.bam
-- Running step: isoform_identification @ Mon Apr 20 19:52:58 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Mon Apr 20 19:52:58 2026 -------------------
Realignment complete for the following samples:
sample1 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459342cf7880/sample1_realign2transcript.bam
sample2 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459342cf7880/sample2_realign2transcript.bam
sample3 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459342cf7880/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Mon Apr 20 19:53:07 2026 ----------
19:53:07 Mon Apr 20 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//RtmpdnWSzD/file1459346bbbc98/config_file_83347.json
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Mon Apr 20 19:53:07 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459346bbbc98/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
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 @ Mon Apr 20 19:53:08 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459346bbbc98/matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459346bbbc98/align2genome.bam
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: isoform_identification @ Mon Apr 20 19:53:08 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---
Detected 2 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Mon Apr 20 19:53:11 2026 -------------------
Checking for fastq file(s) /Library/Frameworks/R.framework/Versions/4.6/Resources/library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459346bbbc98/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459346bbbc98/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459346bbbc98/matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459346bbbc98/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 @ Mon Apr 20 19:53:11 2026 ----------
2026-04-20T23:53:11.380350Z  INFO oarfish: setting user-provided filter parameters.
2026-04-20T23:53:11.380689Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459346bbbc98/realign2transcript.bam, contains 5 reference sequences.
2026-04-20T23:53:11.380760Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-20T23:53:11.380807Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-20T23:53:11.380872Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-20T23:53:11.380885Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-04-20T23:53:11.386457Z  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//RtmpdnWSzD/file145937d78f459/config_file_83347.json
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Mon Apr 20 19:53:11 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145937d78f459/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
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 @ Mon Apr 20 19:53:11 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145937d78f459/matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145937d78f459/align2genome.bam
-- Running step: isoform_identification @ Mon Apr 20 19:53:19 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 @ Mon Apr 20 19:53:22 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//RtmpdnWSzD/file145937d78f459/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145937d78f459/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145937d78f459/matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145937d78f459/realign2transcript.bam
-- Running step: transcript_quantification @ Mon Apr 20 19:53:31 2026 ----------
2026-04-20T23:53:31.016720Z  INFO oarfish: setting user-provided filter parameters.
2026-04-20T23:53:31.017078Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145937d78f459/realign2transcript.bam, contains 5 reference sequences.
2026-04-20T23:53:31.017092Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-20T23:53:31.017107Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-20T23:53:31.017156Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-20T23:53:31.017174Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-04-20T23:53:31.021837Z  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//RtmpdnWSzD/file1459313a618d2/config_file_83347.json
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Mon Apr 20 19:53:31 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459313a618d2/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
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 @ Mon Apr 20 19:53:31 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459313a618d2/matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459313a618d2/align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: isoform_identification @ Mon Apr 20 19:53:31 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 @ Mon Apr 20 19:53:34 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//RtmpdnWSzD/file1459313a618d2/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459313a618d2/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459313a618d2/matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459313a618d2/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 @ Mon Apr 20 19:53:34 2026 ----------
19:53:34 Mon Apr 20 2026 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Inputs:  ['/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459342cf7880/sample3_realign2transcript.bam', '/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459342cf7880/sample2_realign2transcript.bam', '/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459342cf7880/sample1_realign2transcript.bam'] /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459342cf7880/transcript_assembly.fa.fai 5 0.4 0.4
	Counter({'counted_reads': 895, 'not_enough_coverage': 5})
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
i Writing configuration to: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145931e01747a/config_file_83347.json
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Mon Apr 20 19:53:35 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145931e01747a/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
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 @ Mon Apr 20 19:53:35 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145931e01747a/matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145931e01747a/align2genome.bam
-- Running step: isoform_identification @ Mon Apr 20 19:53:43 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 @ Mon Apr 20 19:53:46 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//RtmpdnWSzD/file145931e01747a/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145931e01747a/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145931e01747a/matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145931e01747a/realign2transcript.bam
-- Running step: transcript_quantification @ Mon Apr 20 19:53:54 2026 ----------
19:53:54 Mon Apr 20 2026 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
	Counter({'counted_reads': 354, 'not_enough_coverage': 12, 'unmapped': 6})
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
i Writing configuration to: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145934fed838/config_file_83347.json
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Mon Apr 20 19:53:55 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145934fed838/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
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 @ Mon Apr 20 19:53:55 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145934fed838/matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145934fed838/align2genome.bam
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: isoform_identification @ Mon Apr 20 19:53:55 2026 -------------
	Counter({'counted_reads': 354, 'not_enough_coverage': 12, 'unmapped': 6})
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Mon Apr 20 19:53:55 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//RtmpdnWSzD/file145934fed838/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145934fed838/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145934fed838/matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145934fed838/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 @ Mon Apr 20 19:53:55 2026 ----------
2026-04-20T23:53:55.720731Z  INFO oarfish: setting user-provided filter parameters.
2026-04-20T23:53:55.721064Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145934fed838/realign2transcript.bam, contains 10 reference sequences.
2026-04-20T23:53:55.721107Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-20T23:53:55.721148Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-20T23:53:55.721258Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-20T23:53:55.721276Z  INFO oarfish: parsed reference information for 10 transcripts.
2026-04-20T23:53:55.726311Z  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//RtmpdnWSzD/file145935c155d7b/config_file_83347.json
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Mon Apr 20 19:53:55 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145935c155d7b/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
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 @ Mon Apr 20 19:53:56 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145935c155d7b/matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145935c155d7b/align2genome.bam
-- Running step: isoform_identification @ Mon Apr 20 19:54:04 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Mon Apr 20 19:54:04 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//RtmpdnWSzD/file145935c155d7b/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145935c155d7b/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145935c155d7b/matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145935c155d7b/realign2transcript.bam
-- Running step: transcript_quantification @ Mon Apr 20 19:54:12 2026 ----------
2026-04-20T23:54:12.431295Z  INFO oarfish: setting user-provided filter parameters.
2026-04-20T23:54:12.431548Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145935c155d7b/realign2transcript.bam, contains 10 reference sequences.
2026-04-20T23:54:12.431564Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-20T23:54:12.431578Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-20T23:54:12.431649Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-20T23:54:12.431659Z  INFO oarfish: parsed reference information for 10 transcripts.
2026-04-20T23:54:12.441266Z  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//RtmpdnWSzD/file145937d937e49/config_file_83347.json
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Mon Apr 20 19:54:12 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145937d937e49/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
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 @ Mon Apr 20 19:54:12 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145937d937e49/matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145937d937e49/align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: isoform_identification @ Mon Apr 20 19:54:13 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Mon Apr 20 19:54:13 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//RtmpdnWSzD/file145937d937e49/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145937d937e49/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145937d937e49/matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145937d937e49/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 @ Mon Apr 20 19:54:13 2026 ----------
19:54:13 Mon Apr 20 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//RtmpdnWSzD/file1459371557078/config_file_83347.json
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Mon Apr 20 19:54:13 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459371557078/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
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 @ Mon Apr 20 19:54:14 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459371557078/matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459371557078/align2genome.bam
-- Running step: isoform_identification @ Mon Apr 20 19:54:22 2026 -------------
	Counter({'counted_reads': 368, 'unmapped': 4})
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Mon Apr 20 19:54:22 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//RtmpdnWSzD/file1459371557078/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459371557078/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459371557078/matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459371557078/realign2transcript.bam
-- Running step: transcript_quantification @ Mon Apr 20 19:54:30 2026 ----------
19:54:30 Mon Apr 20 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//RtmpdnWSzD/file145934ab81f04/config_file_83347.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 @ Mon Apr 20 19:54:30 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145934ab81f04/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
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145934ab81f04/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145934ab81f04/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145934ab81f04/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//RtmpdnWSzD/file145934ab81f04/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
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145934ab81f04/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//RtmpdnWSzD/file145934ab81f04/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
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145934ab81f04/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//RtmpdnWSzD/file145934ab81f04/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
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145934ab81f04/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 @ Mon Apr 20 19:54:31 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145934ab81f04/sampleA_matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145934ab81f04/sampleA_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145934ab81f04/sample1_matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145934ab81f04/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145934ab81f04/sample2_matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145934ab81f04/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145934ab81f04/sample3_matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145934ab81f04/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Mon Apr 20 19:54:33 2026 ----------------
19:54:33 Mon Apr 20 2026 quantify genes 
Using BAM(s):
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145934ab81f04/sampleA_align2genome.bam',
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145934ab81f04/sample1_align2genome.bam',
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145934ab81f04/sample2_align2genome.bam',
and
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145934ab81f04/sample3_align2genome.bam'
	Counter({'counted_reads': 368, 'unmapped': 4})
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145934ab81f04/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 44.68gene_group/s]
2026-04-20 19:54:33.894 R[83347:275150940] +[NSXPCSharedListener endpointForReply:withListenerName:replyErrorCode:]: an error occurred while attempting to obtain endpoint for listener 'ClientCallsAuxiliary': Connection interrupted
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 393447.15Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145934ab81f04/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 89.29gene_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, 1221262.52Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145934ab81f04/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 73.35gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 883383.32Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145934ab81f04/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 77.93gene_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, 718399.56Read/s]
-- Running step: isoform_identification @ Mon Apr 20 19:54:34 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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  |                                                                            
  |==================                                                    |  25%
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Detected 7 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Mon Apr 20 19:54:42 2026 -------------------
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145934ab81f04/fastq, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145934ab81f04/fastq/sample1.fq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145934ab81f04/fastq/sample2.fq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145934ab81f04/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145934ab81f04/sampleA_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145934ab81f04/sample1_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145934ab81f04/sample2_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145934ab81f04/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145934ab81f04/sampleA_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145934ab81f04/sample1_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145934ab81f04/sample2_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145934ab81f04/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145934ab81f04/sampleA_matched_reads_dedup.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145934ab81f04/sampleA_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145934ab81f04/sample1_matched_reads_dedup.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145934ab81f04/sample1_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145934ab81f04/sample2_matched_reads_dedup.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145934ab81f04/sample2_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145934ab81f04/sample3_matched_reads_dedup.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145934ab81f04/sample3_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

-- Running step: transcript_quantification @ Mon Apr 20 19:54:43 2026 ----------
2026-04-20T23:54:43.245964Z  INFO oarfish: setting user-provided filter parameters.
2026-04-20T23:54:43.246153Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145934ab81f04/sampleA_realign2transcript.bam, contains 6 reference sequences.
2026-04-20T23:54:43.246165Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-20T23:54:43.246178Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-20T23:54:43.246246Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-20T23:54:43.246261Z  INFO oarfish: parsed reference information for 6 transcripts.
2026-04-20T23:54:43.255909Z  INFO oarfish::single_cell: Processed 100 cells.
2026-04-20T23:54:43.363285Z  INFO oarfish: setting user-provided filter parameters.
2026-04-20T23:54:43.363521Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145934ab81f04/sample1_realign2transcript.bam, contains 6 reference sequences.
2026-04-20T23:54:43.363560Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-20T23:54:43.363585Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-20T23:54:43.363644Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-20T23:54:43.363655Z  INFO oarfish: parsed reference information for 6 transcripts.
2026-04-20T23:54:43.367481Z  INFO oarfish::single_cell: Processed 100 cells.
2026-04-20T23:54:43.485712Z  INFO oarfish: setting user-provided filter parameters.
2026-04-20T23:54:43.486012Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145934ab81f04/sample2_realign2transcript.bam, contains 6 reference sequences.
2026-04-20T23:54:43.486104Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-20T23:54:43.486139Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-20T23:54:43.486263Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-20T23:54:43.486297Z  INFO oarfish: parsed reference information for 6 transcripts.
2026-04-20T23:54:43.491068Z  INFO oarfish::single_cell: Processed 100 cells.
2026-04-20T23:54:43.616008Z  INFO oarfish: setting user-provided filter parameters.
2026-04-20T23:54:43.616256Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145934ab81f04/sample3_realign2transcript.bam, contains 6 reference sequences.
2026-04-20T23:54:43.616267Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-20T23:54:43.616280Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-20T23:54:43.616332Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-20T23:54:43.616339Z  INFO oarfish: parsed reference information for 6 transcripts.
2026-04-20T23:54:43.621515Z  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//RtmpdnWSzD/file145936f8dbbac/config_file_83347.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 @ Mon Apr 20 19:54:44 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936f8dbbac/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
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936f8dbbac/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936f8dbbac/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936f8dbbac/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//RtmpdnWSzD/file145936f8dbbac/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
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936f8dbbac/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//RtmpdnWSzD/file145936f8dbbac/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
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936f8dbbac/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//RtmpdnWSzD/file145936f8dbbac/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
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936f8dbbac/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 @ Mon Apr 20 19:54:44 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936f8dbbac/sampleA_matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936f8dbbac/sampleA_align2genome.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936f8dbbac/sample1_matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936f8dbbac/sample1_align2genome.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936f8dbbac/sample2_matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936f8dbbac/sample2_align2genome.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936f8dbbac/sample3_matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936f8dbbac/sample3_align2genome.bam
-- Running step: gene_quantification @ Mon Apr 20 19:54:54 2026 ----------------
19:54:54 Mon Apr 20 2026 quantify genes 
Using BAM(s):
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936f8dbbac/sampleA_align2genome.bam',
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936f8dbbac/sample1_align2genome.bam',
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936f8dbbac/sample2_align2genome.bam',
and
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936f8dbbac/sample3_align2genome.bam'
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936f8dbbac/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 40.46gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 378779.76Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936f8dbbac/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 56.86gene_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, 1302093.63Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936f8dbbac/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 75.90gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1405974.79Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936f8dbbac/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 59.07gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1133350.63Read/s]
-- Running step: isoform_identification @ Mon Apr 20 19:54:54 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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  |                                                                            
  |==================                                                    |  25%
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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 @ Mon Apr 20 19:55:02 2026 -------------------
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936f8dbbac/fastq, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936f8dbbac/fastq/sample1.fq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936f8dbbac/fastq/sample2.fq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936f8dbbac/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936f8dbbac/sampleA_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936f8dbbac/sample1_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936f8dbbac/sample2_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936f8dbbac/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936f8dbbac/sampleA_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936f8dbbac/sample1_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936f8dbbac/sample2_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936f8dbbac/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936f8dbbac/sampleA_matched_reads_dedup.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936f8dbbac/sampleA_realign2transcript.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936f8dbbac/sample1_matched_reads_dedup.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936f8dbbac/sample1_realign2transcript.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936f8dbbac/sample2_matched_reads_dedup.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936f8dbbac/sample2_realign2transcript.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936f8dbbac/sample3_matched_reads_dedup.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936f8dbbac/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Mon Apr 20 19:55:12 2026 ----------
2026-04-20T23:55:12.079383Z  INFO oarfish: setting user-provided filter parameters.
2026-04-20T23:55:12.079666Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936f8dbbac/sampleA_realign2transcript.bam, contains 6 reference sequences.
2026-04-20T23:55:12.079704Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-20T23:55:12.079732Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-20T23:55:12.079844Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-20T23:55:12.079854Z  INFO oarfish: parsed reference information for 6 transcripts.
2026-04-20T23:55:12.088553Z  INFO oarfish::single_cell: Processed 100 cells.
2026-04-20T23:55:12.225483Z  INFO oarfish: setting user-provided filter parameters.
2026-04-20T23:55:12.225743Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936f8dbbac/sample1_realign2transcript.bam, contains 6 reference sequences.
2026-04-20T23:55:12.225774Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-20T23:55:12.225791Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-20T23:55:12.225855Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-20T23:55:12.225864Z  INFO oarfish: parsed reference information for 6 transcripts.
2026-04-20T23:55:12.228048Z  INFO oarfish::single_cell: Processed 100 cells.
2026-04-20T23:55:12.352829Z  INFO oarfish: setting user-provided filter parameters.
2026-04-20T23:55:12.353088Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936f8dbbac/sample2_realign2transcript.bam, contains 6 reference sequences.
2026-04-20T23:55:12.353103Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-20T23:55:12.353117Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-20T23:55:12.353169Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-20T23:55:12.353182Z  INFO oarfish: parsed reference information for 6 transcripts.
2026-04-20T23:55:12.357119Z  INFO oarfish::single_cell: Processed 100 cells.
2026-04-20T23:55:12.479085Z  INFO oarfish: setting user-provided filter parameters.
2026-04-20T23:55:12.479309Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936f8dbbac/sample3_realign2transcript.bam, contains 6 reference sequences.
2026-04-20T23:55:12.479322Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-20T23:55:12.479340Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-20T23:55:12.479386Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-20T23:55:12.479395Z  INFO oarfish: parsed reference information for 6 transcripts.
2026-04-20T23:55:12.483143Z  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//RtmpdnWSzD/file1459316963692/config_file_83347.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 @ Mon Apr 20 19:55:12 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459316963692/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
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459316963692/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459316963692/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459316963692/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//RtmpdnWSzD/file1459316963692/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
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459316963692/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//RtmpdnWSzD/file1459316963692/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
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459316963692/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//RtmpdnWSzD/file1459316963692/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
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459316963692/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 @ Mon Apr 20 19:55:13 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459316963692/sampleA_matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459316963692/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//RtmpdnWSzD/file1459316963692/sample1_matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459316963692/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//RtmpdnWSzD/file1459316963692/sample2_matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459316963692/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//RtmpdnWSzD/file1459316963692/sample3_matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459316963692/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 @ Mon Apr 20 19:55:15 2026 ----------------
19:55:15 Mon Apr 20 2026 quantify genes 
Using BAM(s):
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459316963692/sampleA_align2genome.bam',
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459316963692/sample1_align2genome.bam',
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459316963692/sample2_align2genome.bam',
and
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459316963692/sample3_align2genome.bam'
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459316963692/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 48.34gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 418125.85Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459316963692/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 95.24gene_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, 1393456.48Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459316963692/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 92.48gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1376806.72Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459316963692/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 38.60gene_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, 1007277.62Read/s]
-- Running step: isoform_identification @ Mon Apr 20 19:55:15 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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  |                                                                            
  |==================                                                    |  25%
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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 @ Mon Apr 20 19:55:22 2026 -------------------
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459316963692/fastq, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459316963692/fastq/sample1.fq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459316963692/fastq/sample2.fq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459316963692/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459316963692/sampleA_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459316963692/sample1_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459316963692/sample2_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459316963692/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459316963692/sampleA_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459316963692/sample1_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459316963692/sample2_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459316963692/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459316963692/sampleA_matched_reads_dedup.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459316963692/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//RtmpdnWSzD/file1459316963692/sample1_matched_reads_dedup.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459316963692/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//RtmpdnWSzD/file1459316963692/sample2_matched_reads_dedup.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459316963692/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//RtmpdnWSzD/file1459316963692/sample3_matched_reads_dedup.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459316963692/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 @ Mon Apr 20 19:55:23 2026 ----------
19:55:23 Mon Apr 20 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//RtmpdnWSzD/file1459316963692/sample3_realign2transcript.bam...
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459316963692/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459316963692/sample3_realign2transcript.bamdone
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459316963692/sampleA_realign2transcript.bam...
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459316963692/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459316963692/sampleA_realign2transcript.bamdone
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459316963692/sample2_realign2transcript.bam...
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459316963692/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459316963692/sample2_realign2transcript.bamdone
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459316963692/sample1_realign2transcript.bam...
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459316963692/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459316963692/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//RtmpdnWSzD/file1459341357860/config_file_83347.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 @ Mon Apr 20 19:55:24 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459341357860/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
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459341357860/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459341357860/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459341357860/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//RtmpdnWSzD/file1459341357860/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
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459341357860/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//RtmpdnWSzD/file1459341357860/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
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459341357860/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//RtmpdnWSzD/file1459341357860/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
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459341357860/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 @ Mon Apr 20 19:55:25 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459341357860/sampleA_matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459341357860/sampleA_align2genome.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459341357860/sample1_matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459341357860/sample1_align2genome.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459341357860/sample2_matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459341357860/sample2_align2genome.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459341357860/sample3_matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459341357860/sample3_align2genome.bam
-- Running step: gene_quantification @ Mon Apr 20 19:55:34 2026 ----------------
19:55:34 Mon Apr 20 2026 quantify genes 
Using BAM(s):
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459341357860/sampleA_align2genome.bam',
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459341357860/sample1_align2genome.bam',
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459341357860/sample2_align2genome.bam',
and
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459341357860/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//RtmpdnWSzD/file1459341357860/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 48.22gene_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, 411722.95Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459341357860/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 88.32gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1245635.54Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459341357860/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 89.13gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1336276.28Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459341357860/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 69.48gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1064220.03Read/s]
-- Running step: isoform_identification @ Mon Apr 20 19:55:35 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

  |                                                                            
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  |                                                                            
  |==================                                                    |  25%
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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 @ Mon Apr 20 19:55:42 2026 -------------------
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459341357860/fastq, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459341357860/fastq/sample1.fq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459341357860/fastq/sample2.fq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459341357860/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459341357860/sampleA_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459341357860/sample1_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459341357860/sample2_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459341357860/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459341357860/sampleA_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459341357860/sample1_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459341357860/sample2_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459341357860/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459341357860/sampleA_matched_reads_dedup.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459341357860/sampleA_realign2transcript.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459341357860/sample1_matched_reads_dedup.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459341357860/sample1_realign2transcript.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459341357860/sample2_matched_reads_dedup.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459341357860/sample2_realign2transcript.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459341357860/sample3_matched_reads_dedup.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459341357860/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Mon Apr 20 19:55:51 2026 ----------
19:55:51 Mon Apr 20 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//RtmpdnWSzD/file1459341357860/sample3_realign2transcript.bam...
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459341357860/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459341357860/sample3_realign2transcript.bamdone
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459341357860/sampleA_realign2transcript.bam...
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459341357860/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459341357860/sampleA_realign2transcript.bamdone
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459341357860/sample2_realign2transcript.bam...
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459341357860/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459341357860/sample2_realign2transcript.bamdone
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459341357860/sample1_realign2transcript.bam...
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459341357860/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459341357860/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//RtmpdnWSzD/file145937413b0a2/config_file_83347.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 @ Mon Apr 20 19:55:51 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145937413b0a2/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
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145937413b0a2/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145937413b0a2/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145937413b0a2/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//RtmpdnWSzD/file145937413b0a2/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
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145937413b0a2/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//RtmpdnWSzD/file145937413b0a2/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
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145937413b0a2/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//RtmpdnWSzD/file145937413b0a2/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
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145937413b0a2/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 @ Mon Apr 20 19:55:52 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145937413b0a2/sampleA_matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145937413b0a2/sampleA_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145937413b0a2/sample1_matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145937413b0a2/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145937413b0a2/sample2_matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145937413b0a2/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145937413b0a2/sample3_matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145937413b0a2/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Mon Apr 20 19:55:54 2026 ----------------
19:55:54 Mon Apr 20 2026 quantify genes 
Using BAM(s):
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145937413b0a2/sampleA_align2genome.bam',
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145937413b0a2/sample1_align2genome.bam',
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145937413b0a2/sample2_align2genome.bam',
and
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145937413b0a2/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//RtmpdnWSzD/file145937413b0a2/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 46.76gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 402169.29Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145937413b0a2/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 84.15gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1233328.63Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145937413b0a2/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 83.79gene_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, 1378073.33Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145937413b0a2/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 74.22gene_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, 978514.37Read/s]
-- Running step: isoform_identification @ Mon Apr 20 19:55:54 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Mon Apr 20 19:55:54 2026 -------------------
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145937413b0a2/fastq, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145937413b0a2/fastq/sample1.fq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145937413b0a2/fastq/sample2.fq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145937413b0a2/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145937413b0a2/sampleA_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145937413b0a2/sample1_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145937413b0a2/sample2_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145937413b0a2/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145937413b0a2/sampleA_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145937413b0a2/sample1_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145937413b0a2/sample2_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145937413b0a2/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145937413b0a2/sampleA_matched_reads_dedup.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145937413b0a2/sampleA_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145937413b0a2/sample1_matched_reads_dedup.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145937413b0a2/sample1_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145937413b0a2/sample2_matched_reads_dedup.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145937413b0a2/sample2_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145937413b0a2/sample3_matched_reads_dedup.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145937413b0a2/sample3_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

-- Running step: transcript_quantification @ Mon Apr 20 19:55:58 2026 ----------
2026-04-20T23:55:58.254991Z  INFO oarfish: setting user-provided filter parameters.
2026-04-20T23:55:58.255243Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145937413b0a2/sampleA_realign2transcript.bam, contains 27 reference sequences.
2026-04-20T23:55:58.255256Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-20T23:55:58.255271Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-20T23:55:58.255346Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-20T23:55:58.255357Z  INFO oarfish: parsed reference information for 27 transcripts.
2026-04-20T23:55:58.276451Z  INFO oarfish::single_cell: Processed 100 cells.
2026-04-20T23:55:58.461096Z  INFO oarfish: setting user-provided filter parameters.
2026-04-20T23:55:58.461355Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145937413b0a2/sample1_realign2transcript.bam, contains 27 reference sequences.
2026-04-20T23:55:58.461386Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-20T23:55:58.461402Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-20T23:55:58.461492Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-20T23:55:58.461508Z  INFO oarfish: parsed reference information for 27 transcripts.
2026-04-20T23:55:58.466779Z  INFO oarfish::single_cell: Processed 100 cells.
2026-04-20T23:55:58.668059Z  INFO oarfish: setting user-provided filter parameters.
2026-04-20T23:55:58.668327Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145937413b0a2/sample2_realign2transcript.bam, contains 27 reference sequences.
2026-04-20T23:55:58.668345Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-20T23:55:58.668361Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-20T23:55:58.668442Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-20T23:55:58.668455Z  INFO oarfish: parsed reference information for 27 transcripts.
2026-04-20T23:55:58.677087Z  INFO oarfish::single_cell: Processed 100 cells.
2026-04-20T23:55:58.862869Z  INFO oarfish: setting user-provided filter parameters.
2026-04-20T23:55:58.863136Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145937413b0a2/sample3_realign2transcript.bam, contains 27 reference sequences.
2026-04-20T23:55:58.863153Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-20T23:55:58.863169Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-20T23:55:58.863247Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-20T23:55:58.863258Z  INFO oarfish: parsed reference information for 27 transcripts.
2026-04-20T23:55:58.873711Z  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//RtmpdnWSzD/file1459345e9170f/config_file_83347.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 @ Mon Apr 20 19:55:59 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459345e9170f/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
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459345e9170f/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459345e9170f/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459345e9170f/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//RtmpdnWSzD/file1459345e9170f/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
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459345e9170f/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//RtmpdnWSzD/file1459345e9170f/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
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459345e9170f/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//RtmpdnWSzD/file1459345e9170f/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
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459345e9170f/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 @ Mon Apr 20 19:56:00 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459345e9170f/sampleA_matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459345e9170f/sampleA_align2genome.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459345e9170f/sample1_matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459345e9170f/sample1_align2genome.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459345e9170f/sample2_matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459345e9170f/sample2_align2genome.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459345e9170f/sample3_matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459345e9170f/sample3_align2genome.bam
-- Running step: gene_quantification @ Mon Apr 20 19:56:09 2026 ----------------
19:56:09 Mon Apr 20 2026 quantify genes 
Using BAM(s):
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459345e9170f/sampleA_align2genome.bam',
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459345e9170f/sample1_align2genome.bam',
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459345e9170f/sample2_align2genome.bam',
and
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459345e9170f/sample3_align2genome.bam'
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459345e9170f/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 45.68gene_group/s]
/Library/Frameworks/R.framework/Versions/4.6/Resources/library/FLAMES/python/count_gene.py:702: 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, 420372.03Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459345e9170f/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 80.80gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1245635.54Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459345e9170f/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 87.66gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1418720.06Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459345e9170f/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 74.68gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1040770.22Read/s]
-- Running step: isoform_identification @ Mon Apr 20 19:56:09 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Mon Apr 20 19:56:10 2026 -------------------
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459345e9170f/fastq, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459345e9170f/fastq/sample1.fq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459345e9170f/fastq/sample2.fq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459345e9170f/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459345e9170f/sampleA_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459345e9170f/sample1_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459345e9170f/sample2_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459345e9170f/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459345e9170f/sampleA_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459345e9170f/sample1_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459345e9170f/sample2_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459345e9170f/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459345e9170f/sampleA_matched_reads_dedup.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459345e9170f/sampleA_realign2transcript.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459345e9170f/sample1_matched_reads_dedup.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459345e9170f/sample1_realign2transcript.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459345e9170f/sample2_matched_reads_dedup.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459345e9170f/sample2_realign2transcript.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459345e9170f/sample3_matched_reads_dedup.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459345e9170f/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Mon Apr 20 19:56:21 2026 ----------
2026-04-20T23:56:21.748668Z  INFO oarfish: setting user-provided filter parameters.
2026-04-20T23:56:21.748993Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459345e9170f/sampleA_realign2transcript.bam, contains 28 reference sequences.
2026-04-20T23:56:21.749017Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-20T23:56:21.749037Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-20T23:56:21.749126Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-20T23:56:21.749145Z  INFO oarfish: parsed reference information for 28 transcripts.
2026-04-20T23:56:21.771602Z  INFO oarfish::single_cell: Processed 100 cells.
2026-04-20T23:56:22.032020Z  INFO oarfish: setting user-provided filter parameters.
2026-04-20T23:56:22.032487Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459345e9170f/sample1_realign2transcript.bam, contains 28 reference sequences.
2026-04-20T23:56:22.032504Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-20T23:56:22.032519Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-20T23:56:22.032607Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-20T23:56:22.032618Z  INFO oarfish: parsed reference information for 28 transcripts.
2026-04-20T23:56:22.044026Z  INFO oarfish::single_cell: Processed 100 cells.
2026-04-20T23:56:22.313561Z  INFO oarfish: setting user-provided filter parameters.
2026-04-20T23:56:22.313931Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459345e9170f/sample2_realign2transcript.bam, contains 28 reference sequences.
2026-04-20T23:56:22.313950Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-20T23:56:22.313969Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-20T23:56:22.314090Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-20T23:56:22.314177Z  INFO oarfish: parsed reference information for 28 transcripts.
2026-04-20T23:56:22.324899Z  INFO oarfish::single_cell: Processed 100 cells.
2026-04-20T23:56:22.552984Z  INFO oarfish: setting user-provided filter parameters.
2026-04-20T23:56:22.553318Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459345e9170f/sample3_realign2transcript.bam, contains 28 reference sequences.
2026-04-20T23:56:22.553419Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-20T23:56:22.553479Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-20T23:56:22.553675Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-20T23:56:22.553704Z  INFO oarfish: parsed reference information for 28 transcripts.
2026-04-20T23:56:22.570307Z  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//RtmpdnWSzD/file145936afb67b/config_file_83347.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 @ Mon Apr 20 19:56:23 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936afb67b/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
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936afb67b/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936afb67b/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936afb67b/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//RtmpdnWSzD/file145936afb67b/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
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936afb67b/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//RtmpdnWSzD/file145936afb67b/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
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936afb67b/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//RtmpdnWSzD/file145936afb67b/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
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936afb67b/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 @ Mon Apr 20 19:56:23 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936afb67b/sampleA_matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936afb67b/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//RtmpdnWSzD/file145936afb67b/sample1_matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936afb67b/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//RtmpdnWSzD/file145936afb67b/sample2_matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936afb67b/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//RtmpdnWSzD/file145936afb67b/sample3_matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936afb67b/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 @ Mon Apr 20 19:56:25 2026 ----------------
19:56:25 Mon Apr 20 2026 quantify genes 
Using BAM(s):
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936afb67b/sampleA_align2genome.bam',
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936afb67b/sample1_align2genome.bam',
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936afb67b/sample2_align2genome.bam',
and
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936afb67b/sample3_align2genome.bam'
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936afb67b/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 48.12gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 432812.98Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936afb67b/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 87.94gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1370687.58Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936afb67b/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 89.05gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1327143.40Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936afb67b/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 66.82gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 694881.38Read/s]
-- Running step: isoform_identification @ Mon Apr 20 19:56:25 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Mon Apr 20 19:56:26 2026 -------------------
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936afb67b/fastq, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936afb67b/fastq/sample1.fq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936afb67b/fastq/sample2.fq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936afb67b/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936afb67b/sampleA_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936afb67b/sample1_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936afb67b/sample2_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936afb67b/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936afb67b/sampleA_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936afb67b/sample1_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936afb67b/sample2_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936afb67b/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936afb67b/sampleA_matched_reads_dedup.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936afb67b/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//RtmpdnWSzD/file145936afb67b/sample1_matched_reads_dedup.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936afb67b/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//RtmpdnWSzD/file145936afb67b/sample2_matched_reads_dedup.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936afb67b/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//RtmpdnWSzD/file145936afb67b/sample3_matched_reads_dedup.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936afb67b/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 @ Mon Apr 20 19:56:27 2026 ----------
19:56:27 Mon Apr 20 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//RtmpdnWSzD/file145936afb67b/sample3_realign2transcript.bam...
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936afb67b/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936afb67b/sample3_realign2transcript.bamdone
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936afb67b/sampleA_realign2transcript.bam...
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936afb67b/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936afb67b/sampleA_realign2transcript.bamdone
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936afb67b/sample2_realign2transcript.bam...
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936afb67b/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936afb67b/sample2_realign2transcript.bamdone
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936afb67b/sample1_realign2transcript.bam...
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936afb67b/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file145936afb67b/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//RtmpdnWSzD/file1459368e3ed0d/config_file_83347.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 @ Mon Apr 20 19:56:28 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459368e3ed0d/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
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459368e3ed0d/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459368e3ed0d/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459368e3ed0d/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//RtmpdnWSzD/file1459368e3ed0d/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
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459368e3ed0d/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//RtmpdnWSzD/file1459368e3ed0d/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
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459368e3ed0d/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//RtmpdnWSzD/file1459368e3ed0d/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
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459368e3ed0d/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 @ Mon Apr 20 19:56:29 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459368e3ed0d/sampleA_matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459368e3ed0d/sampleA_align2genome.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459368e3ed0d/sample1_matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459368e3ed0d/sample1_align2genome.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459368e3ed0d/sample2_matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459368e3ed0d/sample2_align2genome.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459368e3ed0d/sample3_matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459368e3ed0d/sample3_align2genome.bam
-- Running step: gene_quantification @ Mon Apr 20 19:56:39 2026 ----------------
19:56:39 Mon Apr 20 2026 quantify genes 
Using BAM(s):
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459368e3ed0d/sampleA_align2genome.bam',
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459368e3ed0d/sample1_align2genome.bam',
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459368e3ed0d/sample2_align2genome.bam',
and
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459368e3ed0d/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//RtmpdnWSzD/file1459368e3ed0d/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 40.23gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 393270.07Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459368e3ed0d/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 84.50gene_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, 1319959.72Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459368e3ed0d/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 93.63gene_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, 1288651.84Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459368e3ed0d/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 68.22gene_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, 1031352.41Read/s]
-- Running step: isoform_identification @ Mon Apr 20 19:56:39 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Mon Apr 20 19:56:40 2026 -------------------
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459368e3ed0d/fastq, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459368e3ed0d/fastq/sample1.fq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459368e3ed0d/fastq/sample2.fq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459368e3ed0d/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459368e3ed0d/sampleA_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459368e3ed0d/sample1_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459368e3ed0d/sample2_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459368e3ed0d/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459368e3ed0d/sampleA_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459368e3ed0d/sample1_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459368e3ed0d/sample2_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459368e3ed0d/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459368e3ed0d/sampleA_matched_reads_dedup.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459368e3ed0d/sampleA_realign2transcript.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459368e3ed0d/sample1_matched_reads_dedup.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459368e3ed0d/sample1_realign2transcript.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459368e3ed0d/sample2_matched_reads_dedup.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459368e3ed0d/sample2_realign2transcript.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459368e3ed0d/sample3_matched_reads_dedup.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459368e3ed0d/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Mon Apr 20 19:56:49 2026 ----------
19:56:49 Mon Apr 20 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//RtmpdnWSzD/file1459368e3ed0d/sample3_realign2transcript.bam...
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459368e3ed0d/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459368e3ed0d/sample3_realign2transcript.bamdone
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459368e3ed0d/sampleA_realign2transcript.bam...
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459368e3ed0d/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459368e3ed0d/sampleA_realign2transcript.bamdone
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459368e3ed0d/sample2_realign2transcript.bam...
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459368e3ed0d/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459368e3ed0d/sample2_realign2transcript.bamdone
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459368e3ed0d/sample1_realign2transcript.bam...
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459368e3ed0d/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//RtmpdnWSzD/file1459368e3ed0d/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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  |                                                                      |   0%
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  |=======================                                               |  33%
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  |===============================================                       |  67%
  |                                                                            
  |======================================================================| 100%

[ FAIL 0 | WARN 194 | SKIP 0 | PASS 98 ]

[ FAIL 0 | WARN 194 | 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 
321.087  21.271 348.535 
/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 '