##############################################################################
##############################################################################
###
### 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_2.5.2.tar.gz
###
##############################################################################
##############################################################################


* using log directory ‘/Users/biocbuild/bbs-3.23-bioc/meat/FLAMES.Rcheck’
* using R Under development (unstable) (2026-03-20 r89666)
* 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: 2026-03-24 00:04:18 UTC
* using option ‘--no-vignettes’
* checking for file ‘FLAMES/DESCRIPTION’ ... OK
* this is package ‘FLAMES’ version ‘2.5.2’
* package encoding: UTF-8
* checking package namespace information ... OK
* checking package dependencies ... INFO
Imports includes 47 non-default packages.
Importing from so many packages makes the package vulnerable to any of
them becoming unavailable.  Move as many as possible to Suggests and
use conditionally.
* checking if this is a source package ... OK
* checking if there is a namespace ... OK
* checking for hidden files and directories ... NOTE
Found the following hidden files and directories:
  .dockerignore
These were most likely included in error. See section ‘Package
structure’ in the ‘Writing R Extensions’ manual.
* checking for portable file names ... OK
* checking for sufficient/correct file permissions ... OK
* checking whether package ‘FLAMES’ can be installed ... NOTE
Found the following notes/warnings:
  Non-staged installation was used
See ‘/Users/biocbuild/bbs-3.23-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 10.9Mb
  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
There are ::: calls to the package's namespace in its code. A package
  almost never needs to use ::: for its own objects:
  'blaze' 'find_barcode' 'gene_quantification' 'isoform_identification'
  'minimap2_align' 'transcript_quantification'
* checking S3 generic/method consistency ... OK
* checking replacement functions ... OK
* checking foreign function calls ... OK
* checking R code for possible problems ... NOTE
BulkPipeline: no visible global function definition for ‘new’
BulkPipeline: no visible global function definition for ‘setNames’
MultiSampleSCPipeline: no visible global function definition for ‘new’
MultiSampleSCPipeline: no visible global function definition for
  ‘setNames’
SingleCellPipeline: no visible global function definition for ‘new’
SingleCellPipeline: no visible global function definition for
  ‘setNames’
addRowRanges: no visible global function definition for ‘head’
addRowRanges: no visible global function definition for ‘as’
add_gene_counts: no visible global function definition for ‘as’
cache_dir: no visible global function definition for ‘packageVersion’
chisq_test_by_gene: no visible global function definition for
  ‘chisq.test’
create_sce_from_dir: no visible global function definition for
  ‘setNames’
create_spe: no visible binding for global variable ‘barcode’
create_spe: no visible binding for global variable ‘in_tissue’
download_oarfish: no visible global function definition for
  ‘download.file’
download_oarfish: no visible global function definition for ‘unzip’
filter_coverage: no visible global function definition for
  ‘starts_with’
filter_coverage: no visible binding for global variable ‘filter_res’
find_variants: no visible global function definition for ‘setNames’
find_variants_grange: no visible binding for global variable
  ‘which_label’
find_variants_grange: no visible binding for global variable
  ‘nucleotide’
find_variants_grange: no visible binding for global variable ‘pos’
find_variants_grange: no visible binding for global variable ‘count’
find_variants_grange: no visible binding for global variable
  ‘counts_no_ins’
find_variants_grange: no visible binding for global variable ‘ref’
generate_sc_sce: no visible binding for global variable ‘FSM_match’
get_coverage: no visible binding for global variable ‘Freq’
homopolymer_pct : <anonymous>: no visible binding for global variable
  ‘Freq’
homopolymer_pct : <anonymous>: no visible binding for global variable
  ‘pct’
plot_coverage: no visible binding for global variable ‘tr_length’
plot_coverage: no visible binding for global variable ‘read_counts’
plot_coverage: no visible binding for global variable ‘total_counts’
plot_coverage: no visible binding for global variable ‘cumpct’
plot_coverage: no visible binding for global variable ‘length_bin’
plot_coverage: no visible binding for global variable ‘min_length’
plot_coverage: no visible binding for global variable ‘max_length’
plot_coverage: no visible global function definition for ‘head’
plot_coverage: no visible binding for global variable ‘transcript’
plot_demultiplex_raw: no visible binding for global variable ‘Sample’
plot_demultiplex_raw: no visible binding for global variable
  ‘CellBarcode’
plot_demultiplex_raw: no visible binding for global variable ‘UMI’
plot_demultiplex_raw: no visible binding for global variable
  ‘UMI_count’
plot_demultiplex_raw: no visible binding for global variable
  ‘barcode_rank’
plot_demultiplex_raw: no visible binding for global variable
  ‘FlankEditDist’
plot_demultiplex_raw: no visible binding for global variable ‘n_reads’
plot_demultiplex_raw: no visible binding for global variable
  ‘BarcodeEditDist’
plot_demultiplex_raw: no visible binding for global variable ‘total
  reads’
plot_demultiplex_raw: no visible binding for global variable
  ‘demultiplexed reads’
plot_demultiplex_raw: no visible binding for global variable ‘single
  match reads’
plot_demultiplex_raw: no visible binding for global variable
  ‘undemultiplexted reads’
plot_demultiplex_raw: no visible binding for global variable
  ‘multi-matching reads’
plot_demultiplex_raw: no visible binding for global variable ‘Type’
plot_demultiplex_raw: no visible binding for global variable ‘Reads’
plot_demultiplex_raw: no visible binding for global variable ‘input’
plot_demultiplex_raw: no visible binding for global variable ‘output’
plot_demultiplex_raw: no visible binding for global variable
  ‘read1_with_adapter’
plot_demultiplex_raw: no visible binding for global variable ‘Count’
plot_flagstat: no visible global function definition for ‘everything’
plot_flagstat: no visible binding for global variable ‘name’
plot_flagstat: no visible binding for global variable ‘value’
plot_isoform_reduced_dim: no visible binding for global variable ‘x’
plot_isoform_reduced_dim: no visible binding for global variable ‘y’
plot_isoform_reduced_dim: no visible binding for global variable ‘expr’
plot_spatial: no visible binding for global variable ‘imageX’
plot_spatial: no visible binding for global variable ‘imageY’
plot_spatial_feature: no visible binding for global variable ‘imageX’
plot_spatial_feature: no visible binding for global variable ‘imageY’
plot_spatial_feature: no visible binding for global variable ‘x’
plot_spatial_feature: no visible binding for global variable ‘y’
plot_spatial_feature: no visible global function definition for
  ‘scale_alpha_continuous’
plot_spatial_feature: no visible global function definition for
  ‘scale_colour_gradient’
plot_spatial_isoform: no visible global function definition for ‘head’
plot_spatial_pie: no visible global function definition for ‘setNames’
plot_spatial_pie: no visible global function definition for ‘head’
plot_spatial_pie: no visible binding for global variable ‘imageX’
plot_spatial_pie: no visible binding for global variable ‘imageY’
sc_gene_entropy: no visible global function definition for ‘as’
sc_genotype: no visible binding for global variable ‘allele’
sc_genotype: no visible binding for global variable ‘allele_count’
sc_genotype: no visible binding for global variable ‘barcode’
sc_genotype: no visible binding for global variable ‘pct’
sc_mutations: no visible binding for global variable ‘mutation_index’
sc_mutations: no visible binding for global variable ‘bam_index’
sc_plot_genotype: no visible global function definition for ‘setNames’
sc_plot_genotype: no visible binding for global variable ‘barcode’
sc_plot_genotype: no visible binding for global variable ‘genotype’
sc_plot_genotype: no visible binding for global variable ‘x’
sc_plot_genotype: no visible binding for global variable ‘y’
sc_transcript_usage_chisq: no visible global function definition for
  ‘as’
sc_transcript_usage_chisq: no visible binding for global variable
  ‘p.value’
sc_transcript_usage_chisq: no visible binding for global variable
  ‘adj.p.value’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘total’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘test’
sc_transcript_usage_permutation: no visible global function definition
  for ‘as’
sc_transcript_usage_permutation : <anonymous>: no visible global
  function definition for ‘as’
sc_transcript_usage_permutation : <anonymous> : <anonymous>: no visible
  global function definition for ‘na.omit’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘transcript’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘p.value’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘adj.p.value’
variant_count_tb: no visible binding for global variable ‘barcode’
variant_count_tb: no visible binding for global variable ‘allele_count’
variant_count_tb: no visible binding for global variable
  ‘cell_total_reads’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘expect_cell_number’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘fastq’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘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_file’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible global
  function definition for ‘setNames’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘j’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘genome_bam’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘minimap2’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘samtools’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘threads’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘outdir’
plot_durations,FLAMES.Pipeline: no visible binding for global variable
  ‘step’
plot_durations,FLAMES.Pipeline: no visible binding for global variable
  ‘duration’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘j’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘transcriptome_assembly’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘transcriptome_bam’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘minimap2’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘samtools’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘outdir’
resume_FLAMES,FLAMES.Pipeline : <anonymous>: no visible global function
  definition for ‘capture.output’
run_FLAMES,FLAMES.Pipeline : <anonymous>: no visible global function
  definition for ‘capture.output’
Undefined global functions or variables:
  BarcodeEditDist CellBarcode Count FSM_match FlankEditDist Freq Reads
  Sample Type UMI UMI_count adj.p.value allele allele_count as
  bam_index barcode barcode_rank barcodes_file capture.output
  cell_total_reads chisq.test count counts_no_ins cumpct demultiplexed
  reads demultiplexed_fastq download.file duration everything
  expect_cell_number expr fastq filter_res genome_bam genotype head
  imageX imageY in_tissue input j length_bin max_length min_length
  minimap2 multi-matching reads mutation_index n_reads na.omit name new
  nucleotide outdir output p.value packageVersion pct pos
  read1_with_adapter read_counts ref samtools scale_alpha_continuous
  scale_colour_gradient setNames single match reads starts_with step
  test threads total total reads total_counts tr_length transcript
  transcriptome_assembly transcriptome_bam undemultiplexted reads unzip
  value which_label x y
Consider adding
  importFrom("base", "match", "single")
  importFrom("methods", "as", "new")
  importFrom("stats", "chisq.test", "na.omit", "setNames", "step")
  importFrom("utils", "capture.output", "download.file", "head",
             "packageVersion", "unzip")
to your NAMESPACE file (and ensure that your DESCRIPTION Imports field
contains 'methods').
* checking Rd files ... OK
* checking Rd metadata ... OK
* checking Rd cross-references ... OK
* checking for missing documentation entries ... OK
* checking for code/documentation mismatches ... OK
* checking Rd \usage sections ... OK
* checking Rd contents ... OK
* checking for unstated dependencies in examples ... OK
* checking contents of ‘data’ directory ... OK
* checking data for non-ASCII characters ... OK
* checking data for ASCII and uncompressed saves ... OK
* checking line endings in shell scripts ... OK
* checking line endings in C/C++/Fortran sources/headers ... OK
* checking line endings in Makefiles ... OK
* checking compilation flags in Makevars ... OK
* checking for GNU extensions in Makefiles ... INFO
GNU make is a SystemRequirements.
* checking for portable use of $(BLAS_LIBS) and $(LAPACK_LIBS) ... OK
* checking use of PKG_*FLAGS in Makefiles ... OK
* checking include directives in Makefiles ... OK
* checking compiled code ... NOTE
Note: information on .o files is not available
File ‘/Library/Frameworks/R.framework/Versions/4.6/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     10.722  0.189  11.387
sc_long_multisample_pipeline  8.888  1.521   7.065
find_variants                 8.064  0.250   8.248
MultiSampleSCPipeline         6.517  0.978   8.693
sc_plot_genotype              5.783  0.208   5.339
sc_DTU_analysis               4.671  0.753   4.254
* checking for unstated dependencies in ‘tests’ ... OK
* checking tests ...
  Running ‘testthat.R’
 OK
* checking for unstated dependencies in vignettes ... OK
* checking package vignettes ... OK
* checking running R code from vignettes ... SKIPPED
* checking re-building of vignette outputs ... SKIPPED
* checking PDF version of manual ... OK
* DONE

Status: 5 NOTEs
See
  ‘/Users/biocbuild/bbs-3.23-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 ‘2.5.2’
** 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 -std=gnu23 -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 278 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 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 v0.9.2 (available: v0.10.0)
      Adding tabled v0.18.0 (available: v0.20.0)
      Adding typed-builder v0.21.2 (available: v0.23.2)
      Adding wasip2 v1.0.2+wasi-0.2.9 (requires Rust 1.87.0)
      Adding wasip3 v0.4.0+wasi-0.3.0-rc-2026-01-06 (requires Rust 1.87.0)
      Adding wit-bindgen v0.51.0 (requires Rust 1.87.0)
      Adding wit-bindgen-core v0.51.0 (requires Rust 1.87.0)
      Adding wit-bindgen-rust v0.51.0 (requires Rust 1.87.0)
   Compiling libc v0.2.183
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   Compiling statrs v0.18.0
   Compiling oarfish v0.8.0
    Finished `release` profile [optimized] target(s) in 32.26s
  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 Under development (unstable) (2026-03-20 r89666) -- "Unsuffered Consequences"
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")
Writing configuration parameters to:  /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc13a69bd79/config_file_69569.json 
Writing configuration parameters to:  /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc13a69bd79/config_file_69569.json 
Writing configuration parameters to:  /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc13a69bd79/config_file_69569.json 
Writing configuration parameters to:  /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc11849a6a7/config_file_69569.json 
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc179589690/bc_allow.tsv
Number of known barcodes: 143
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 reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
Skipping TSO trimming...
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc14e2e0f62/bc_allow.tsv
Number of known barcodes: 143
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 reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
Skipping TSO trimming...
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc14e2e0f62/bc_allow.tsv
Number of known barcodes: 143
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1f0d4a30/musc_rps24_1.fastq
Searching for barcodes...
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1f0d4a30/musc_rps24_2.fastq
Searching for barcodes...
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1f0d4a30/musc_rps24_3.fastq
Searching for barcodes...
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1f0d4a30/musc_rps24_4.fastq
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
Skipping TSO trimming...
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc134f7c5c3/bc_allow.tsv
Number of known barcodes: 143
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 reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
Skipping TSO trimming...
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1649eb0d0/config_file_69569.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 Mar 23 20:08:38 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1649eb0d0/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1649eb0d0/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1649eb0d0/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Mon Mar 23 20:08:39 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 4 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Mon Mar 23 20:08:49 2026 -------------------
Realigning sample sample1 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1649eb0d0/sample1_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample2 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1649eb0d0/sample2_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample3 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1649eb0d0/sample3_realign2transcript.bam
Skipped sorting BAM files.

-- Running step: transcript_quantification @ Mon Mar 23 20:08:50 2026 ----------
2026-03-24T00:08:50.234176Z  INFO oarfish: setting user-provided filter parameters.
2026-03-24T00:08:50.234468Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1649eb0d0/sample1_realign2transcript.bam, contains 5 reference sequences.
2026-03-24T00:08:50.234495Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-03-24T00:08:50.234500Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-03-24T00:08:50.234563Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-03-24T00:08:50.234579Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-03-24T00:08:50.236710Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-03-24T00:08:50.236895Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 253   │
│ aligned fraction too low        │ 4     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 1     │
│ reads with valid best alignment │ 96    │
╰─────────────────────────────────┴───────╯

2026-03-24T00:08:50.236973Z  INFO oarfish::bulk: Total number of alignment records : 116
2026-03-24T00:08:50.236986Z  INFO oarfish::bulk: number of aligned reads : 96
2026-03-24T00:08:50.236994Z  INFO oarfish::bulk: number of unique alignments : 86
2026-03-24T00:08:50.240960Z  INFO oarfish: oarfish completed successfully.
2026-03-24T00:08:50.266758Z  INFO oarfish: setting user-provided filter parameters.
2026-03-24T00:08:50.267128Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1649eb0d0/sample2_realign2transcript.bam, contains 5 reference sequences.
2026-03-24T00:08:50.267185Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-03-24T00:08:50.267198Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-03-24T00:08:50.267295Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-03-24T00:08:50.267318Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-03-24T00:08:50.269499Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-03-24T00:08:50.269688Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 251   │
│ aligned fraction too low        │ 5     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 2     │
│ reads with valid best alignment │ 95    │
╰─────────────────────────────────┴───────╯

2026-03-24T00:08:50.269736Z  INFO oarfish::bulk: Total number of alignment records : 114
2026-03-24T00:08:50.269742Z  INFO oarfish::bulk: number of aligned reads : 95
2026-03-24T00:08:50.269746Z  INFO oarfish::bulk: number of unique alignments : 82
2026-03-24T00:08:50.272185Z  INFO oarfish: oarfish completed successfully.
2026-03-24T00:08:50.301129Z  INFO oarfish: setting user-provided filter parameters.
2026-03-24T00:08:50.301496Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1649eb0d0/sample3_realign2transcript.bam, contains 5 reference sequences.
2026-03-24T00:08:50.301554Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-03-24T00:08:50.301565Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-03-24T00:08:50.301762Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-03-24T00:08:50.301779Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-03-24T00:08:50.305505Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2026-03-24T00:08:50.305800Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 398   │
│ aligned fraction too low        │ 12    │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 5     │
│ reads with valid best alignment │ 179   │
╰─────────────────────────────────┴───────╯

2026-03-24T00:08:50.305888Z  INFO oarfish::bulk: Total number of alignment records : 239
2026-03-24T00:08:50.305898Z  INFO oarfish::bulk: number of aligned reads : 179
2026-03-24T00:08:50.305905Z  INFO oarfish::bulk: number of unique alignments : 143
2026-03-24T00:08:50.308912Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc145c5a91c/config_file_69569.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 Mar 23 20:08:50 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc145c5a91c/sample1_align2genome.bam
sample2 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc145c5a91c/sample2_align2genome.bam
sample3 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc145c5a91c/sample3_align2genome.bam
-- Running step: isoform_identification @ Mon Mar 23 20:09:01 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

  |                                                                            
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Detected 4 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Mon Mar 23 20:09:08 2026 -------------------
Realignment complete for the following samples:
sample1 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc145c5a91c/sample1_realign2transcript.bam
sample2 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc145c5a91c/sample2_realign2transcript.bam
sample3 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc145c5a91c/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Mon Mar 23 20:09:18 2026 ----------
2026-03-24T00:09:18.758602Z  INFO oarfish: setting user-provided filter parameters.
2026-03-24T00:09:18.759074Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc145c5a91c/sample1_realign2transcript.bam, contains 5 reference sequences.
2026-03-24T00:09:18.759107Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-03-24T00:09:18.759113Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-03-24T00:09:18.759176Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-03-24T00:09:18.759189Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-03-24T00:09:18.761315Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-03-24T00:09:18.761416Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 253   │
│ aligned fraction too low        │ 4     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 1     │
│ reads with valid best alignment │ 96    │
╰─────────────────────────────────┴───────╯

2026-03-24T00:09:18.761455Z  INFO oarfish::bulk: Total number of alignment records : 116
2026-03-24T00:09:18.761460Z  INFO oarfish::bulk: number of aligned reads : 96
2026-03-24T00:09:18.761463Z  INFO oarfish::bulk: number of unique alignments : 86
2026-03-24T00:09:18.765171Z  INFO oarfish: oarfish completed successfully.
2026-03-24T00:09:18.797668Z  INFO oarfish: setting user-provided filter parameters.
2026-03-24T00:09:18.798243Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc145c5a91c/sample2_realign2transcript.bam, contains 5 reference sequences.
2026-03-24T00:09:18.798265Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-03-24T00:09:18.798270Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-03-24T00:09:18.798321Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-03-24T00:09:18.798329Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-03-24T00:09:18.800284Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-03-24T00:09:18.800462Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 251   │
│ aligned fraction too low        │ 5     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 2     │
│ reads with valid best alignment │ 95    │
╰─────────────────────────────────┴───────╯

2026-03-24T00:09:18.800512Z  INFO oarfish::bulk: Total number of alignment records : 114
2026-03-24T00:09:18.800528Z  INFO oarfish::bulk: number of aligned reads : 95
2026-03-24T00:09:18.800541Z  INFO oarfish::bulk: number of unique alignments : 82
2026-03-24T00:09:18.804045Z  INFO oarfish: oarfish completed successfully.
2026-03-24T00:09:18.835466Z  INFO oarfish: setting user-provided filter parameters.
2026-03-24T00:09:18.835956Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc145c5a91c/sample3_realign2transcript.bam, contains 5 reference sequences.
2026-03-24T00:09:18.836023Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-03-24T00:09:18.836038Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-03-24T00:09:18.836139Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-03-24T00:09:18.836196Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-03-24T00:09:18.839431Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2026-03-24T00:09:18.839632Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 398   │
│ aligned fraction too low        │ 12    │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 5     │
│ reads with valid best alignment │ 179   │
╰─────────────────────────────────┴───────╯

2026-03-24T00:09:18.839682Z  INFO oarfish::bulk: Total number of alignment records : 239
2026-03-24T00:09:18.839692Z  INFO oarfish::bulk: number of aligned reads : 179
2026-03-24T00:09:18.839700Z  INFO oarfish::bulk: number of unique alignments : 143
2026-03-24T00:09:18.842829Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc14f22df14/config_file_69569.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 Mar 23 20:09:19 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc14f22df14/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc14f22df14/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc14f22df14/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Mon Mar 23 20:09:19 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 4 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Mon Mar 23 20:09:26 2026 -------------------
Realigning sample sample1 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc14f22df14/sample1_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample2 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc14f22df14/sample2_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample3 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc14f22df14/sample3_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Mon Mar 23 20:09:26 2026 ----------
20:09:26 Mon Mar 23 2026 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Inputs:  ['/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc14f22df14/sample3_realign2transcript.bam', '/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc14f22df14/sample2_realign2transcript.bam', '/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc14f22df14/sample1_realign2transcript.bam'] /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc14f22df14/transcript_assembly.fa.fai 5 0.4 0.4
	Counter({'counted_reads': 375, 'not_enough_coverage': 16, 'unmapped': 2})
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc16985e9fe/config_file_69569.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 Mar 23 20:09:27 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc16985e9fe/sample1_align2genome.bam
sample2 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc16985e9fe/sample2_align2genome.bam
sample3 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc16985e9fe/sample3_align2genome.bam
-- Running step: isoform_identification @ Mon Mar 23 20:09:37 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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Detected 4 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Mon Mar 23 20:09:44 2026 -------------------
Realignment complete for the following samples:
sample1 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc16985e9fe/sample1_realign2transcript.bam
sample2 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc16985e9fe/sample2_realign2transcript.bam
sample3 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc16985e9fe/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Mon Mar 23 20:09:54 2026 ----------
20:09:54 Mon Mar 23 2026 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc124a57345/config_file_69569.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 Mar 23 20:09:55 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc124a57345/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc124a57345/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc124a57345/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Mon Mar 23 20:09:55 2026 -------------
Inputs:  ['/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc16985e9fe/sample3_realign2transcript.bam', '/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc16985e9fe/sample2_realign2transcript.bam', '/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc16985e9fe/sample1_realign2transcript.bam'] /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc16985e9fe/transcript_assembly.fa.fai 5 0.4 0.4
	Counter({'counted_reads': 375, 'not_enough_coverage': 16, 'unmapped': 2})
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Mon Mar 23 20:09:55 2026 -------------------
Realigning sample sample1 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc124a57345/sample1_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample2 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc124a57345/sample2_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample3 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc124a57345/sample3_realign2transcript.bam
Skipped sorting BAM files.

-- Running step: transcript_quantification @ Mon Mar 23 20:09:56 2026 ----------
2026-03-24T00:09:56.577488Z  INFO oarfish: setting user-provided filter parameters.
2026-03-24T00:09:56.577849Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc124a57345/sample1_realign2transcript.bam, contains 10 reference sequences.
2026-03-24T00:09:56.577874Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-03-24T00:09:56.577880Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-03-24T00:09:56.577950Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-03-24T00:09:56.577963Z  INFO oarfish: parsed reference information for 10 transcripts.
2026-03-24T00:09:56.585864Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-03-24T00:09:56.586052Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 707   │
│ aligned fraction too low        │ 2     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 98    │
╰─────────────────────────────────┴───────╯

2026-03-24T00:09:56.586143Z  INFO oarfish::bulk: Total number of alignment records : 125
2026-03-24T00:09:56.586157Z  INFO oarfish::bulk: number of aligned reads : 98
2026-03-24T00:09:56.586166Z  INFO oarfish::bulk: number of unique alignments : 86
2026-03-24T00:09:56.590154Z  INFO oarfish: oarfish completed successfully.
2026-03-24T00:09:56.615826Z  INFO oarfish: setting user-provided filter parameters.
2026-03-24T00:09:56.616097Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc124a57345/sample2_realign2transcript.bam, contains 10 reference sequences.
2026-03-24T00:09:56.616158Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-03-24T00:09:56.616167Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-03-24T00:09:56.616294Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-03-24T00:09:56.616315Z  INFO oarfish: parsed reference information for 10 transcripts.
2026-03-24T00:09:56.618879Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-03-24T00:09:56.618975Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 701   │
│ aligned fraction too low        │ 3     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 97    │
╰─────────────────────────────────┴───────╯

2026-03-24T00:09:56.619012Z  INFO oarfish::bulk: Total number of alignment records : 136
2026-03-24T00:09:56.619017Z  INFO oarfish::bulk: number of aligned reads : 97
2026-03-24T00:09:56.619021Z  INFO oarfish::bulk: number of unique alignments : 79
2026-03-24T00:09:56.620520Z  INFO oarfish: oarfish completed successfully.
2026-03-24T00:09:56.650246Z  INFO oarfish: setting user-provided filter parameters.
2026-03-24T00:09:56.650588Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc124a57345/sample3_realign2transcript.bam, contains 10 reference sequences.
2026-03-24T00:09:56.650606Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-03-24T00:09:56.650612Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-03-24T00:09:56.650675Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-03-24T00:09:56.650684Z  INFO oarfish: parsed reference information for 10 transcripts.
2026-03-24T00:09:56.655095Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-03-24T00:09:56.655182Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 1060  │
│ aligned fraction too low        │ 6     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 187   │
╰─────────────────────────────────┴───────╯

2026-03-24T00:09:56.655206Z  INFO oarfish::bulk: Total number of alignment records : 272
2026-03-24T00:09:56.655210Z  INFO oarfish::bulk: number of aligned reads : 187
2026-03-24T00:09:56.655213Z  INFO oarfish::bulk: number of unique alignments : 140
2026-03-24T00:09:56.657602Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc12a9a72b/config_file_69569.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 Mar 23 20:09:56 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc12a9a72b/sample1_align2genome.bam
sample2 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc12a9a72b/sample2_align2genome.bam
sample3 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc12a9a72b/sample3_align2genome.bam
-- Running step: isoform_identification @ Mon Mar 23 20:10:07 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Mon Mar 23 20:10:07 2026 -------------------
Realignment complete for the following samples:
sample1 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc12a9a72b/sample1_realign2transcript.bam
sample2 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc12a9a72b/sample2_realign2transcript.bam
sample3 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc12a9a72b/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Mon Mar 23 20:10:17 2026 ----------
2026-03-24T00:10:17.742025Z  INFO oarfish: setting user-provided filter parameters.
2026-03-24T00:10:17.742413Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc12a9a72b/sample1_realign2transcript.bam, contains 10 reference sequences.
2026-03-24T00:10:17.742489Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-03-24T00:10:17.742498Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-03-24T00:10:17.742683Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-03-24T00:10:17.742697Z  INFO oarfish: parsed reference information for 10 transcripts.
2026-03-24T00:10:17.744635Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-03-24T00:10:17.744892Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 707   │
│ aligned fraction too low        │ 2     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 98    │
╰─────────────────────────────────┴───────╯

2026-03-24T00:10:17.744950Z  INFO oarfish::bulk: Total number of alignment records : 125
2026-03-24T00:10:17.744960Z  INFO oarfish::bulk: number of aligned reads : 98
2026-03-24T00:10:17.744968Z  INFO oarfish::bulk: number of unique alignments : 86
2026-03-24T00:10:17.748065Z  INFO oarfish: oarfish completed successfully.
2026-03-24T00:10:17.779029Z  INFO oarfish: setting user-provided filter parameters.
2026-03-24T00:10:17.779429Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc12a9a72b/sample2_realign2transcript.bam, contains 10 reference sequences.
2026-03-24T00:10:17.779464Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-03-24T00:10:17.779470Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-03-24T00:10:17.779534Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-03-24T00:10:17.779548Z  INFO oarfish: parsed reference information for 10 transcripts.
2026-03-24T00:10:17.781581Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-03-24T00:10:17.781650Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 701   │
│ aligned fraction too low        │ 3     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 97    │
╰─────────────────────────────────┴───────╯

2026-03-24T00:10:17.781688Z  INFO oarfish::bulk: Total number of alignment records : 136
2026-03-24T00:10:17.781692Z  INFO oarfish::bulk: number of aligned reads : 97
2026-03-24T00:10:17.781695Z  INFO oarfish::bulk: number of unique alignments : 79
2026-03-24T00:10:17.784004Z  INFO oarfish: oarfish completed successfully.
2026-03-24T00:10:17.811892Z  INFO oarfish: setting user-provided filter parameters.
2026-03-24T00:10:17.812421Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc12a9a72b/sample3_realign2transcript.bam, contains 10 reference sequences.
2026-03-24T00:10:17.812450Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-03-24T00:10:17.812456Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-03-24T00:10:17.812517Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-03-24T00:10:17.812528Z  INFO oarfish: parsed reference information for 10 transcripts.
2026-03-24T00:10:17.817251Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-03-24T00:10:17.817352Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 1060  │
│ aligned fraction too low        │ 6     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 187   │
╰─────────────────────────────────┴───────╯

2026-03-24T00:10:17.817379Z  INFO oarfish::bulk: Total number of alignment records : 272
2026-03-24T00:10:17.817382Z  INFO oarfish::bulk: number of aligned reads : 187
2026-03-24T00:10:17.817385Z  INFO oarfish::bulk: number of unique alignments : 140
2026-03-24T00:10:17.819173Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1aad16ce/config_file_69569.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 Mar 23 20:10:18 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1aad16ce/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1aad16ce/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1aad16ce/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Mon Mar 23 20:10:18 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Mon Mar 23 20:10:18 2026 -------------------
Realigning sample sample1 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1aad16ce/sample1_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample2 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1aad16ce/sample2_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample3 -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1aad16ce/sample3_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Mon Mar 23 20:10:19 2026 ----------
20:10:19 Mon Mar 23 2026 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Inputs:  ['/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1aad16ce/sample3_realign2transcript.bam', '/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1aad16ce/sample2_realign2transcript.bam', '/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1aad16ce/sample1_realign2transcript.bam'] /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1aad16ce/transcript_assembly.fa.fai 5 0.4 0.4
	Counter({'counted_reads': 391, 'not_enough_coverage': 2})
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc131b46038/config_file_69569.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 Mar 23 20:10:19 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc131b46038/sample1_align2genome.bam
sample2 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc131b46038/sample2_align2genome.bam
sample3 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc131b46038/sample3_align2genome.bam
-- Running step: isoform_identification @ Mon Mar 23 20:10:30 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Mon Mar 23 20:10:30 2026 -------------------
Realignment complete for the following samples:
sample1 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc131b46038/sample1_realign2transcript.bam
sample2 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc131b46038/sample2_realign2transcript.bam
sample3 ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc131b46038/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Mon Mar 23 20:10:39 2026 ----------
20:10:39 Mon Mar 23 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)
Writing configuration parameters to:  /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc11f173313/config_file_69569.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 Mar 23 20:10:40 2026 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc11f173313/bc_allow.tsv
Number of known barcodes: 143
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 reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Mon Mar 23 20:10:40 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc11f173313/matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc11f173313/align2genome.bam
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: isoform_identification @ Mon Mar 23 20:10:40 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 Mar 23 20:10:44 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//Rtmp8H0zq1/file10fc11f173313/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc11f173313/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc11f173313/matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc11f173313/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 Mar 23 20:10:44 2026 ----------
2026-03-24T00:10:44.608002Z  INFO oarfish: setting user-provided filter parameters.
2026-03-24T00:10:44.608241Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc11f173313/realign2transcript.bam, contains 5 reference sequences.
2026-03-24T00:10:44.608298Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-03-24T00:10:44.608339Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-03-24T00:10:44.608436Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-03-24T00:10:44.608459Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-03-24T00:10:44.616246Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc149b0be7c/config_file_69569.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 Mar 23 20:10:44 2026 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc149b0be7c/bc_allow.tsv
Number of known barcodes: 143
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 reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Mon Mar 23 20:10:45 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc149b0be7c/matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc149b0be7c/align2genome.bam
-- Running step: isoform_identification @ Mon Mar 23 20:10:54 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---
Detected 2 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Mon Mar 23 20:10:58 2026 -------------------
Checking for fastq file(s) /Library/Frameworks/R.framework/Versions/4.6/Resources/library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc149b0be7c/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc149b0be7c/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc149b0be7c/matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc149b0be7c/realign2transcript.bam
-- Running step: transcript_quantification @ Mon Mar 23 20:11:07 2026 ----------
2026-03-24T00:11:07.994997Z  INFO oarfish: setting user-provided filter parameters.
2026-03-24T00:11:07.995512Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc149b0be7c/realign2transcript.bam, contains 5 reference sequences.
2026-03-24T00:11:07.995538Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-03-24T00:11:07.995543Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-03-24T00:11:07.995589Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-03-24T00:11:07.995597Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-03-24T00:11:08.040255Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc147bb5ba8/config_file_69569.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 Mar 23 20:11:08 2026 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc147bb5ba8/bc_allow.tsv
Number of known barcodes: 143
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 reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Mon Mar 23 20:11:08 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc147bb5ba8/matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc147bb5ba8/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 Mar 23 20:11:08 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 Mar 23 20:11:12 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//Rtmp8H0zq1/file10fc147bb5ba8/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc147bb5ba8/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc147bb5ba8/matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc147bb5ba8/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 Mar 23 20:11:12 2026 ----------
20:11:12 Mon Mar 23 2026 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Inputs:  ['/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc131b46038/sample3_realign2transcript.bam', '/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc131b46038/sample2_realign2transcript.bam', '/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc131b46038/sample1_realign2transcript.bam'] /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc131b46038/transcript_assembly.fa.fai 5 0.4 0.4
	Counter({'counted_reads': 391, 'not_enough_coverage': 2})
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc11559c22d/config_file_69569.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 Mar 23 20:11:13 2026 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc11559c22d/bc_allow.tsv
Number of known barcodes: 143
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 reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Mon Mar 23 20:11:13 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc11559c22d/matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc11559c22d/align2genome.bam
-- Running step: isoform_identification @ Mon Mar 23 20:11:23 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 Mar 23 20:11:26 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//Rtmp8H0zq1/file10fc11559c22d/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc11559c22d/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc11559c22d/matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc11559c22d/realign2transcript.bam
-- Running step: transcript_quantification @ Mon Mar 23 20:11:35 2026 ----------
20:11:35 Mon Mar 23 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)
Writing configuration parameters to:  /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc159b9658d/config_file_69569.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 Mar 23 20:11:36 2026 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc159b9658d/bc_allow.tsv
Number of known barcodes: 143
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 reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Mon Mar 23 20:11:36 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc159b9658d/matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc159b9658d/align2genome.bam
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: isoform_identification @ Mon Mar 23 20:11:36 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 Mar 23 20:11:36 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//Rtmp8H0zq1/file10fc159b9658d/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc159b9658d/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc159b9658d/matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc159b9658d/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 Mar 23 20:11:36 2026 ----------
2026-03-24T00:11:36.977806Z  INFO oarfish: setting user-provided filter parameters.
2026-03-24T00:11:36.978186Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc159b9658d/realign2transcript.bam, contains 10 reference sequences.
2026-03-24T00:11:36.978217Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-03-24T00:11:36.978237Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-03-24T00:11:36.978360Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-03-24T00:11:36.978386Z  INFO oarfish: parsed reference information for 10 transcripts.
2026-03-24T00:11:36.994804Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1274c8638/config_file_69569.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 Mar 23 20:11:37 2026 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1274c8638/bc_allow.tsv
Number of known barcodes: 143
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 reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Mon Mar 23 20:11:37 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1274c8638/matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1274c8638/align2genome.bam
-- Running step: isoform_identification @ Mon Mar 23 20:11:46 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Mon Mar 23 20:11:47 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//Rtmp8H0zq1/file10fc1274c8638/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1274c8638/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1274c8638/matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1274c8638/realign2transcript.bam
-- Running step: transcript_quantification @ Mon Mar 23 20:11:56 2026 ----------
2026-03-24T00:11:56.678197Z  INFO oarfish: setting user-provided filter parameters.
2026-03-24T00:11:56.678753Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1274c8638/realign2transcript.bam, contains 10 reference sequences.
2026-03-24T00:11:56.678784Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-03-24T00:11:56.678795Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-03-24T00:11:56.678856Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-03-24T00:11:56.678870Z  INFO oarfish: parsed reference information for 10 transcripts.
2026-03-24T00:11:56.731772Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc164ca1349/config_file_69569.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 Mar 23 20:11:57 2026 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc164ca1349/bc_allow.tsv
Number of known barcodes: 143
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 reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Mon Mar 23 20:11:57 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc164ca1349/matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc164ca1349/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 Mar 23 20:11:57 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Mon Mar 23 20:11:57 2026 -------------------
Checking for fastq file(s) /Library/Frameworks/R.framework/Versions/4.6/Resources/library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc164ca1349/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc164ca1349/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc164ca1349/matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc164ca1349/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 Mar 23 20:11:57 2026 ----------
20:11:57 Mon Mar 23 2026 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc152bbf9bc/config_file_69569.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 Mar 23 20:11:58 2026 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc152bbf9bc/bc_allow.tsv
Number of known barcodes: 143
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 reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Mon Mar 23 20:11:58 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc152bbf9bc/matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc152bbf9bc/align2genome.bam
-- Running step: isoform_identification @ Mon Mar 23 20:12:07 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 Mar 23 20:12:07 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//Rtmp8H0zq1/file10fc152bbf9bc/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc152bbf9bc/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc152bbf9bc/matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc152bbf9bc/realign2transcript.bam
-- Running step: transcript_quantification @ Mon Mar 23 20:12:17 2026 ----------
20:12:17 Mon Mar 23 2026 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc12d8f9f61/config_file_69569.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 Mar 23 20:12:17 2026 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc12d8f9f61/bc_allow.tsv
Number of known barcodes: 143
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc12d8f9f61/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc12d8f9f61/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc12d8f9f61/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc12d8f9f61/bc_allow.tsv
Number of known barcodes: 143
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc12d8f9f61/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 92
Number of reads with exactly one barcode match: 91
Number of chimera reads: 1
All done!
Reads	Barcodes
4	1
3	9
2	9
1	44
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc12d8f9f61/bc_allow.tsv
Number of known barcodes: 143
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc12d8f9f61/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 95
Number of reads with exactly one barcode match: 94
Number of chimera reads: 0
All done!
Reads	Barcodes
4	2
3	3
2	16
1	47
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc12d8f9f61/bc_allow.tsv
Number of known barcodes: 143
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc12d8f9f61/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Mon Mar 23 20:12:18 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc12d8f9f61/sampleA_matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc12d8f9f61/sampleA_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc12d8f9f61/sample1_matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc12d8f9f61/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc12d8f9f61/sample2_matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc12d8f9f61/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc12d8f9f61/sample3_matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc12d8f9f61/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Mon Mar 23 20:12:19 2026 ----------------
20:12:19 Mon Mar 23 2026 quantify genes 
Using BAM(s):
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc12d8f9f61/sampleA_align2genome.bam',
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc12d8f9f61/sample1_align2genome.bam',
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc12d8f9f61/sample2_align2genome.bam',
and
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc12d8f9f61/sample3_align2genome.bam'
	Counter({'counted_reads': 368, 'unmapped': 4})
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc12d8f9f61/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 69.44gene_group/s]
2026-03-23 20:12:20.883 R[69569:29500676] +[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, 895682.92Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc12d8f9f61/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 157.21gene_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, 2784322.89Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc12d8f9f61/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 70.44gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1748792.53Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc12d8f9f61/sample3_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, 1156857.90Read/s]
-- Running step: isoform_identification @ Mon Mar 23 20:12:21 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

  |                                                                            
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  |                                                                            
  |==================                                                    |  25%
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  |====================================================                  |  75%
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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 Mar 23 20:12:31 2026 -------------------
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc12d8f9f61/fastq, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc12d8f9f61/fastq/sample1.fq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc12d8f9f61/fastq/sample2.fq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc12d8f9f61/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc12d8f9f61/sampleA_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc12d8f9f61/sample1_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc12d8f9f61/sample2_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc12d8f9f61/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc12d8f9f61/sampleA_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc12d8f9f61/sample1_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc12d8f9f61/sample2_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc12d8f9f61/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc12d8f9f61/sampleA_matched_reads_dedup.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc12d8f9f61/sampleA_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc12d8f9f61/sample1_matched_reads_dedup.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc12d8f9f61/sample1_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc12d8f9f61/sample2_matched_reads_dedup.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc12d8f9f61/sample2_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc12d8f9f61/sample3_matched_reads_dedup.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc12d8f9f61/sample3_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

-- Running step: transcript_quantification @ Mon Mar 23 20:12:32 2026 ----------
2026-03-24T00:12:32.089229Z  INFO oarfish: setting user-provided filter parameters.
2026-03-24T00:12:32.089721Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc12d8f9f61/sampleA_realign2transcript.bam, contains 5 reference sequences.
2026-03-24T00:12:32.089812Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-03-24T00:12:32.089836Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-03-24T00:12:32.089996Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-03-24T00:12:32.090039Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-03-24T00:12:32.095191Z  INFO oarfish::single_cell: Processed 100 cells.
2026-03-24T00:12:32.214700Z  INFO oarfish: setting user-provided filter parameters.
2026-03-24T00:12:32.215060Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc12d8f9f61/sample1_realign2transcript.bam, contains 5 reference sequences.
2026-03-24T00:12:32.215076Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-03-24T00:12:32.215160Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-03-24T00:12:32.215291Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-03-24T00:12:32.215323Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-03-24T00:12:32.384388Z  INFO oarfish: setting user-provided filter parameters.
2026-03-24T00:12:32.384754Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc12d8f9f61/sample2_realign2transcript.bam, contains 5 reference sequences.
2026-03-24T00:12:32.384787Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-03-24T00:12:32.384799Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-03-24T00:12:32.384882Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-03-24T00:12:32.384902Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-03-24T00:12:32.521224Z  INFO oarfish: setting user-provided filter parameters.
2026-03-24T00:12:32.521526Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc12d8f9f61/sample3_realign2transcript.bam, contains 5 reference sequences.
2026-03-24T00:12:32.521647Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-03-24T00:12:32.521667Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-03-24T00:12:32.521775Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-03-24T00:12:32.521802Z  INFO oarfish: parsed reference information for 5 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc11c3461b2/config_file_69569.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 Mar 23 20:12:32 2026 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc11c3461b2/bc_allow.tsv
Number of known barcodes: 143
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc11c3461b2/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc11c3461b2/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc11c3461b2/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc11c3461b2/bc_allow.tsv
Number of known barcodes: 143
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc11c3461b2/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 92
Number of reads with exactly one barcode match: 91
Number of chimera reads: 1
All done!
Reads	Barcodes
4	1
3	9
2	9
1	44
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc11c3461b2/bc_allow.tsv
Number of known barcodes: 143
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc11c3461b2/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 95
Number of reads with exactly one barcode match: 94
Number of chimera reads: 0
All done!
Reads	Barcodes
4	2
3	3
2	16
1	47
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc11c3461b2/bc_allow.tsv
Number of known barcodes: 143
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc11c3461b2/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Mon Mar 23 20:12:33 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc11c3461b2/sampleA_matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc11c3461b2/sampleA_align2genome.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc11c3461b2/sample1_matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc11c3461b2/sample1_align2genome.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc11c3461b2/sample2_matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc11c3461b2/sample2_align2genome.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc11c3461b2/sample3_matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc11c3461b2/sample3_align2genome.bam
-- Running step: gene_quantification @ Mon Mar 23 20:12:43 2026 ----------------
20:12:43 Mon Mar 23 2026 quantify genes 
Using BAM(s):
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc11c3461b2/sampleA_align2genome.bam',
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc11c3461b2/sample1_align2genome.bam',
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc11c3461b2/sample2_align2genome.bam',
and
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc11c3461b2/sample3_align2genome.bam'
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc11c3461b2/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 46.83gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 695203.87Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc11c3461b2/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 153.14gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 3703906.75Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc11c3461b2/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 57.88gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 2045602.81Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc11c3461b2/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 53.41gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1800130.47Read/s]
-- Running step: isoform_identification @ Mon Mar 23 20:12:43 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 Mar 23 20:12:51 2026 -------------------
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc11c3461b2/fastq, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc11c3461b2/fastq/sample1.fq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc11c3461b2/fastq/sample2.fq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc11c3461b2/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc11c3461b2/sampleA_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc11c3461b2/sample1_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc11c3461b2/sample2_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc11c3461b2/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc11c3461b2/sampleA_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc11c3461b2/sample1_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc11c3461b2/sample2_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc11c3461b2/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc11c3461b2/sampleA_matched_reads_dedup.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc11c3461b2/sampleA_realign2transcript.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc11c3461b2/sample1_matched_reads_dedup.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc11c3461b2/sample1_realign2transcript.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc11c3461b2/sample2_matched_reads_dedup.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc11c3461b2/sample2_realign2transcript.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc11c3461b2/sample3_matched_reads_dedup.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc11c3461b2/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Mon Mar 23 20:13:01 2026 ----------
2026-03-24T00:13:01.206228Z  INFO oarfish: setting user-provided filter parameters.
2026-03-24T00:13:01.206616Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc11c3461b2/sampleA_realign2transcript.bam, contains 5 reference sequences.
2026-03-24T00:13:01.206670Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-03-24T00:13:01.206684Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-03-24T00:13:01.206787Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-03-24T00:13:01.206809Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-03-24T00:13:01.211661Z  INFO oarfish::single_cell: Processed 100 cells.
2026-03-24T00:13:01.393096Z  INFO oarfish: setting user-provided filter parameters.
2026-03-24T00:13:01.393464Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc11c3461b2/sample1_realign2transcript.bam, contains 5 reference sequences.
2026-03-24T00:13:01.393482Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-03-24T00:13:01.393488Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-03-24T00:13:01.393535Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-03-24T00:13:01.393545Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-03-24T00:13:01.528421Z  INFO oarfish: setting user-provided filter parameters.
2026-03-24T00:13:01.528704Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc11c3461b2/sample2_realign2transcript.bam, contains 5 reference sequences.
2026-03-24T00:13:01.528770Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-03-24T00:13:01.528786Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-03-24T00:13:01.528883Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-03-24T00:13:01.528904Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-03-24T00:13:01.672330Z  INFO oarfish: setting user-provided filter parameters.
2026-03-24T00:13:01.672647Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc11c3461b2/sample3_realign2transcript.bam, contains 5 reference sequences.
2026-03-24T00:13:01.672713Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-03-24T00:13:01.672734Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-03-24T00:13:01.672830Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-03-24T00:13:01.672852Z  INFO oarfish: parsed reference information for 5 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc17effa9c1/config_file_69569.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 Mar 23 20:13:02 2026 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc17effa9c1/bc_allow.tsv
Number of known barcodes: 143
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc17effa9c1/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc17effa9c1/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc17effa9c1/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc17effa9c1/bc_allow.tsv
Number of known barcodes: 143
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc17effa9c1/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 92
Number of reads with exactly one barcode match: 91
Number of chimera reads: 1
All done!
Reads	Barcodes
4	1
3	9
2	9
1	44
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc17effa9c1/bc_allow.tsv
Number of known barcodes: 143
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc17effa9c1/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 95
Number of reads with exactly one barcode match: 94
Number of chimera reads: 0
All done!
Reads	Barcodes
4	2
3	3
2	16
1	47
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc17effa9c1/bc_allow.tsv
Number of known barcodes: 143
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc17effa9c1/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Mon Mar 23 20:13:02 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc17effa9c1/sampleA_matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc17effa9c1/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//Rtmp8H0zq1/file10fc17effa9c1/sample1_matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc17effa9c1/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//Rtmp8H0zq1/file10fc17effa9c1/sample2_matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc17effa9c1/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//Rtmp8H0zq1/file10fc17effa9c1/sample3_matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc17effa9c1/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 Mar 23 20:13:03 2026 ----------------
20:13:03 Mon Mar 23 2026 quantify genes 
Using BAM(s):
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc17effa9c1/sampleA_align2genome.bam',
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc17effa9c1/sample1_align2genome.bam',
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc17effa9c1/sample2_align2genome.bam',
and
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc17effa9c1/sample3_align2genome.bam'
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc17effa9c1/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 74.08gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 831675.13Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc17effa9c1/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 168.19gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1916957.95Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc17effa9c1/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 51.74gene_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, 2527907.43Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc17effa9c1/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 41.01gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1368362.26Read/s]
-- Running step: isoform_identification @ Mon Mar 23 20:13:03 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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  |                                                                            
  |==================                                                    |  25%
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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 Mar 23 20:13:11 2026 -------------------
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc17effa9c1/fastq, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc17effa9c1/fastq/sample1.fq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc17effa9c1/fastq/sample2.fq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc17effa9c1/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc17effa9c1/sampleA_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc17effa9c1/sample1_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc17effa9c1/sample2_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc17effa9c1/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc17effa9c1/sampleA_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc17effa9c1/sample1_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc17effa9c1/sample2_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc17effa9c1/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc17effa9c1/sampleA_matched_reads_dedup.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc17effa9c1/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//Rtmp8H0zq1/file10fc17effa9c1/sample1_matched_reads_dedup.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc17effa9c1/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//Rtmp8H0zq1/file10fc17effa9c1/sample2_matched_reads_dedup.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc17effa9c1/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//Rtmp8H0zq1/file10fc17effa9c1/sample3_matched_reads_dedup.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc17effa9c1/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 Mar 23 20:13:12 2026 ----------
20:13:12 Mon Mar 23 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//Rtmp8H0zq1/file10fc17effa9c1/sample3_realign2transcript.bam...
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc17effa9c1/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc17effa9c1/sample3_realign2transcript.bamdone
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc17effa9c1/sampleA_realign2transcript.bam...
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc17effa9c1/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc17effa9c1/sampleA_realign2transcript.bamdone
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc17effa9c1/sample2_realign2transcript.bam...
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc17effa9c1/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc17effa9c1/sample2_realign2transcript.bamdone
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc17effa9c1/sample1_realign2transcript.bam...
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc17effa9c1/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc17effa9c1/sample1_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc15032b4ed/config_file_69569.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 Mar 23 20:13:12 2026 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc15032b4ed/bc_allow.tsv
Number of known barcodes: 143
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc15032b4ed/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc15032b4ed/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc15032b4ed/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc15032b4ed/bc_allow.tsv
Number of known barcodes: 143
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc15032b4ed/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 92
Number of reads with exactly one barcode match: 91
Number of chimera reads: 1
All done!
Reads	Barcodes
4	1
3	9
2	9
1	44
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc15032b4ed/bc_allow.tsv
Number of known barcodes: 143
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc15032b4ed/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 95
Number of reads with exactly one barcode match: 94
Number of chimera reads: 0
All done!
Reads	Barcodes
4	2
3	3
2	16
1	47
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc15032b4ed/bc_allow.tsv
Number of known barcodes: 143
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc15032b4ed/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Mon Mar 23 20:13:13 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc15032b4ed/sampleA_matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc15032b4ed/sampleA_align2genome.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc15032b4ed/sample1_matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc15032b4ed/sample1_align2genome.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc15032b4ed/sample2_matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc15032b4ed/sample2_align2genome.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc15032b4ed/sample3_matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc15032b4ed/sample3_align2genome.bam
-- Running step: gene_quantification @ Mon Mar 23 20:13:23 2026 ----------------
20:13:23 Mon Mar 23 2026 quantify genes 
Using BAM(s):
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc15032b4ed/sampleA_align2genome.bam',
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc15032b4ed/sample1_align2genome.bam',
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc15032b4ed/sample2_align2genome.bam',
and
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc15032b4ed/sample3_align2genome.bam'
	Counter({'counted_reads': 168, 'not_enough_coverage': 6, 'unmapped': 2})
	Counter({'counted_reads': 347, 'not_enough_coverage': 9, 'unmapped': 2})
	Counter({'counted_reads': 92, 'not_enough_coverage': 3})
	Counter({'counted_reads': 89, 'not_enough_coverage': 2})
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc15032b4ed/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 72.10gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 900915.89Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc15032b4ed/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 153.49gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 2352122.03Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc15032b4ed/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 56.77gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 2765232.07Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc15032b4ed/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 113.69gene_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, 1207063.43Read/s]
-- Running step: isoform_identification @ Mon Mar 23 20:13:24 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 Mar 23 20:13:32 2026 -------------------
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc15032b4ed/fastq, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc15032b4ed/fastq/sample1.fq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc15032b4ed/fastq/sample2.fq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc15032b4ed/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc15032b4ed/sampleA_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc15032b4ed/sample1_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc15032b4ed/sample2_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc15032b4ed/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc15032b4ed/sampleA_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc15032b4ed/sample1_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc15032b4ed/sample2_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc15032b4ed/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc15032b4ed/sampleA_matched_reads_dedup.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc15032b4ed/sampleA_realign2transcript.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc15032b4ed/sample1_matched_reads_dedup.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc15032b4ed/sample1_realign2transcript.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc15032b4ed/sample2_matched_reads_dedup.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc15032b4ed/sample2_realign2transcript.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc15032b4ed/sample3_matched_reads_dedup.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc15032b4ed/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Mon Mar 23 20:13:41 2026 ----------
20:13:41 Mon Mar 23 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//Rtmp8H0zq1/file10fc15032b4ed/sample3_realign2transcript.bam...
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc15032b4ed/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc15032b4ed/sample3_realign2transcript.bamdone
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc15032b4ed/sampleA_realign2transcript.bam...
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc15032b4ed/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc15032b4ed/sampleA_realign2transcript.bamdone
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc15032b4ed/sample2_realign2transcript.bam...
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc15032b4ed/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc15032b4ed/sample2_realign2transcript.bamdone
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc15032b4ed/sample1_realign2transcript.bam...
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc15032b4ed/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc15032b4ed/sample1_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1510bc72/config_file_69569.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 Mar 23 20:13:42 2026 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1510bc72/bc_allow.tsv
Number of known barcodes: 143
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1510bc72/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1510bc72/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1510bc72/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1510bc72/bc_allow.tsv
Number of known barcodes: 143
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1510bc72/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 92
Number of reads with exactly one barcode match: 91
Number of chimera reads: 1
All done!
Reads	Barcodes
4	1
3	9
2	9
1	44
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1510bc72/bc_allow.tsv
Number of known barcodes: 143
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1510bc72/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 95
Number of reads with exactly one barcode match: 94
Number of chimera reads: 0
All done!
Reads	Barcodes
4	2
3	3
2	16
1	47
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1510bc72/bc_allow.tsv
Number of known barcodes: 143
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1510bc72/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Mon Mar 23 20:13:42 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1510bc72/sampleA_matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1510bc72/sampleA_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1510bc72/sample1_matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1510bc72/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1510bc72/sample2_matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1510bc72/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1510bc72/sample3_matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1510bc72/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Mon Mar 23 20:13:43 2026 ----------------
20:13:43 Mon Mar 23 2026 quantify genes 
Using BAM(s):
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1510bc72/sampleA_align2genome.bam',
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1510bc72/sample1_align2genome.bam',
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1510bc72/sample2_align2genome.bam',
and
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1510bc72/sample3_align2genome.bam'
	Counter({'counted_reads': 168, 'not_enough_coverage': 6, 'unmapped': 2})
	Counter({'counted_reads': 347, 'not_enough_coverage': 9, 'unmapped': 2})
	Counter({'counted_reads': 92, 'not_enough_coverage': 3})
	Counter({'counted_reads': 89, 'not_enough_coverage': 2})
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1510bc72/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 53.12gene_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, 820803.13Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1510bc72/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 159.95gene_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, 959707.12Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1510bc72/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 155.15gene_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, 2799935.91Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1510bc72/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 87.20gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1397356.08Read/s]
-- Running step: isoform_identification @ Mon Mar 23 20:13:44 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Mon Mar 23 20:13:44 2026 -------------------
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1510bc72/fastq, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1510bc72/fastq/sample1.fq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1510bc72/fastq/sample2.fq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1510bc72/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1510bc72/sampleA_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1510bc72/sample1_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1510bc72/sample2_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1510bc72/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1510bc72/sampleA_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1510bc72/sample1_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1510bc72/sample2_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1510bc72/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1510bc72/sampleA_matched_reads_dedup.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1510bc72/sampleA_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1510bc72/sample1_matched_reads_dedup.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1510bc72/sample1_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1510bc72/sample2_matched_reads_dedup.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1510bc72/sample2_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1510bc72/sample3_matched_reads_dedup.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1510bc72/sample3_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

-- Running step: transcript_quantification @ Mon Mar 23 20:13:45 2026 ----------
2026-03-24T00:13:45.315286Z  INFO oarfish: setting user-provided filter parameters.
2026-03-24T00:13:45.315683Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1510bc72/sampleA_realign2transcript.bam, contains 14 reference sequences.
2026-03-24T00:13:45.315753Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-03-24T00:13:45.315773Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-03-24T00:13:45.315934Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-03-24T00:13:45.315964Z  INFO oarfish: parsed reference information for 14 transcripts.
2026-03-24T00:13:45.329056Z  INFO oarfish::single_cell: Processed 100 cells.
2026-03-24T00:13:45.652115Z  INFO oarfish: setting user-provided filter parameters.
2026-03-24T00:13:45.652571Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1510bc72/sample1_realign2transcript.bam, contains 14 reference sequences.
2026-03-24T00:13:45.652601Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-03-24T00:13:45.652610Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-03-24T00:13:45.652672Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-03-24T00:13:45.652685Z  INFO oarfish: parsed reference information for 14 transcripts.
2026-03-24T00:13:45.882788Z  INFO oarfish: setting user-provided filter parameters.
2026-03-24T00:13:45.883138Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1510bc72/sample2_realign2transcript.bam, contains 14 reference sequences.
2026-03-24T00:13:45.883190Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-03-24T00:13:45.883219Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-03-24T00:13:45.883364Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-03-24T00:13:45.883401Z  INFO oarfish: parsed reference information for 14 transcripts.
2026-03-24T00:13:46.112943Z  INFO oarfish: setting user-provided filter parameters.
2026-03-24T00:13:46.113300Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1510bc72/sample3_realign2transcript.bam, contains 14 reference sequences.
2026-03-24T00:13:46.113337Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-03-24T00:13:46.113353Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-03-24T00:13:46.113471Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-03-24T00:13:46.113499Z  INFO oarfish: parsed reference information for 14 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc125015ce7/config_file_69569.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 Mar 23 20:13:46 2026 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc125015ce7/bc_allow.tsv
Number of known barcodes: 143
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc125015ce7/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc125015ce7/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc125015ce7/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc125015ce7/bc_allow.tsv
Number of known barcodes: 143
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc125015ce7/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 92
Number of reads with exactly one barcode match: 91
Number of chimera reads: 1
All done!
Reads	Barcodes
4	1
3	9
2	9
1	44
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc125015ce7/bc_allow.tsv
Number of known barcodes: 143
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc125015ce7/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 95
Number of reads with exactly one barcode match: 94
Number of chimera reads: 0
All done!
Reads	Barcodes
4	2
3	3
2	16
1	47
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc125015ce7/bc_allow.tsv
Number of known barcodes: 143
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc125015ce7/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Mon Mar 23 20:13:47 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc125015ce7/sampleA_matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc125015ce7/sampleA_align2genome.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc125015ce7/sample1_matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc125015ce7/sample1_align2genome.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc125015ce7/sample2_matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc125015ce7/sample2_align2genome.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc125015ce7/sample3_matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc125015ce7/sample3_align2genome.bam
-- Running step: gene_quantification @ Mon Mar 23 20:13:56 2026 ----------------
20:13:56 Mon Mar 23 2026 quantify genes 
Using BAM(s):
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc125015ce7/sampleA_align2genome.bam',
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc125015ce7/sample1_align2genome.bam',
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc125015ce7/sample2_align2genome.bam',
and
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc125015ce7/sample3_align2genome.bam'
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc125015ce7/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 66.83gene_group/s]
/Library/Frameworks/R.framework/Versions/4.6/Resources/library/FLAMES/python/count_gene.py:705: RuntimeWarning: More than 20 figures have been opened. Figures created through the pyplot interface (`matplotlib.pyplot.figure`) are retained until explicitly closed and may consume too much memory. (To control this warning, see the rcParam `figure.max_open_warning`). Consider using `matplotlib.pyplot.close()`.
  plt.figure(figsize=(8, 6))
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 763155.75Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc125015ce7/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 76.50gene_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, 2059261.59Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc125015ce7/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 82.32gene_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, 2117052.29Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc125015ce7/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 99.56gene_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, 1417377.67Read/s]
-- Running step: isoform_identification @ Mon Mar 23 20:13:57 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Mon Mar 23 20:13:57 2026 -------------------
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc125015ce7/fastq, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc125015ce7/fastq/sample1.fq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc125015ce7/fastq/sample2.fq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc125015ce7/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc125015ce7/sampleA_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc125015ce7/sample1_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc125015ce7/sample2_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc125015ce7/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc125015ce7/sampleA_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc125015ce7/sample1_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc125015ce7/sample2_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc125015ce7/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc125015ce7/sampleA_matched_reads_dedup.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc125015ce7/sampleA_realign2transcript.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc125015ce7/sample1_matched_reads_dedup.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc125015ce7/sample1_realign2transcript.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc125015ce7/sample2_matched_reads_dedup.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc125015ce7/sample2_realign2transcript.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc125015ce7/sample3_matched_reads_dedup.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc125015ce7/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Mon Mar 23 20:14:07 2026 ----------
2026-03-24T00:14:08.028348Z  INFO oarfish: setting user-provided filter parameters.
2026-03-24T00:14:08.028744Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc125015ce7/sampleA_realign2transcript.bam, contains 14 reference sequences.
2026-03-24T00:14:08.028774Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-03-24T00:14:08.028786Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-03-24T00:14:08.028897Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-03-24T00:14:08.028919Z  INFO oarfish: parsed reference information for 14 transcripts.
2026-03-24T00:14:08.038911Z  INFO oarfish::single_cell: Processed 100 cells.
2026-03-24T00:14:08.354155Z  INFO oarfish: setting user-provided filter parameters.
2026-03-24T00:14:08.354453Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc125015ce7/sample1_realign2transcript.bam, contains 14 reference sequences.
2026-03-24T00:14:08.354477Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-03-24T00:14:08.354484Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-03-24T00:14:08.354563Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-03-24T00:14:08.354573Z  INFO oarfish: parsed reference information for 14 transcripts.
2026-03-24T00:14:08.717441Z  INFO oarfish: setting user-provided filter parameters.
2026-03-24T00:14:08.717982Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc125015ce7/sample2_realign2transcript.bam, contains 14 reference sequences.
2026-03-24T00:14:08.718034Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-03-24T00:14:08.718047Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-03-24T00:14:08.718178Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-03-24T00:14:08.718203Z  INFO oarfish: parsed reference information for 14 transcripts.
2026-03-24T00:14:08.971030Z  INFO oarfish: setting user-provided filter parameters.
2026-03-24T00:14:08.971498Z  INFO oarfish::alignment_parser: read header from BAM file /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc125015ce7/sample3_realign2transcript.bam, contains 14 reference sequences.
2026-03-24T00:14:08.971578Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-03-24T00:14:08.971594Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-03-24T00:14:08.971732Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-03-24T00:14:08.971765Z  INFO oarfish: parsed reference information for 14 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc111748f6c/config_file_69569.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 Mar 23 20:14:09 2026 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc111748f6c/bc_allow.tsv
Number of known barcodes: 143
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc111748f6c/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc111748f6c/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc111748f6c/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc111748f6c/bc_allow.tsv
Number of known barcodes: 143
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc111748f6c/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 92
Number of reads with exactly one barcode match: 91
Number of chimera reads: 1
All done!
Reads	Barcodes
4	1
3	9
2	9
1	44
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc111748f6c/bc_allow.tsv
Number of known barcodes: 143
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc111748f6c/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 95
Number of reads with exactly one barcode match: 94
Number of chimera reads: 0
All done!
Reads	Barcodes
4	2
3	3
2	16
1	47
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc111748f6c/bc_allow.tsv
Number of known barcodes: 143
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc111748f6c/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Mon Mar 23 20:14:10 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc111748f6c/sampleA_matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc111748f6c/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//Rtmp8H0zq1/file10fc111748f6c/sample1_matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc111748f6c/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//Rtmp8H0zq1/file10fc111748f6c/sample2_matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc111748f6c/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//Rtmp8H0zq1/file10fc111748f6c/sample3_matched_reads.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc111748f6c/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 Mar 23 20:14:11 2026 ----------------
20:14:11 Mon Mar 23 2026 quantify genes 
Using BAM(s):
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc111748f6c/sampleA_align2genome.bam',
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc111748f6c/sample1_align2genome.bam',
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc111748f6c/sample2_align2genome.bam',
and
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc111748f6c/sample3_align2genome.bam'
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc111748f6c/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 60.03gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 493122.65Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc111748f6c/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 124.92gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 2615555.00Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc111748f6c/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 101.29gene_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, 3221431.64Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc111748f6c/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 95.09gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1416611.73Read/s]
-- Running step: isoform_identification @ Mon Mar 23 20:14:11 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Mon Mar 23 20:14:11 2026 -------------------
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc111748f6c/fastq, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc111748f6c/fastq/sample1.fq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc111748f6c/fastq/sample2.fq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc111748f6c/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc111748f6c/sampleA_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc111748f6c/sample1_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc111748f6c/sample2_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc111748f6c/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc111748f6c/sampleA_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc111748f6c/sample1_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc111748f6c/sample2_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc111748f6c/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc111748f6c/sampleA_matched_reads_dedup.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc111748f6c/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//Rtmp8H0zq1/file10fc111748f6c/sample1_matched_reads_dedup.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc111748f6c/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//Rtmp8H0zq1/file10fc111748f6c/sample2_matched_reads_dedup.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc111748f6c/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//Rtmp8H0zq1/file10fc111748f6c/sample3_matched_reads_dedup.fastq.gz -> /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc111748f6c/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 Mar 23 20:14:12 2026 ----------
20:14:12 Mon Mar 23 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//Rtmp8H0zq1/file10fc111748f6c/sample3_realign2transcript.bam...
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc111748f6c/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc111748f6c/sample3_realign2transcript.bamdone
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc111748f6c/sampleA_realign2transcript.bam...
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc111748f6c/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc111748f6c/sampleA_realign2transcript.bamdone
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc111748f6c/sample2_realign2transcript.bam...
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc111748f6c/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc111748f6c/sample2_realign2transcript.bamdone
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc111748f6c/sample1_realign2transcript.bam...
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc111748f6c/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc111748f6c/sample1_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1248409cc/config_file_69569.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 Mar 23 20:14:13 2026 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1248409cc/bc_allow.tsv
Number of known barcodes: 143
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1248409cc/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1248409cc/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1248409cc/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1248409cc/bc_allow.tsv
Number of known barcodes: 143
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1248409cc/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 92
Number of reads with exactly one barcode match: 91
Number of chimera reads: 1
All done!
Reads	Barcodes
4	1
3	9
2	9
1	44
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1248409cc/bc_allow.tsv
Number of known barcodes: 143
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1248409cc/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 95
Number of reads with exactly one barcode match: 94
Number of chimera reads: 0
All done!
Reads	Barcodes
4	2
3	3
2	16
1	47
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1248409cc/bc_allow.tsv
Number of known barcodes: 143
Processing file: /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1248409cc/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Mon Mar 23 20:14:14 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1248409cc/sampleA_matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1248409cc/sampleA_align2genome.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1248409cc/sample1_matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1248409cc/sample1_align2genome.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1248409cc/sample2_matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1248409cc/sample2_align2genome.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1248409cc/sample3_matched_reads.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1248409cc/sample3_align2genome.bam
-- Running step: gene_quantification @ Mon Mar 23 20:14:24 2026 ----------------
20:14:24 Mon Mar 23 2026 quantify genes 
Using BAM(s):
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1248409cc/sampleA_align2genome.bam',
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1248409cc/sample1_align2genome.bam',
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1248409cc/sample2_align2genome.bam',
and
'/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1248409cc/sample3_align2genome.bam'
	Counter({'counted_reads': 176})
	Counter({'counted_reads': 358})
	Counter({'counted_reads': 95})
	Counter({'counted_reads': 91})
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1248409cc/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 65.57gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 604889.53Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1248409cc/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 59.62gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 3346875.20Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1248409cc/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 164.69gene_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, 2204280.01Read/s]
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1248409cc/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 94.85gene_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, 1808825.25Read/s]
-- Running step: isoform_identification @ Mon Mar 23 20:14:24 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Mon Mar 23 20:14:24 2026 -------------------
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1248409cc/fastq, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1248409cc/fastq/sample1.fq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1248409cc/fastq/sample2.fq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1248409cc/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1248409cc/sampleA_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1248409cc/sample1_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1248409cc/sample2_matched_reads.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1248409cc/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1248409cc/sampleA_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1248409cc/sample1_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1248409cc/sample2_matched_reads_dedup.fastq.gz, /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1248409cc/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1248409cc/sampleA_matched_reads_dedup.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1248409cc/sampleA_realign2transcript.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1248409cc/sample1_matched_reads_dedup.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1248409cc/sample1_realign2transcript.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1248409cc/sample2_matched_reads_dedup.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1248409cc/sample2_realign2transcript.bam
/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1248409cc/sample3_matched_reads_dedup.fastq.gz ->/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1248409cc/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Mon Mar 23 20:14:34 2026 ----------
20:14:34 Mon Mar 23 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//Rtmp8H0zq1/file10fc1248409cc/sample3_realign2transcript.bam...
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1248409cc/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1248409cc/sample3_realign2transcript.bamdone
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1248409cc/sampleA_realign2transcript.bam...
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1248409cc/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1248409cc/sampleA_realign2transcript.bamdone
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1248409cc/sample2_realign2transcript.bam...
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1248409cc/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1248409cc/sample2_realign2transcript.bamdone
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1248409cc/sample1_realign2transcript.bam...
parsing /var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1248409cc/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/var/folders/r0/l4fjk6cj5xj0j3brt4bplpl40000gt/T//Rtmp8H0zq1/file10fc1248409cc/sample1_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
[ FAIL 0 | WARN 182 | SKIP 0 | PASS 59 ]

[ FAIL 0 | WARN 182 | SKIP 0 | PASS 59 ]
	Counter({'counted_reads': 176})
	Counter({'counted_reads': 358})
	Counter({'counted_reads': 95})
	Counter({'counted_reads': 91})
> 
> proc.time()
   user  system elapsed 
326.953  24.154 367.203 
/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 '