##############################################################################
##############################################################################
###
### 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_2.4.1.tar.gz
###
##############################################################################
##############################################################################


* using log directory ‘/Users/biocbuild/bbs-3.22-bioc/meat/FLAMES.Rcheck’
* using R version 4.5.2 (2025-10-31)
* using platform: x86_64-apple-darwin20
* R was compiled by
    Apple clang version 14.0.0 (clang-1400.0.29.202)
    GNU Fortran (GCC) 14.2.0
* running under: macOS Monterey 12.7.6
* using session charset: UTF-8
* using option ‘--no-vignettes’
* checking for file ‘FLAMES/DESCRIPTION’ ... OK
* this is package ‘FLAMES’ version ‘2.4.1’
* package encoding: UTF-8
* checking package namespace information ... OK
* checking package dependencies ... INFO
Imports includes 47 non-default packages.
Importing from so many packages makes the package vulnerable to any of
them becoming unavailable.  Move as many as possible to Suggests and
use conditionally.
* checking if this is a source package ... OK
* checking if there is a namespace ... OK
* checking for hidden files and directories ... NOTE
Found the following hidden files and directories:
  .dockerignore
These were most likely included in error. See section ‘Package
structure’ in the ‘Writing R Extensions’ manual.
* checking for portable file names ... OK
* checking for sufficient/correct file permissions ... OK
* checking whether package ‘FLAMES’ can be installed ... NOTE
Found the following notes/warnings:
  Non-staged installation was used
See ‘/Users/biocbuild/bbs-3.22-bioc/meat/FLAMES.Rcheck/00install.out’ for details.
* used C++ compiler: ‘Apple clang version 14.0.0 (clang-1400.0.29.202)’
* used SDK: ‘MacOSX11.3.1.sdk’
* checking C++ specification ... OK
* checking installed package size ... INFO
  installed size is  5.5Mb
  sub-directories of 1Mb or more:
    data   1.8Mb
    libs   1.8Mb
* checking package directory ... OK
* checking ‘build’ directory ... OK
* checking DESCRIPTION meta-information ... OK
* checking top-level files ... OK
* checking for left-over files ... OK
* checking index information ... OK
* checking package subdirectories ... OK
* checking code files for non-ASCII characters ... OK
* checking R files for syntax errors ... OK
* checking whether the package can be loaded ... OK
* checking whether the package can be loaded with stated dependencies ... OK
* checking whether the package can be unloaded cleanly ... OK
* checking whether the namespace can be loaded with stated dependencies ... OK
* checking whether the namespace can be unloaded cleanly ... OK
* checking dependencies in R code ... NOTE
There are ::: calls to the package's namespace in its code. A package
  almost never needs to use ::: for its own objects:
  'blaze' 'find_barcode' 'gene_quantification' 'isoform_identification'
  'minimap2_align' 'transcript_quantification'
* checking S3 generic/method consistency ... OK
* checking replacement functions ... OK
* checking foreign function calls ... OK
* checking R code for possible problems ... NOTE
BulkPipeline: no visible global function definition for ‘new’
BulkPipeline: no visible global function definition for ‘setNames’
MultiSampleSCPipeline: no visible global function definition for ‘new’
MultiSampleSCPipeline: no visible global function definition for
  ‘setNames’
SingleCellPipeline: no visible global function definition for ‘new’
SingleCellPipeline: no visible global function definition for
  ‘setNames’
addRowRanges: no visible global function definition for ‘head’
addRowRanges: no visible global function definition for ‘as’
add_gene_counts: no visible global function definition for ‘as’
cache_dir: no visible global function definition for ‘packageVersion’
chisq_test_by_gene: no visible global function definition for
  ‘chisq.test’
create_sce_from_dir: no visible global function definition for
  ‘setNames’
create_spe: no visible binding for global variable ‘barcode’
create_spe: no visible binding for global variable ‘in_tissue’
download_oarfish: no visible global function definition for
  ‘download.file’
download_oarfish: no visible global function definition for ‘unzip’
filter_coverage: no visible global function definition for
  ‘starts_with’
filter_coverage: no visible binding for global variable ‘filter_res’
find_variants: no visible global function definition for ‘setNames’
find_variants_grange: no visible binding for global variable
  ‘which_label’
find_variants_grange: no visible binding for global variable
  ‘nucleotide’
find_variants_grange: no visible binding for global variable ‘pos’
find_variants_grange: no visible binding for global variable ‘count’
find_variants_grange: no visible binding for global variable
  ‘counts_no_ins’
find_variants_grange: no visible binding for global variable ‘ref’
generate_sc_sce: no visible binding for global variable ‘FSM_match’
get_coverage: no visible binding for global variable ‘Freq’
homopolymer_pct : <anonymous>: no visible binding for global variable
  ‘Freq’
homopolymer_pct : <anonymous>: no visible binding for global variable
  ‘pct’
plot_coverage: no visible binding for global variable ‘tr_length’
plot_coverage: no visible binding for global variable ‘read_counts’
plot_coverage: no visible binding for global variable ‘total_counts’
plot_coverage: no visible binding for global variable ‘cumpct’
plot_coverage: no visible binding for global variable ‘length_bin’
plot_coverage: no visible binding for global variable ‘min_length’
plot_coverage: no visible binding for global variable ‘max_length’
plot_coverage: no visible global function definition for ‘head’
plot_coverage: no visible binding for global variable ‘transcript’
plot_demultiplex_raw: no visible binding for global variable ‘Sample’
plot_demultiplex_raw: no visible binding for global variable
  ‘CellBarcode’
plot_demultiplex_raw: no visible binding for global variable ‘UMI’
plot_demultiplex_raw: no visible binding for global variable
  ‘UMI_count’
plot_demultiplex_raw: no visible binding for global variable
  ‘barcode_rank’
plot_demultiplex_raw: no visible binding for global variable
  ‘FlankEditDist’
plot_demultiplex_raw: no visible binding for global variable ‘n_reads’
plot_demultiplex_raw: no visible binding for global variable
  ‘BarcodeEditDist’
plot_demultiplex_raw: no visible binding for global variable ‘total
  reads’
plot_demultiplex_raw: no visible binding for global variable
  ‘demultiplexed reads’
plot_demultiplex_raw: no visible binding for global variable ‘single
  match reads’
plot_demultiplex_raw: no visible binding for global variable
  ‘undemultiplexted reads’
plot_demultiplex_raw: no visible binding for global variable
  ‘multi-matching reads’
plot_demultiplex_raw: no visible binding for global variable ‘Type’
plot_demultiplex_raw: no visible binding for global variable ‘Reads’
plot_demultiplex_raw: no visible binding for global variable ‘input’
plot_demultiplex_raw: no visible binding for global variable ‘output’
plot_demultiplex_raw: no visible binding for global variable
  ‘read1_with_adapter’
plot_demultiplex_raw: no visible binding for global variable ‘Count’
plot_flagstat: no visible global function definition for ‘everything’
plot_flagstat: no visible binding for global variable ‘name’
plot_flagstat: no visible binding for global variable ‘value’
plot_isoform_reduced_dim: no visible binding for global variable ‘x’
plot_isoform_reduced_dim: no visible binding for global variable ‘y’
plot_isoform_reduced_dim: no visible binding for global variable ‘expr’
plot_spatial: no visible binding for global variable ‘imageX’
plot_spatial: no visible binding for global variable ‘imageY’
plot_spatial_feature: no visible binding for global variable ‘imageX’
plot_spatial_feature: no visible binding for global variable ‘imageY’
plot_spatial_feature: no visible binding for global variable ‘x’
plot_spatial_feature: no visible binding for global variable ‘y’
plot_spatial_feature: no visible global function definition for
  ‘scale_alpha_continuous’
plot_spatial_feature: no visible global function definition for
  ‘scale_colour_gradient’
plot_spatial_isoform: no visible global function definition for ‘head’
plot_spatial_pie: no visible global function definition for ‘setNames’
plot_spatial_pie: no visible global function definition for ‘head’
plot_spatial_pie: no visible binding for global variable ‘imageX’
plot_spatial_pie: no visible binding for global variable ‘imageY’
sc_gene_entropy: no visible global function definition for ‘as’
sc_genotype: no visible binding for global variable ‘allele’
sc_genotype: no visible binding for global variable ‘allele_count’
sc_genotype: no visible binding for global variable ‘barcode’
sc_genotype: no visible binding for global variable ‘pct’
sc_mutations: no visible binding for global variable ‘mutation_index’
sc_mutations: no visible binding for global variable ‘bam_index’
sc_plot_genotype: no visible global function definition for ‘setNames’
sc_plot_genotype: no visible binding for global variable ‘barcode’
sc_plot_genotype: no visible binding for global variable ‘genotype’
sc_plot_genotype: no visible binding for global variable ‘x’
sc_plot_genotype: no visible binding for global variable ‘y’
sc_transcript_usage_chisq: no visible global function definition for
  ‘as’
sc_transcript_usage_chisq: no visible binding for global variable
  ‘p.value’
sc_transcript_usage_chisq: no visible binding for global variable
  ‘adj.p.value’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘total’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘test’
sc_transcript_usage_permutation: no visible global function definition
  for ‘as’
sc_transcript_usage_permutation : <anonymous>: no visible global
  function definition for ‘as’
sc_transcript_usage_permutation : <anonymous> : <anonymous>: no visible
  global function definition for ‘na.omit’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘transcript’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘p.value’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘adj.p.value’
variant_count_tb: no visible binding for global variable ‘barcode’
variant_count_tb: no visible binding for global variable ‘allele_count’
variant_count_tb: no visible binding for global variable
  ‘cell_total_reads’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘expect_cell_number’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘fastq’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘demultiplexed_fastq’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘barcodes_file’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘outdir’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible global
  function definition for ‘setNames’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline : <anonymous>: no
  visible global function definition for ‘setNames’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘expect_cell_number’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘fastq’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘demultiplexed_fastq’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘barcodes_file’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘outdir’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible global
  function definition for ‘setNames’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘j’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘genome_bam’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘minimap2’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘samtools’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘threads’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘outdir’
plot_durations,FLAMES.Pipeline: no visible binding for global variable
  ‘step’
plot_durations,FLAMES.Pipeline: no visible binding for global variable
  ‘duration’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘j’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘transcriptome_assembly’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘transcriptome_bam’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘minimap2’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘samtools’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘outdir’
resume_FLAMES,FLAMES.Pipeline : <anonymous>: no visible global function
  definition for ‘capture.output’
run_FLAMES,FLAMES.Pipeline : <anonymous>: no visible global function
  definition for ‘capture.output’
Undefined global functions or variables:
  BarcodeEditDist CellBarcode Count FSM_match FlankEditDist Freq Reads
  Sample Type UMI UMI_count adj.p.value allele allele_count as
  bam_index barcode barcode_rank barcodes_file capture.output
  cell_total_reads chisq.test count counts_no_ins cumpct demultiplexed
  reads demultiplexed_fastq download.file duration everything
  expect_cell_number expr fastq filter_res genome_bam genotype head
  imageX imageY in_tissue input j length_bin max_length min_length
  minimap2 multi-matching reads mutation_index n_reads na.omit name new
  nucleotide outdir output p.value packageVersion pct pos
  read1_with_adapter read_counts ref samtools scale_alpha_continuous
  scale_colour_gradient setNames single match reads starts_with step
  test threads total total reads total_counts tr_length transcript
  transcriptome_assembly transcriptome_bam undemultiplexted reads unzip
  value which_label x y
Consider adding
  importFrom("base", "match", "single")
  importFrom("methods", "as", "new")
  importFrom("stats", "chisq.test", "na.omit", "setNames", "step")
  importFrom("utils", "capture.output", "download.file", "head",
             "packageVersion", "unzip")
to your NAMESPACE file (and ensure that your DESCRIPTION Imports field
contains 'methods').
* checking Rd files ... OK
* checking Rd metadata ... OK
* checking Rd cross-references ... OK
* checking for missing documentation entries ... OK
* checking for code/documentation mismatches ... OK
* checking Rd \usage sections ... OK
* checking Rd contents ... OK
* checking for unstated dependencies in examples ... OK
* checking contents of ‘data’ directory ... OK
* checking data for non-ASCII characters ... OK
* checking data for ASCII and uncompressed saves ... OK
* checking line endings in shell scripts ... OK
* checking line endings in C/C++/Fortran sources/headers ... OK
* checking line endings in Makefiles ... OK
* checking compilation flags in Makevars ... OK
* checking for GNU extensions in Makefiles ... INFO
GNU make is a SystemRequirements.
* checking for portable use of $(BLAS_LIBS) and $(LAPACK_LIBS) ... OK
* checking use of PKG_*FLAGS in Makefiles ... OK
* checking include directives in Makefiles ... OK
* checking compiled code ... NOTE
Note: information on .o files is not available
File ‘/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/FLAMES/libs/FLAMES.so’:
  Found ‘___assert_rtn’, possibly from ‘assert’ (C)
  Found ‘___stderrp’, possibly from ‘stderr’ (C)
  Found ‘___stdoutp’, possibly from ‘stdout’ (C)
  Found ‘_abort’, possibly from ‘abort’ (C)
  Found ‘_exit’, possibly from ‘exit’ (C)

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

See ‘Writing portable packages’ in the ‘Writing R Extensions’ manual.
* checking files in ‘vignettes’ ... OK
* checking examples ... OK
Examples with CPU (user + system) or elapsed time > 5s
                               user system elapsed
plot_isoform_reduced_dim     45.187  0.516  47.370
find_variants                45.155  0.416  45.659
sc_long_multisample_pipeline 27.647  3.806  24.659
MultiSampleSCPipeline        22.271  2.834  34.607
plot_isoform_heatmap         17.624  0.514  18.546
sc_DTU_analysis              15.744  1.697  14.661
sc_plot_genotype             16.965  0.401  15.606
create_sce_from_dir          11.886  1.984  11.988
blaze                        11.980  1.584  16.009
sc_long_pipeline              9.224  1.194   7.874
plot_isoforms                 8.338  0.038   8.460
bulk_long_pipeline            6.937  1.290   6.140
BulkPipeline                  7.234  0.773  22.942
SingleCellPipeline            6.778  0.341   4.558
plot_demultiplex              6.223  0.530   6.841
create_se_from_dir            5.691  0.952   7.026
plot_coverage                 6.228  0.300   6.637
sc_genotype                   5.133  1.145   4.845
plot_durations                5.087  0.445   5.898
sc_mutations                  4.596  0.655   4.271
resume_FLAMES                 4.786  0.458   5.418
experiment                    4.500  0.462   5.115
run_FLAMES                    4.502  0.458   5.179
* checking for unstated dependencies in ‘tests’ ... OK
* checking tests ...
  Running ‘testthat.R’
 ERROR
Running the tests in ‘tests/testthat.R’ failed.
Last 13 lines of output:
  experiment is NULL for run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
  ── Failure ('test-run_FLAMES.R:29:11'): run_FLAMES() completes and returns experiment for all pipeline types and option combinations ──
  Expected `is.null(experiment(result))` to be FALSE.
  Differences:
  `actual`:   TRUE 
  `expected`: FALSE
  
  experiment is NULL for run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
  
  [ FAIL 12 | WARN 106 | SKIP 0 | PASS 47 ]
  Error:
  ! Test failures.
  Execution halted
  /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 '
* 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: 1 ERROR, 5 NOTEs
See
  ‘/Users/biocbuild/bbs-3.22-bioc/meat/FLAMES.Rcheck/00check.log’
for details.

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


* installing to library ‘/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library’
* installing *source* package ‘FLAMES’ ...
** this is package ‘FLAMES’ version ‘2.4.1’
** using non-staged installation via StagedInstall field
** libs
/bin/sh: rustc: command not found
using C++ compiler: ‘Apple clang version 14.0.0 (clang-1400.0.29.202)’
using C++17
using SDK: ‘MacOSX11.3.1.sdk’
/bin/sh: rustc: command not found
clang++ -arch x86_64 -std=gnu++17 -I"/Library/Frameworks/R.framework/Resources/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/Rcpp/include' -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/Rhtslib/include' -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/testthat/include' -I/opt/R/x86_64/include    -fPIC  -falign-functions=64 -Wall -g -O2   -c RcppExports.cpp -o RcppExports.o
clang++ -arch x86_64 -std=gnu++17 -I"/Library/Frameworks/R.framework/Resources/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/Rcpp/include' -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/Rhtslib/include' -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/testthat/include' -I/opt/R/x86_64/include    -fPIC  -falign-functions=64 -Wall -g -O2   -c RcppFunctions.cpp -o RcppFunctions.o
clang++ -arch x86_64 -std=gnu++17 -I"/Library/Frameworks/R.framework/Resources/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/Rcpp/include' -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/Rhtslib/include' -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/testthat/include' -I/opt/R/x86_64/include    -fPIC  -falign-functions=64 -Wall -g -O2   -c classes/BamRecord.cpp -o classes/BamRecord.o
clang++ -arch x86_64 -std=gnu++17 -I"/Library/Frameworks/R.framework/Resources/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/Rcpp/include' -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/Rhtslib/include' -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/testthat/include' -I/opt/R/x86_64/include    -fPIC  -falign-functions=64 -Wall -g -O2   -c classes/GFFRecord.cpp -o classes/GFFRecord.o
clang++ -arch x86_64 -std=gnu++17 -I"/Library/Frameworks/R.framework/Resources/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/Rcpp/include' -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/Rhtslib/include' -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/testthat/include' -I/opt/R/x86_64/include    -fPIC  -falign-functions=64 -Wall -g -O2   -c classes/GeneAnnotationParser.cpp -o classes/GeneAnnotationParser.o
clang++ -arch x86_64 -std=gnu++17 -I"/Library/Frameworks/R.framework/Resources/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/Rcpp/include' -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/Rhtslib/include' -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/testthat/include' -I/opt/R/x86_64/include    -fPIC  -falign-functions=64 -Wall -g -O2   -c classes/Isoforms.cpp -o classes/Isoforms.o
clang++ -arch x86_64 -std=gnu++17 -I"/Library/Frameworks/R.framework/Resources/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/Rcpp/include' -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/Rhtslib/include' -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/testthat/include' -I/opt/R/x86_64/include    -fPIC  -falign-functions=64 -Wall -g -O2   -c classes/junctions.cpp -o classes/junctions.o
clang++ -arch x86_64 -std=gnu++17 -I"/Library/Frameworks/R.framework/Resources/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/Rcpp/include' -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/Rhtslib/include' -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/testthat/include' -I/opt/R/x86_64/include    -fPIC  -falign-functions=64 -Wall -g -O2   -c main-functions/find_isoform.cpp -o main-functions/find_isoform.o
clang++ -arch x86_64 -std=gnu++17 -I"/Library/Frameworks/R.framework/Resources/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/Rcpp/include' -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/Rhtslib/include' -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/testthat/include' -I/opt/R/x86_64/include    -fPIC  -falign-functions=64 -Wall -g -O2   -c main-functions/flexiplex.cpp -o main-functions/flexiplex.o
clang++ -arch x86_64 -std=gnu++17 -I"/Library/Frameworks/R.framework/Resources/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/Rcpp/include' -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/Rhtslib/include' -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/testthat/include' -I/opt/R/x86_64/include    -fPIC  -falign-functions=64 -Wall -g -O2   -c main-functions/get_transcript_seq.cpp -o main-functions/get_transcript_seq.o
clang++ -arch x86_64 -std=gnu++17 -I"/Library/Frameworks/R.framework/Resources/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/Rcpp/include' -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/Rhtslib/include' -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/testthat/include' -I/opt/R/x86_64/include    -fPIC  -falign-functions=64 -Wall -g -O2   -c main-functions/group_bam2isoform.cpp -o main-functions/group_bam2isoform.o
clang++ -arch x86_64 -std=gnu++17 -I"/Library/Frameworks/R.framework/Resources/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/Rcpp/include' -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/Rhtslib/include' -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/testthat/include' -I/opt/R/x86_64/include    -fPIC  -falign-functions=64 -Wall -g -O2   -c main-functions/pileup_readid.cpp -o main-functions/pileup_readid.o
main-functions/pileup_readid.cpp:86:16: warning: unused variable 'end' [-Wunused-variable]
  unsigned int end;
               ^
1 warning generated.
clang++ -arch x86_64 -std=gnu++17 -I"/Library/Frameworks/R.framework/Resources/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/Rcpp/include' -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/Rhtslib/include' -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/testthat/include' -I/opt/R/x86_64/include    -fPIC  -falign-functions=64 -Wall -g -O2   -c tests/test-junctions.cpp -o tests/test-junctions.o
clang++ -arch x86_64 -std=gnu++17 -I"/Library/Frameworks/R.framework/Resources/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/Rcpp/include' -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/Rhtslib/include' -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/testthat/include' -I/opt/R/x86_64/include    -fPIC  -falign-functions=64 -Wall -g -O2   -c tests/test-parsing.cpp -o tests/test-parsing.o
clang++ -arch x86_64 -std=gnu++17 -I"/Library/Frameworks/R.framework/Resources/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/Rcpp/include' -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/Rhtslib/include' -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/testthat/include' -I/opt/R/x86_64/include    -fPIC  -falign-functions=64 -Wall -g -O2   -c utility/cigars.cpp -o utility/cigars.o
clang++ -arch x86_64 -std=gnu++17 -I"/Library/Frameworks/R.framework/Resources/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/Rcpp/include' -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/Rhtslib/include' -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/testthat/include' -I/opt/R/x86_64/include    -fPIC  -falign-functions=64 -Wall -g -O2   -c utility/edlib-1.2.7/edlib.cpp -o utility/edlib-1.2.7/edlib.o
clang -arch x86_64 -I"/Library/Frameworks/R.framework/Resources/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/Rcpp/include' -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/Rhtslib/include' -I'/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/testthat/include' -I/opt/R/x86_64/include    -fPIC  -falign-functions=64 -Wall -g -O2  -c utility/bam.c -o utility/bam.o
clang++ -arch x86_64 -std=gnu++17 -dynamiclib -Wl,-headerpad_max_install_names -undefined dynamic_lookup -L/Library/Frameworks/R.framework/Resources/lib -L/opt/R/x86_64/lib -o FLAMES.so RcppExports.o RcppFunctions.o classes/BamRecord.o classes/GFFRecord.o classes/GeneAnnotationParser.o classes/Isoforms.o classes/junctions.o main-functions/find_isoform.o main-functions/flexiplex.o main-functions/get_transcript_seq.o main-functions/group_bam2isoform.o main-functions/pileup_readid.o tests/test-junctions.o tests/test-parsing.o utility/cigars.o utility/edlib-1.2.7/edlib.o utility/bam.o -pthread -lz /Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/Rhtslib/usrlib/libhts.a -lcurl -lbz2 -llzma -lz -F/Library/Frameworks/R.framework/.. -framework R
if test -e "/usr/bin/strip" & test -e "/bin/uname" & [[ `uname` == "Linux" ]] ; then /usr/bin/strip --strip-debug FLAMES.so; fi
Building for x86_64
(cd submodule/minimap2 && make -f Makefile CFLAGS="-falign-functions=64 -Wall -g -O2  -Wno-unused-result" minimap2)
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  main.c -o main.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  kthread.c -o kthread.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  kalloc.c -o kalloc.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  misc.c -o misc.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  bseq.c -o bseq.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  sketch.c -o sketch.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  sdust.c -o sdust.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  options.c -o options.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  index.c -o index.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  lchain.c -o lchain.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  align.c -o align.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  hit.c -o hit.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  seed.c -o seed.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  jump.c -o jump.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  map.c -o map.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  format.c -o format.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  pe.c -o pe.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  esterr.c -o esterr.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -DHAVE_KALLOC  splitidx.c -o splitidx.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -msse2 -DHAVE_KALLOC  ksw2_ll_sse.c -o ksw2_ll_sse.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -msse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH  ksw2_extz2_sse.c -o ksw2_extz2_sse41.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -msse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH  ksw2_extd2_sse.c -o ksw2_extd2_sse41.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -msse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH  ksw2_exts2_sse.c -o ksw2_exts2_sse41.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -msse2 -mno-sse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH -DKSW_SSE2_ONLY  ksw2_extz2_sse.c -o ksw2_extz2_sse2.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -msse2 -mno-sse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH -DKSW_SSE2_ONLY  ksw2_extd2_sse.c -o ksw2_extd2_sse2.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -msse2 -mno-sse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH -DKSW_SSE2_ONLY  ksw2_exts2_sse.c -o ksw2_exts2_sse2.o
cc -c -falign-functions=64 -Wall -g -O2  -Wno-unused-result -msse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH  ksw2_dispatch.c -o ksw2_dispatch.o
ar -csru libminimap2.a kthread.o kalloc.o misc.o bseq.o sketch.o sdust.o options.o index.o lchain.o align.o hit.o seed.o jump.o map.o format.o pe.o esterr.o splitidx.o ksw2_ll_sse.o ksw2_extz2_sse41.o ksw2_extd2_sse41.o ksw2_exts2_sse41.o ksw2_extz2_sse2.o ksw2_extd2_sse2.o ksw2_exts2_sse2.o ksw2_dispatch.o
cc -falign-functions=64 -Wall -g -O2  -Wno-unused-result main.o -o minimap2 -L. -lminimap2 -lm -lz -lpthread
echo "Installing binary to /Users/biocbuild/bbs-3.22-bioc/meat/FLAMES/src/../inst/bin"
Installing binary to /Users/biocbuild/bbs-3.22-bioc/meat/FLAMES/src/../inst/bin
mkdir -p ../inst/bin
cp submodule/minimap2/minimap2 ../inst/bin/
Cargo not found, skipping oarfish installation.
installing to /Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/FLAMES/libs
** R
** data
** inst
** byte-compile and prepare package for lazy loading
** help
*** installing help indices
*** copying figures
** building package indices
** installing vignettes
** testing if installed package can be loaded
* DONE (FLAMES)

R version 4.5.2 (2025-10-31) -- "[Not] Part in a Rumble"
Copyright (C) 2025 The R Foundation for Statistical Computing
Platform: x86_64-apple-darwin20

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

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

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

> # This file is part of the standard setup for testthat.
> # It is recommended that you do not modify it.
> #
> # Where should you do additional test configuration?
> # Learn more about the roles of various files in:
> # * https://r-pkgs.org/tests.html
> # * https://testthat.r-lib.org/reference/test_package.html#special-files
> 
> library(testthat)
> library(FLAMES)
> 
> test_check("FLAMES")
Writing configuration parameters to:  /tmp/Rtmpusyr8Y/fileb7571aa3bd42/config_file_46935.json 
Writing configuration parameters to:  /tmp/Rtmpusyr8Y/fileb7571aa3bd42/config_file_46935.json 
Writing configuration parameters to:  /tmp/Rtmpusyr8Y/fileb7571aa3bd42/config_file_46935.json 
Writing configuration parameters to:  /tmp/Rtmpusyr8Y/fileb75758b96f19/config_file_46935.json 
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpusyr8Y/fileb7573f3d7504/bc_allow.tsv
Number of known barcodes: 143
Processing file: /Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
Skipping TSO trimming...
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpusyr8Y/fileb75746d726e8/bc_allow.tsv
Number of known barcodes: 143
Processing file: /Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
Skipping TSO trimming...
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpusyr8Y/fileb75746d726e8/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpusyr8Y/fileb7577e9da598/musc_rps24_1.fastq
Searching for barcodes...
Processing file: /tmp/Rtmpusyr8Y/fileb7577e9da598/musc_rps24_2.fastq
Searching for barcodes...
Processing file: /tmp/Rtmpusyr8Y/fileb7577e9da598/musc_rps24_3.fastq
Searching for barcodes...
Processing file: /tmp/Rtmpusyr8Y/fileb7577e9da598/musc_rps24_4.fastq
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
Skipping TSO trimming...
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpusyr8Y/fileb7576455d3e1/bc_allow.tsv
Number of known barcodes: 143
Processing file: /Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
Skipping TSO trimming...
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpusyr8Y/fileb75778cdaf25/config_file_46935.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Fri Dec 19 04:35:14 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/Rtmpusyr8Y/fileb75778cdaf25/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/Rtmpusyr8Y/fileb75778cdaf25/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/Rtmpusyr8Y/fileb75778cdaf25/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Fri Dec 19 04:35:16 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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Saving _problems/test-run_FLAMES-32.R
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpusyr8Y/fileb7573192c2f3/config_file_46935.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Fri Dec 19 04:35:29 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/Rtmpusyr8Y/fileb7573192c2f3/sample1_align2genome.bam
sample2 ->/tmp/Rtmpusyr8Y/fileb7573192c2f3/sample2_align2genome.bam
sample3 ->/tmp/Rtmpusyr8Y/fileb7573192c2f3/sample3_align2genome.bam
-- Running step: isoform_identification @ Fri Dec 19 04:36:10 2025 -------------
Unzipping annotation file for bambu
Saving _problems/test-run_FLAMES-32.R
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpusyr8Y/fileb7577f7e4784/config_file_46935.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Fri Dec 19 04:36:12 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/Rtmpusyr8Y/fileb7577f7e4784/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/Rtmpusyr8Y/fileb7577f7e4784/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/Rtmpusyr8Y/fileb7577f7e4784/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Fri Dec 19 04:36:14 2025 -------------
Unzipping annotation file for bambu
Saving _problems/test-run_FLAMES-32.R
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpusyr8Y/fileb7576e76852f/config_file_46935.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Fri Dec 19 04:36:14 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/Rtmpusyr8Y/fileb7576e76852f/sample1_align2genome.bam
sample2 ->/tmp/Rtmpusyr8Y/fileb7576e76852f/sample2_align2genome.bam
sample3 ->/tmp/Rtmpusyr8Y/fileb7576e76852f/sample3_align2genome.bam
-- Running step: isoform_identification @ Fri Dec 19 04:36:53 2025 -------------
Unzipping annotation file for bambu
Saving _problems/test-run_FLAMES-32.R
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpusyr8Y/fileb7577c4f15c7/config_file_46935.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Fri Dec 19 04:36:54 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/Rtmpusyr8Y/fileb7577c4f15c7/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/Rtmpusyr8Y/fileb7577c4f15c7/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/Rtmpusyr8Y/fileb7577c4f15c7/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Fri Dec 19 04:36:56 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri Dec 19 04:36:59 2025 -------------------
Realigning sample sample1 -> /tmp/Rtmpusyr8Y/fileb7577c4f15c7/sample1_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample2 -> /tmp/Rtmpusyr8Y/fileb7577c4f15c7/sample2_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample3 -> /tmp/Rtmpusyr8Y/fileb7577c4f15c7/sample3_realign2transcript.bam
Skipped sorting BAM files.

-- Running step: transcript_quantification @ Fri Dec 19 04:37:01 2025 ----------
2025-12-19T09:37:01.456954Z  INFO oarfish: setting user-provided filter parameters.
2025-12-19T09:37:01.458857Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpusyr8Y/fileb7577c4f15c7/sample1_realign2transcript.bam, contains 10 reference sequences.
2025-12-19T09:37:01.458938Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-12-19T09:37:01.458981Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-12-19T09:37:01.459259Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-12-19T09:37:01.459314Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-12-19T09:37:01.475709Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-12-19T09:37:01.476223Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 707   │
│ aligned fraction too low        │ 2     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 98    │
╰─────────────────────────────────┴───────╯

2025-12-19T09:37:01.476356Z  INFO oarfish::bulk: Total number of alignment records : 125
2025-12-19T09:37:01.476389Z  INFO oarfish::bulk: number of aligned reads : 98
2025-12-19T09:37:01.476411Z  INFO oarfish::bulk: number of unique alignments : 86
2025-12-19T09:37:01.486946Z  INFO oarfish: oarfish completed successfully.
2025-12-19T09:37:01.535213Z  INFO oarfish: setting user-provided filter parameters.
2025-12-19T09:37:01.540571Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpusyr8Y/fileb7577c4f15c7/sample2_realign2transcript.bam, contains 10 reference sequences.
2025-12-19T09:37:01.540658Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-12-19T09:37:01.540687Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-12-19T09:37:01.540932Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-12-19T09:37:01.540975Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-12-19T09:37:01.573231Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-12-19T09:37:01.574055Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 701   │
│ aligned fraction too low        │ 3     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 97    │
╰─────────────────────────────────┴───────╯

2025-12-19T09:37:01.574169Z  INFO oarfish::bulk: Total number of alignment records : 136
2025-12-19T09:37:01.574199Z  INFO oarfish::bulk: number of aligned reads : 97
2025-12-19T09:37:01.574222Z  INFO oarfish::bulk: number of unique alignments : 79
2025-12-19T09:37:01.583284Z  INFO oarfish: oarfish completed successfully.
2025-12-19T09:37:01.625744Z  INFO oarfish: setting user-provided filter parameters.
2025-12-19T09:37:01.627474Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpusyr8Y/fileb7577c4f15c7/sample3_realign2transcript.bam, contains 10 reference sequences.
2025-12-19T09:37:01.627543Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-12-19T09:37:01.627579Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-12-19T09:37:01.627839Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-12-19T09:37:01.627915Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-12-19T09:37:01.644526Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-12-19T09:37:01.645157Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 1060  │
│ aligned fraction too low        │ 6     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 187   │
╰─────────────────────────────────┴───────╯

2025-12-19T09:37:01.645254Z  INFO oarfish::bulk: Total number of alignment records : 272
2025-12-19T09:37:01.645283Z  INFO oarfish::bulk: number of aligned reads : 187
2025-12-19T09:37:01.645303Z  INFO oarfish::bulk: number of unique alignments : 140
2025-12-19T09:37:01.665992Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpusyr8Y/fileb75741eeafb6/config_file_46935.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Fri Dec 19 04:37:02 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/Rtmpusyr8Y/fileb75741eeafb6/sample1_align2genome.bam
sample2 ->/tmp/Rtmpusyr8Y/fileb75741eeafb6/sample2_align2genome.bam
sample3 ->/tmp/Rtmpusyr8Y/fileb75741eeafb6/sample3_align2genome.bam
-- Running step: isoform_identification @ Fri Dec 19 04:37:41 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri Dec 19 04:37:42 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/Rtmpusyr8Y/fileb75741eeafb6/sample1_realign2transcript.bam
sample2 ->/tmp/Rtmpusyr8Y/fileb75741eeafb6/sample2_realign2transcript.bam
sample3 ->/tmp/Rtmpusyr8Y/fileb75741eeafb6/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Fri Dec 19 04:38:22 2025 ----------
2025-12-19T09:38:22.446129Z  INFO oarfish: setting user-provided filter parameters.
2025-12-19T09:38:22.449842Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpusyr8Y/fileb75741eeafb6/sample1_realign2transcript.bam, contains 10 reference sequences.
2025-12-19T09:38:22.449941Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-12-19T09:38:22.449985Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-12-19T09:38:22.450260Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-12-19T09:38:22.450318Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-12-19T09:38:22.511024Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-12-19T09:38:22.511580Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 707   │
│ aligned fraction too low        │ 2     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 98    │
╰─────────────────────────────────┴───────╯

2025-12-19T09:38:22.511712Z  INFO oarfish::bulk: Total number of alignment records : 125
2025-12-19T09:38:22.511749Z  INFO oarfish::bulk: number of aligned reads : 98
2025-12-19T09:38:22.511775Z  INFO oarfish::bulk: number of unique alignments : 86
2025-12-19T09:38:22.521360Z  INFO oarfish: oarfish completed successfully.
2025-12-19T09:38:22.563948Z  INFO oarfish: setting user-provided filter parameters.
2025-12-19T09:38:22.566973Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpusyr8Y/fileb75741eeafb6/sample2_realign2transcript.bam, contains 10 reference sequences.
2025-12-19T09:38:22.567058Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-12-19T09:38:22.567089Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-12-19T09:38:22.567324Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-12-19T09:38:22.567370Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-12-19T09:38:22.594080Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-12-19T09:38:22.594697Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 701   │
│ aligned fraction too low        │ 3     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 97    │
╰─────────────────────────────────┴───────╯

2025-12-19T09:38:22.594796Z  INFO oarfish::bulk: Total number of alignment records : 136
2025-12-19T09:38:22.594829Z  INFO oarfish::bulk: number of aligned reads : 97
2025-12-19T09:38:22.594856Z  INFO oarfish::bulk: number of unique alignments : 79
2025-12-19T09:38:22.604158Z  INFO oarfish: oarfish completed successfully.
2025-12-19T09:38:22.644214Z  INFO oarfish: setting user-provided filter parameters.
2025-12-19T09:38:22.645073Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpusyr8Y/fileb75741eeafb6/sample3_realign2transcript.bam, contains 10 reference sequences.
2025-12-19T09:38:22.645194Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-12-19T09:38:22.645231Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-12-19T09:38:22.645633Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-12-19T09:38:22.645699Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-12-19T09:38:22.674925Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-12-19T09:38:22.675462Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 1060  │
│ aligned fraction too low        │ 6     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 187   │
╰─────────────────────────────────┴───────╯

2025-12-19T09:38:22.675565Z  INFO oarfish::bulk: Total number of alignment records : 272
2025-12-19T09:38:22.675599Z  INFO oarfish::bulk: number of aligned reads : 187
2025-12-19T09:38:22.675624Z  INFO oarfish::bulk: number of unique alignments : 140
2025-12-19T09:38:22.685210Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpusyr8Y/fileb75777c21521/config_file_46935.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Fri Dec 19 04:38:23 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/Rtmpusyr8Y/fileb75777c21521/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/Rtmpusyr8Y/fileb75777c21521/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/Rtmpusyr8Y/fileb75777c21521/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Fri Dec 19 04:38:25 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri Dec 19 04:38:27 2025 -------------------
Realigning sample sample1 -> /tmp/Rtmpusyr8Y/fileb75777c21521/sample1_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample2 -> /tmp/Rtmpusyr8Y/fileb75777c21521/sample2_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample3 -> /tmp/Rtmpusyr8Y/fileb75777c21521/sample3_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Fri Dec 19 04:38:28 2025 ----------
04:38:28 Fri Dec 19 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpusyr8Y/fileb75772fcf9a2/config_file_46935.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Fri Dec 19 04:38:32 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/Rtmpusyr8Y/fileb75772fcf9a2/sample1_align2genome.bam
sample2 ->/tmp/Rtmpusyr8Y/fileb75772fcf9a2/sample2_align2genome.bam
sample3 ->/tmp/Rtmpusyr8Y/fileb75772fcf9a2/sample3_align2genome.bam
-- Running step: isoform_identification @ Fri Dec 19 04:39:13 2025 -------------
Inputs:  ['/tmp/Rtmpusyr8Y/fileb75777c21521/sample3_realign2transcript.bam', '/tmp/Rtmpusyr8Y/fileb75777c21521/sample2_realign2transcript.bam', '/tmp/Rtmpusyr8Y/fileb75777c21521/sample1_realign2transcript.bam'] /tmp/Rtmpusyr8Y/fileb75777c21521/transcript_assembly.fa.fai 5 0.4 0.4
	Counter({'counted_reads': 391, 'not_enough_coverage': 2})
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri Dec 19 04:39:14 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/Rtmpusyr8Y/fileb75772fcf9a2/sample1_realign2transcript.bam
sample2 ->/tmp/Rtmpusyr8Y/fileb75772fcf9a2/sample2_realign2transcript.bam
sample3 ->/tmp/Rtmpusyr8Y/fileb75772fcf9a2/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Fri Dec 19 04:39:54 2025 ----------
04:39:54 Fri Dec 19 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Inputs:  ['/tmp/Rtmpusyr8Y/fileb75772fcf9a2/sample3_realign2transcript.bam', '/tmp/Rtmpusyr8Y/fileb75772fcf9a2/sample2_realign2transcript.bam', '/tmp/Rtmpusyr8Y/fileb75772fcf9a2/sample1_realign2transcript.bam'] /tmp/Rtmpusyr8Y/fileb75772fcf9a2/transcript_assembly.fa.fai 5 0.4 0.4
	Counter({'counted_reads': 391, 'not_enough_coverage': 2})
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpusyr8Y/fileb7572f4025cc/config_file_46935.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Fri Dec 19 04:39:57 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpusyr8Y/fileb7572f4025cc/bc_allow.tsv
Number of known barcodes: 143
Processing file: /Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Fri Dec 19 04:39:58 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmpusyr8Y/fileb7572f4025cc/matched_reads.fastq.gz -> /tmp/Rtmpusyr8Y/fileb7572f4025cc/align2genome.bam
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: isoform_identification @ Fri Dec 19 04:39:58 2025 -------------
Unzipping annotation file for bambu
Saving _problems/test-run_FLAMES-32.R
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpusyr8Y/fileb75732020bad/config_file_46935.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Fri Dec 19 04:39:59 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpusyr8Y/fileb75732020bad/bc_allow.tsv
Number of known barcodes: 143
Processing file: /Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Fri Dec 19 04:40:00 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmpusyr8Y/fileb75732020bad/matched_reads.fastq.gz ->/tmp/Rtmpusyr8Y/fileb75732020bad/align2genome.bam
-- Running step: isoform_identification @ Fri Dec 19 04:40:36 2025 -------------
Unzipping annotation file for bambu
Saving _problems/test-run_FLAMES-32.R
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpusyr8Y/fileb7577579b03/config_file_46935.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Fri Dec 19 04:40:37 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpusyr8Y/fileb7577579b03/bc_allow.tsv
Number of known barcodes: 143
Processing file: /Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Fri Dec 19 04:40:37 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmpusyr8Y/fileb7577579b03/matched_reads.fastq.gz -> /tmp/Rtmpusyr8Y/fileb7577579b03/align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: isoform_identification @ Fri Dec 19 04:40:38 2025 -------------
Unzipping annotation file for bambu
Saving _problems/test-run_FLAMES-32.R
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpusyr8Y/fileb75770895bb6/config_file_46935.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Fri Dec 19 04:40:39 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpusyr8Y/fileb75770895bb6/bc_allow.tsv
Number of known barcodes: 143
Processing file: /Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Fri Dec 19 04:40:39 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmpusyr8Y/fileb75770895bb6/matched_reads.fastq.gz ->/tmp/Rtmpusyr8Y/fileb75770895bb6/align2genome.bam
-- Running step: isoform_identification @ Fri Dec 19 04:41:17 2025 -------------
Unzipping annotation file for bambu
Saving _problems/test-run_FLAMES-32.R
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpusyr8Y/fileb7573e328796/config_file_46935.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Fri Dec 19 04:41:18 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpusyr8Y/fileb7573e328796/bc_allow.tsv
Number of known barcodes: 143
Processing file: /Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Fri Dec 19 04:41:19 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmpusyr8Y/fileb7573e328796/matched_reads.fastq.gz -> /tmp/Rtmpusyr8Y/fileb7573e328796/align2genome.bam
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: isoform_identification @ Fri Dec 19 04:41:19 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri Dec 19 04:41:20 2025 -------------------
Checking for fastq file(s) /Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpusyr8Y/fileb7573e328796/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpusyr8Y/fileb7573e328796/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/Rtmpusyr8Y/fileb7573e328796/matched_reads.fastq.gz -> /tmp/Rtmpusyr8Y/fileb7573e328796/realign2transcript.bam
Sorting BAM files by 8 with CB threads...

[bam_sort_core] merging from 0 files and 8 in-memory blocks...
-- Running step: transcript_quantification @ Fri Dec 19 04:41:21 2025 ----------
2025-12-19T09:41:21.463143Z  INFO oarfish: setting user-provided filter parameters.
2025-12-19T09:41:21.465021Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpusyr8Y/fileb7573e328796/realign2transcript.bam, contains 10 reference sequences.
2025-12-19T09:41:21.465160Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-12-19T09:41:21.465196Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-12-19T09:41:21.465515Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-12-19T09:41:21.465572Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-12-19T09:41:21.574260Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpusyr8Y/fileb7574942b582/config_file_46935.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Fri Dec 19 04:41:23 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpusyr8Y/fileb7574942b582/bc_allow.tsv
Number of known barcodes: 143
Processing file: /Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Fri Dec 19 04:41:24 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmpusyr8Y/fileb7574942b582/matched_reads.fastq.gz ->/tmp/Rtmpusyr8Y/fileb7574942b582/align2genome.bam
-- Running step: isoform_identification @ Fri Dec 19 04:42:01 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri Dec 19 04:42:02 2025 -------------------
Checking for fastq file(s) /Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpusyr8Y/fileb7574942b582/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpusyr8Y/fileb7574942b582/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/Rtmpusyr8Y/fileb7574942b582/matched_reads.fastq.gz ->/tmp/Rtmpusyr8Y/fileb7574942b582/realign2transcript.bam
-- Running step: transcript_quantification @ Fri Dec 19 04:42:38 2025 ----------
2025-12-19T09:42:38.975605Z  INFO oarfish: setting user-provided filter parameters.
2025-12-19T09:42:38.986328Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpusyr8Y/fileb7574942b582/realign2transcript.bam, contains 10 reference sequences.
2025-12-19T09:42:38.986405Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-12-19T09:42:38.986447Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-12-19T09:42:38.986706Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-12-19T09:42:38.986763Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-12-19T09:42:39.087098Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpusyr8Y/fileb757780d3a31/config_file_46935.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Fri Dec 19 04:42:41 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpusyr8Y/fileb757780d3a31/bc_allow.tsv
Number of known barcodes: 143
Processing file: /Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Fri Dec 19 04:42:41 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmpusyr8Y/fileb757780d3a31/matched_reads.fastq.gz -> /tmp/Rtmpusyr8Y/fileb757780d3a31/align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: isoform_identification @ Fri Dec 19 04:42:42 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri Dec 19 04:42:43 2025 -------------------
Checking for fastq file(s) /Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpusyr8Y/fileb757780d3a31/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpusyr8Y/fileb757780d3a31/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/Rtmpusyr8Y/fileb757780d3a31/matched_reads.fastq.gz -> /tmp/Rtmpusyr8Y/fileb757780d3a31/realign2transcript.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 8 threads...

[bam_sort_core] merging from 0 files and 8 in-memory blocks...
Indexing bam files
-- Running step: transcript_quantification @ Fri Dec 19 04:42:43 2025 ----------
04:42:43 Fri Dec 19 2025 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpusyr8Y/fileb7573bd879c8/config_file_46935.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Fri Dec 19 04:42:46 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpusyr8Y/fileb7573bd879c8/bc_allow.tsv
Number of known barcodes: 143
Processing file: /Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Fri Dec 19 04:42:46 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmpusyr8Y/fileb7573bd879c8/matched_reads.fastq.gz ->/tmp/Rtmpusyr8Y/fileb7573bd879c8/align2genome.bam
-- Running step: isoform_identification @ Fri Dec 19 04:43:23 2025 -------------
	Counter({'counted_reads': 368, 'unmapped': 4})
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri Dec 19 04:43:24 2025 -------------------
Checking for fastq file(s) /Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpusyr8Y/fileb7573bd879c8/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpusyr8Y/fileb7573bd879c8/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/Rtmpusyr8Y/fileb7573bd879c8/matched_reads.fastq.gz ->/tmp/Rtmpusyr8Y/fileb7573bd879c8/realign2transcript.bam
-- Running step: transcript_quantification @ Fri Dec 19 04:43:59 2025 ----------
04:43:59 Fri Dec 19 2025 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
	Counter({'counted_reads': 368, 'unmapped': 4})
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpusyr8Y/fileb7571761d59c/config_file_46935.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Fri Dec 19 04:44:03 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpusyr8Y/fileb7571761d59c/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpusyr8Y/fileb7571761d59c/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpusyr8Y/fileb7571761d59c/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpusyr8Y/fileb7571761d59c/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpusyr8Y/fileb7571761d59c/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpusyr8Y/fileb7571761d59c/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 92
Number of reads with exactly one barcode match: 91
Number of chimera reads: 1
All done!
Reads	Barcodes
4	1
3	9
2	9
1	44
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpusyr8Y/fileb7571761d59c/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpusyr8Y/fileb7571761d59c/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 95
Number of reads with exactly one barcode match: 94
Number of chimera reads: 0
All done!
Reads	Barcodes
4	2
3	3
2	16
1	47
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpusyr8Y/fileb7571761d59c/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpusyr8Y/fileb7571761d59c/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Fri Dec 19 04:44:05 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmpusyr8Y/fileb7571761d59c/sampleA_matched_reads.fastq.gz -> /tmp/Rtmpusyr8Y/fileb7571761d59c/sampleA_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmpusyr8Y/fileb7571761d59c/sample1_matched_reads.fastq.gz -> /tmp/Rtmpusyr8Y/fileb7571761d59c/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmpusyr8Y/fileb7571761d59c/sample2_matched_reads.fastq.gz -> /tmp/Rtmpusyr8Y/fileb7571761d59c/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmpusyr8Y/fileb7571761d59c/sample3_matched_reads.fastq.gz -> /tmp/Rtmpusyr8Y/fileb7571761d59c/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Fri Dec 19 04:44:08 2025 ----------------
04:44:08 Fri Dec 19 2025 quantify genes 
Using BAM(s): '/tmp/Rtmpusyr8Y/fileb7571761d59c/sampleA_align2genome.bam',
'/tmp/Rtmpusyr8Y/fileb7571761d59c/sample1_align2genome.bam',
'/tmp/Rtmpusyr8Y/fileb7571761d59c/sample2_align2genome.bam', and
'/tmp/Rtmpusyr8Y/fileb7571761d59c/sample3_align2genome.bam'
parsing /tmp/Rtmpusyr8Y/fileb7571761d59c/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  7.29gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  7.26gene_group/s]
2025-12-19 04:44:12.169 R[46935:142299255] XType: com.apple.fonts is not accessible.
2025-12-19 04:44:12.169 R[46935:142299255] XType: XTFontStaticRegistry is enabled.
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 200140.48Read/s]
parsing /tmp/Rtmpusyr8Y/fileb7571761d59c/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 15.95gene_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, 652586.51Read/s]
parsing /tmp/Rtmpusyr8Y/fileb7571761d59c/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 15.92gene_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, 718153.55Read/s]
parsing /tmp/Rtmpusyr8Y/fileb7571761d59c/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 11.78gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 349408.86Read/s]
-- Running step: isoform_identification @ Fri Dec 19 04:44:13 2025 -------------
Unzipping annotation file for bambu
Saving _problems/test-run_FLAMES-32.R
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpusyr8Y/fileb75745cb37ea/config_file_46935.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Fri Dec 19 04:44:14 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpusyr8Y/fileb75745cb37ea/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpusyr8Y/fileb75745cb37ea/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpusyr8Y/fileb75745cb37ea/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpusyr8Y/fileb75745cb37ea/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpusyr8Y/fileb75745cb37ea/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpusyr8Y/fileb75745cb37ea/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 92
Number of reads with exactly one barcode match: 91
Number of chimera reads: 1
All done!
Reads	Barcodes
4	1
3	9
2	9
1	44
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpusyr8Y/fileb75745cb37ea/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpusyr8Y/fileb75745cb37ea/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 95
Number of reads with exactly one barcode match: 94
Number of chimera reads: 0
All done!
Reads	Barcodes
4	2
3	3
2	16
1	47
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpusyr8Y/fileb75745cb37ea/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpusyr8Y/fileb75745cb37ea/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Fri Dec 19 04:44:16 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmpusyr8Y/fileb75745cb37ea/sampleA_matched_reads.fastq.gz ->/tmp/Rtmpusyr8Y/fileb75745cb37ea/sampleA_align2genome.bam
/tmp/Rtmpusyr8Y/fileb75745cb37ea/sample1_matched_reads.fastq.gz ->/tmp/Rtmpusyr8Y/fileb75745cb37ea/sample1_align2genome.bam
/tmp/Rtmpusyr8Y/fileb75745cb37ea/sample2_matched_reads.fastq.gz ->/tmp/Rtmpusyr8Y/fileb75745cb37ea/sample2_align2genome.bam
/tmp/Rtmpusyr8Y/fileb75745cb37ea/sample3_matched_reads.fastq.gz ->/tmp/Rtmpusyr8Y/fileb75745cb37ea/sample3_align2genome.bam
-- Running step: gene_quantification @ Fri Dec 19 04:44:57 2025 ----------------
04:44:57 Fri Dec 19 2025 quantify genes 
Using BAM(s): '/tmp/Rtmpusyr8Y/fileb75745cb37ea/sampleA_align2genome.bam',
'/tmp/Rtmpusyr8Y/fileb75745cb37ea/sample1_align2genome.bam',
'/tmp/Rtmpusyr8Y/fileb75745cb37ea/sample2_align2genome.bam', and
'/tmp/Rtmpusyr8Y/fileb75745cb37ea/sample3_align2genome.bam'
parsing /tmp/Rtmpusyr8Y/fileb75745cb37ea/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  7.11gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  7.07gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 161570.44Read/s]
parsing /tmp/Rtmpusyr8Y/fileb75745cb37ea/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 17.76gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 713948.39Read/s]
parsing /tmp/Rtmpusyr8Y/fileb75745cb37ea/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 16.18gene_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, 414916.11Read/s]
parsing /tmp/Rtmpusyr8Y/fileb75745cb37ea/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 11.74gene_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, 344540.97Read/s]
-- Running step: isoform_identification @ Fri Dec 19 04:44:59 2025 -------------
Unzipping annotation file for bambu
Saving _problems/test-run_FLAMES-32.R
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpusyr8Y/fileb75747610328/config_file_46935.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Fri Dec 19 04:45:00 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpusyr8Y/fileb75747610328/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpusyr8Y/fileb75747610328/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpusyr8Y/fileb75747610328/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpusyr8Y/fileb75747610328/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpusyr8Y/fileb75747610328/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpusyr8Y/fileb75747610328/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 92
Number of reads with exactly one barcode match: 91
Number of chimera reads: 1
All done!
Reads	Barcodes
4	1
3	9
2	9
1	44
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpusyr8Y/fileb75747610328/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpusyr8Y/fileb75747610328/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 95
Number of reads with exactly one barcode match: 94
Number of chimera reads: 0
All done!
Reads	Barcodes
4	2
3	3
2	16
1	47
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpusyr8Y/fileb75747610328/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpusyr8Y/fileb75747610328/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Fri Dec 19 04:45:03 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmpusyr8Y/fileb75747610328/sampleA_matched_reads.fastq.gz -> /tmp/Rtmpusyr8Y/fileb75747610328/sampleA_align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmpusyr8Y/fileb75747610328/sample1_matched_reads.fastq.gz -> /tmp/Rtmpusyr8Y/fileb75747610328/sample1_align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmpusyr8Y/fileb75747610328/sample2_matched_reads.fastq.gz -> /tmp/Rtmpusyr8Y/fileb75747610328/sample2_align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmpusyr8Y/fileb75747610328/sample3_matched_reads.fastq.gz -> /tmp/Rtmpusyr8Y/fileb75747610328/sample3_align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Fri Dec 19 04:45:06 2025 ----------------
04:45:06 Fri Dec 19 2025 quantify genes 
Using BAM(s): '/tmp/Rtmpusyr8Y/fileb75747610328/sampleA_align2genome.bam',
'/tmp/Rtmpusyr8Y/fileb75747610328/sample1_align2genome.bam',
'/tmp/Rtmpusyr8Y/fileb75747610328/sample2_align2genome.bam', and
'/tmp/Rtmpusyr8Y/fileb75747610328/sample3_align2genome.bam'
parsing /tmp/Rtmpusyr8Y/fileb75747610328/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  7.91gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  7.87gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 200818.92Read/s]
parsing /tmp/Rtmpusyr8Y/fileb75747610328/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 17.71gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 726160.66Read/s]
parsing /tmp/Rtmpusyr8Y/fileb75747610328/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 15.95gene_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, 673675.55Read/s]
parsing /tmp/Rtmpusyr8Y/fileb75747610328/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 12.84gene_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, 373903.87Read/s]
-- Running step: isoform_identification @ Fri Dec 19 04:45:07 2025 -------------
Unzipping annotation file for bambu
Saving _problems/test-run_FLAMES-32.R
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpusyr8Y/fileb7575cf409c9/config_file_46935.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Fri Dec 19 04:45:08 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpusyr8Y/fileb7575cf409c9/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpusyr8Y/fileb7575cf409c9/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpusyr8Y/fileb7575cf409c9/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpusyr8Y/fileb7575cf409c9/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpusyr8Y/fileb7575cf409c9/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpusyr8Y/fileb7575cf409c9/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 92
Number of reads with exactly one barcode match: 91
Number of chimera reads: 1
All done!
Reads	Barcodes
4	1
3	9
2	9
1	44
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpusyr8Y/fileb7575cf409c9/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpusyr8Y/fileb7575cf409c9/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 95
Number of reads with exactly one barcode match: 94
Number of chimera reads: 0
All done!
Reads	Barcodes
4	2
3	3
2	16
1	47
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpusyr8Y/fileb7575cf409c9/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpusyr8Y/fileb7575cf409c9/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Fri Dec 19 04:45:10 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmpusyr8Y/fileb7575cf409c9/sampleA_matched_reads.fastq.gz ->/tmp/Rtmpusyr8Y/fileb7575cf409c9/sampleA_align2genome.bam
/tmp/Rtmpusyr8Y/fileb7575cf409c9/sample1_matched_reads.fastq.gz ->/tmp/Rtmpusyr8Y/fileb7575cf409c9/sample1_align2genome.bam
/tmp/Rtmpusyr8Y/fileb7575cf409c9/sample2_matched_reads.fastq.gz ->/tmp/Rtmpusyr8Y/fileb7575cf409c9/sample2_align2genome.bam
/tmp/Rtmpusyr8Y/fileb7575cf409c9/sample3_matched_reads.fastq.gz ->/tmp/Rtmpusyr8Y/fileb7575cf409c9/sample3_align2genome.bam
-- Running step: gene_quantification @ Fri Dec 19 04:45:50 2025 ----------------
04:45:50 Fri Dec 19 2025 quantify genes 
Using BAM(s): '/tmp/Rtmpusyr8Y/fileb7575cf409c9/sampleA_align2genome.bam',
'/tmp/Rtmpusyr8Y/fileb7575cf409c9/sample1_align2genome.bam',
'/tmp/Rtmpusyr8Y/fileb7575cf409c9/sample2_align2genome.bam', and
'/tmp/Rtmpusyr8Y/fileb7575cf409c9/sample3_align2genome.bam'
parsing /tmp/Rtmpusyr8Y/fileb7575cf409c9/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  7.21gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  7.17gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 184829.73Read/s]
parsing /tmp/Rtmpusyr8Y/fileb7575cf409c9/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 18.07gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 511650.24Read/s]
parsing /tmp/Rtmpusyr8Y/fileb7575cf409c9/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  5.87gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  5.85gene_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, 663865.78Read/s]
parsing /tmp/Rtmpusyr8Y/fileb7575cf409c9/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 11.36gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 368179.78Read/s]
-- Running step: isoform_identification @ Fri Dec 19 04:45:52 2025 -------------
Unzipping annotation file for bambu
Saving _problems/test-run_FLAMES-32.R
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpusyr8Y/fileb7576ae8b05a/config_file_46935.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Fri Dec 19 04:45:54 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpusyr8Y/fileb7576ae8b05a/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpusyr8Y/fileb7576ae8b05a/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpusyr8Y/fileb7576ae8b05a/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpusyr8Y/fileb7576ae8b05a/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpusyr8Y/fileb7576ae8b05a/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpusyr8Y/fileb7576ae8b05a/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 92
Number of reads with exactly one barcode match: 91
Number of chimera reads: 1
All done!
Reads	Barcodes
4	1
3	9
2	9
1	44
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpusyr8Y/fileb7576ae8b05a/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpusyr8Y/fileb7576ae8b05a/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 95
Number of reads with exactly one barcode match: 94
Number of chimera reads: 0
All done!
Reads	Barcodes
4	2
3	3
2	16
1	47
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpusyr8Y/fileb7576ae8b05a/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpusyr8Y/fileb7576ae8b05a/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Fri Dec 19 04:45:56 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmpusyr8Y/fileb7576ae8b05a/sampleA_matched_reads.fastq.gz -> /tmp/Rtmpusyr8Y/fileb7576ae8b05a/sampleA_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmpusyr8Y/fileb7576ae8b05a/sample1_matched_reads.fastq.gz -> /tmp/Rtmpusyr8Y/fileb7576ae8b05a/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmpusyr8Y/fileb7576ae8b05a/sample2_matched_reads.fastq.gz -> /tmp/Rtmpusyr8Y/fileb7576ae8b05a/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmpusyr8Y/fileb7576ae8b05a/sample3_matched_reads.fastq.gz -> /tmp/Rtmpusyr8Y/fileb7576ae8b05a/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Fri Dec 19 04:45:59 2025 ----------------
04:45:59 Fri Dec 19 2025 quantify genes 
Using BAM(s): '/tmp/Rtmpusyr8Y/fileb7576ae8b05a/sampleA_align2genome.bam',
'/tmp/Rtmpusyr8Y/fileb7576ae8b05a/sample1_align2genome.bam',
'/tmp/Rtmpusyr8Y/fileb7576ae8b05a/sample2_align2genome.bam', and
'/tmp/Rtmpusyr8Y/fileb7576ae8b05a/sample3_align2genome.bam'
parsing /tmp/Rtmpusyr8Y/fileb7576ae8b05a/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  7.01gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  6.99gene_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, 197893.07Read/s]
parsing /tmp/Rtmpusyr8Y/fileb7576ae8b05a/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 16.76gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 729697.98Read/s]
parsing /tmp/Rtmpusyr8Y/fileb7576ae8b05a/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 19.09gene_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, 690215.90Read/s]
parsing /tmp/Rtmpusyr8Y/fileb7576ae8b05a/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 11.94gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 270446.71Read/s]
-- Running step: isoform_identification @ Fri Dec 19 04:46:01 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri Dec 19 04:46:02 2025 -------------------
Checking for fastq file(s) /tmp/Rtmpusyr8Y/fileb7576ae8b05a/fastq, /tmp/Rtmpusyr8Y/fileb7576ae8b05a/fastq/sample1.fq.gz, /tmp/Rtmpusyr8Y/fileb7576ae8b05a/fastq/sample2.fq.gz, /tmp/Rtmpusyr8Y/fileb7576ae8b05a/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpusyr8Y/fileb7576ae8b05a/sampleA_matched_reads.fastq.gz, /tmp/Rtmpusyr8Y/fileb7576ae8b05a/sample1_matched_reads.fastq.gz, /tmp/Rtmpusyr8Y/fileb7576ae8b05a/sample2_matched_reads.fastq.gz, /tmp/Rtmpusyr8Y/fileb7576ae8b05a/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpusyr8Y/fileb7576ae8b05a/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmpusyr8Y/fileb7576ae8b05a/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmpusyr8Y/fileb7576ae8b05a/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmpusyr8Y/fileb7576ae8b05a/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/Rtmpusyr8Y/fileb7576ae8b05a/sampleA_matched_reads_dedup.fastq.gz -> /tmp/Rtmpusyr8Y/fileb7576ae8b05a/sampleA_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/Rtmpusyr8Y/fileb7576ae8b05a/sample1_matched_reads_dedup.fastq.gz -> /tmp/Rtmpusyr8Y/fileb7576ae8b05a/sample1_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/Rtmpusyr8Y/fileb7576ae8b05a/sample2_matched_reads_dedup.fastq.gz -> /tmp/Rtmpusyr8Y/fileb7576ae8b05a/sample2_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/Rtmpusyr8Y/fileb7576ae8b05a/sample3_matched_reads_dedup.fastq.gz -> /tmp/Rtmpusyr8Y/fileb7576ae8b05a/sample3_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

-- Running step: transcript_quantification @ Fri Dec 19 04:46:06 2025 ----------
2025-12-19T09:46:06.203093Z  INFO oarfish: setting user-provided filter parameters.
2025-12-19T09:46:06.204113Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpusyr8Y/fileb7576ae8b05a/sampleA_realign2transcript.bam, contains 14 reference sequences.
2025-12-19T09:46:06.204184Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-12-19T09:46:06.204244Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-12-19T09:46:06.204551Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-12-19T09:46:06.204610Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-12-19T09:46:06.342639Z  INFO oarfish::single_cell: Processed 100 cells.
2025-12-19T09:46:07.929063Z  INFO oarfish: setting user-provided filter parameters.
2025-12-19T09:46:07.930002Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpusyr8Y/fileb7576ae8b05a/sample1_realign2transcript.bam, contains 14 reference sequences.
2025-12-19T09:46:07.930070Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-12-19T09:46:07.930107Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-12-19T09:46:07.930375Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-12-19T09:46:07.930428Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-12-19T09:46:09.358376Z  INFO oarfish: setting user-provided filter parameters.
2025-12-19T09:46:09.359334Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpusyr8Y/fileb7576ae8b05a/sample2_realign2transcript.bam, contains 14 reference sequences.
2025-12-19T09:46:09.359402Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-12-19T09:46:09.359440Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-12-19T09:46:09.359740Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-12-19T09:46:09.359796Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-12-19T09:46:10.771142Z  INFO oarfish: setting user-provided filter parameters.
2025-12-19T09:46:10.772127Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpusyr8Y/fileb7576ae8b05a/sample3_realign2transcript.bam, contains 14 reference sequences.
2025-12-19T09:46:10.772200Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-12-19T09:46:10.772244Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-12-19T09:46:10.772564Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-12-19T09:46:10.772623Z  INFO oarfish: parsed reference information for 14 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpusyr8Y/fileb7577cc7818b/config_file_46935.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Fri Dec 19 04:46:12 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpusyr8Y/fileb7577cc7818b/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpusyr8Y/fileb7577cc7818b/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpusyr8Y/fileb7577cc7818b/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpusyr8Y/fileb7577cc7818b/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpusyr8Y/fileb7577cc7818b/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpusyr8Y/fileb7577cc7818b/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 92
Number of reads with exactly one barcode match: 91
Number of chimera reads: 1
All done!
Reads	Barcodes
4	1
3	9
2	9
1	44
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpusyr8Y/fileb7577cc7818b/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpusyr8Y/fileb7577cc7818b/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 95
Number of reads with exactly one barcode match: 94
Number of chimera reads: 0
All done!
Reads	Barcodes
4	2
3	3
2	16
1	47
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpusyr8Y/fileb7577cc7818b/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpusyr8Y/fileb7577cc7818b/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Fri Dec 19 04:46:14 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmpusyr8Y/fileb7577cc7818b/sampleA_matched_reads.fastq.gz ->/tmp/Rtmpusyr8Y/fileb7577cc7818b/sampleA_align2genome.bam
/tmp/Rtmpusyr8Y/fileb7577cc7818b/sample1_matched_reads.fastq.gz ->/tmp/Rtmpusyr8Y/fileb7577cc7818b/sample1_align2genome.bam
/tmp/Rtmpusyr8Y/fileb7577cc7818b/sample2_matched_reads.fastq.gz ->/tmp/Rtmpusyr8Y/fileb7577cc7818b/sample2_align2genome.bam
/tmp/Rtmpusyr8Y/fileb7577cc7818b/sample3_matched_reads.fastq.gz ->/tmp/Rtmpusyr8Y/fileb7577cc7818b/sample3_align2genome.bam
-- Running step: gene_quantification @ Fri Dec 19 04:46:54 2025 ----------------
04:46:54 Fri Dec 19 2025 quantify genes 
Using BAM(s): '/tmp/Rtmpusyr8Y/fileb7577cc7818b/sampleA_align2genome.bam',
'/tmp/Rtmpusyr8Y/fileb7577cc7818b/sample1_align2genome.bam',
'/tmp/Rtmpusyr8Y/fileb7577cc7818b/sample2_align2genome.bam', and
'/tmp/Rtmpusyr8Y/fileb7577cc7818b/sample3_align2genome.bam'
parsing /tmp/Rtmpusyr8Y/fileb7577cc7818b/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  7.47gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  7.44gene_group/s]
/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/FLAMES/python/count_gene.py:705: RuntimeWarning: More than 20 figures have been opened. Figures created through the pyplot interface (`matplotlib.pyplot.figure`) are retained until explicitly closed and may consume too much memory. (To control this warning, see the rcParam `figure.max_open_warning`). Consider using `matplotlib.pyplot.close()`.
  plt.figure(figsize=(8, 6))
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 178186.82Read/s]
parsing /tmp/Rtmpusyr8Y/fileb7577cc7818b/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 16.98gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 684940.88Read/s]
parsing /tmp/Rtmpusyr8Y/fileb7577cc7818b/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 15.85gene_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, 566797.84Read/s]
parsing /tmp/Rtmpusyr8Y/fileb7577cc7818b/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 11.42gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 367122.75Read/s]
-- Running step: isoform_identification @ Fri Dec 19 04:46:56 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri Dec 19 04:46:57 2025 -------------------
Checking for fastq file(s) /tmp/Rtmpusyr8Y/fileb7577cc7818b/fastq, /tmp/Rtmpusyr8Y/fileb7577cc7818b/fastq/sample1.fq.gz, /tmp/Rtmpusyr8Y/fileb7577cc7818b/fastq/sample2.fq.gz, /tmp/Rtmpusyr8Y/fileb7577cc7818b/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpusyr8Y/fileb7577cc7818b/sampleA_matched_reads.fastq.gz, /tmp/Rtmpusyr8Y/fileb7577cc7818b/sample1_matched_reads.fastq.gz, /tmp/Rtmpusyr8Y/fileb7577cc7818b/sample2_matched_reads.fastq.gz, /tmp/Rtmpusyr8Y/fileb7577cc7818b/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpusyr8Y/fileb7577cc7818b/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmpusyr8Y/fileb7577cc7818b/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmpusyr8Y/fileb7577cc7818b/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmpusyr8Y/fileb7577cc7818b/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/Rtmpusyr8Y/fileb7577cc7818b/sampleA_matched_reads_dedup.fastq.gz ->/tmp/Rtmpusyr8Y/fileb7577cc7818b/sampleA_realign2transcript.bam
/tmp/Rtmpusyr8Y/fileb7577cc7818b/sample1_matched_reads_dedup.fastq.gz ->/tmp/Rtmpusyr8Y/fileb7577cc7818b/sample1_realign2transcript.bam
/tmp/Rtmpusyr8Y/fileb7577cc7818b/sample2_matched_reads_dedup.fastq.gz ->/tmp/Rtmpusyr8Y/fileb7577cc7818b/sample2_realign2transcript.bam
/tmp/Rtmpusyr8Y/fileb7577cc7818b/sample3_matched_reads_dedup.fastq.gz ->/tmp/Rtmpusyr8Y/fileb7577cc7818b/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Fri Dec 19 04:47:38 2025 ----------
2025-12-19T09:47:38.555891Z  INFO oarfish: setting user-provided filter parameters.
2025-12-19T09:47:38.556887Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpusyr8Y/fileb7577cc7818b/sampleA_realign2transcript.bam, contains 14 reference sequences.
2025-12-19T09:47:38.556953Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-12-19T09:47:38.556992Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-12-19T09:47:38.557274Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-12-19T09:47:38.557333Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-12-19T09:47:38.725544Z  INFO oarfish::single_cell: Processed 100 cells.
2025-12-19T09:47:40.474011Z  INFO oarfish: setting user-provided filter parameters.
2025-12-19T09:47:40.474940Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpusyr8Y/fileb7577cc7818b/sample1_realign2transcript.bam, contains 14 reference sequences.
2025-12-19T09:47:40.475011Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-12-19T09:47:40.475054Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-12-19T09:47:40.475352Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-12-19T09:47:40.475412Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-12-19T09:47:42.230271Z  INFO oarfish: setting user-provided filter parameters.
2025-12-19T09:47:42.231289Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpusyr8Y/fileb7577cc7818b/sample2_realign2transcript.bam, contains 14 reference sequences.
2025-12-19T09:47:42.231368Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-12-19T09:47:42.231413Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-12-19T09:47:42.231697Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-12-19T09:47:42.231759Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-12-19T09:47:43.633864Z  INFO oarfish: setting user-provided filter parameters.
2025-12-19T09:47:43.636775Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpusyr8Y/fileb7577cc7818b/sample3_realign2transcript.bam, contains 14 reference sequences.
2025-12-19T09:47:43.636877Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-12-19T09:47:43.636921Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-12-19T09:47:43.637230Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-12-19T09:47:43.637292Z  INFO oarfish: parsed reference information for 14 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpusyr8Y/fileb75769cfb9bf/config_file_46935.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Fri Dec 19 04:47:46 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpusyr8Y/fileb75769cfb9bf/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpusyr8Y/fileb75769cfb9bf/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpusyr8Y/fileb75769cfb9bf/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpusyr8Y/fileb75769cfb9bf/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpusyr8Y/fileb75769cfb9bf/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpusyr8Y/fileb75769cfb9bf/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 92
Number of reads with exactly one barcode match: 91
Number of chimera reads: 1
All done!
Reads	Barcodes
4	1
3	9
2	9
1	44
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpusyr8Y/fileb75769cfb9bf/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpusyr8Y/fileb75769cfb9bf/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 95
Number of reads with exactly one barcode match: 94
Number of chimera reads: 0
All done!
Reads	Barcodes
4	2
3	3
2	16
1	47
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpusyr8Y/fileb75769cfb9bf/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpusyr8Y/fileb75769cfb9bf/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Fri Dec 19 04:47:48 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmpusyr8Y/fileb75769cfb9bf/sampleA_matched_reads.fastq.gz -> /tmp/Rtmpusyr8Y/fileb75769cfb9bf/sampleA_align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmpusyr8Y/fileb75769cfb9bf/sample1_matched_reads.fastq.gz -> /tmp/Rtmpusyr8Y/fileb75769cfb9bf/sample1_align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmpusyr8Y/fileb75769cfb9bf/sample2_matched_reads.fastq.gz -> /tmp/Rtmpusyr8Y/fileb75769cfb9bf/sample2_align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmpusyr8Y/fileb75769cfb9bf/sample3_matched_reads.fastq.gz -> /tmp/Rtmpusyr8Y/fileb75769cfb9bf/sample3_align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Fri Dec 19 04:47:51 2025 ----------------
04:47:51 Fri Dec 19 2025 quantify genes 
Using BAM(s): '/tmp/Rtmpusyr8Y/fileb75769cfb9bf/sampleA_align2genome.bam',
'/tmp/Rtmpusyr8Y/fileb75769cfb9bf/sample1_align2genome.bam',
'/tmp/Rtmpusyr8Y/fileb75769cfb9bf/sample2_align2genome.bam', and
'/tmp/Rtmpusyr8Y/fileb75769cfb9bf/sample3_align2genome.bam'
parsing /tmp/Rtmpusyr8Y/fileb75769cfb9bf/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  7.11gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  7.08gene_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, 196002.84Read/s]
parsing /tmp/Rtmpusyr8Y/fileb75769cfb9bf/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 18.01gene_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, 688946.12Read/s]
parsing /tmp/Rtmpusyr8Y/fileb75769cfb9bf/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 16.63gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 662984.32Read/s]
parsing /tmp/Rtmpusyr8Y/fileb75769cfb9bf/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 12.61gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 374250.84Read/s]
-- Running step: isoform_identification @ Fri Dec 19 04:47:53 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri Dec 19 04:47:54 2025 -------------------
Checking for fastq file(s) /tmp/Rtmpusyr8Y/fileb75769cfb9bf/fastq, /tmp/Rtmpusyr8Y/fileb75769cfb9bf/fastq/sample1.fq.gz, /tmp/Rtmpusyr8Y/fileb75769cfb9bf/fastq/sample2.fq.gz, /tmp/Rtmpusyr8Y/fileb75769cfb9bf/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpusyr8Y/fileb75769cfb9bf/sampleA_matched_reads.fastq.gz, /tmp/Rtmpusyr8Y/fileb75769cfb9bf/sample1_matched_reads.fastq.gz, /tmp/Rtmpusyr8Y/fileb75769cfb9bf/sample2_matched_reads.fastq.gz, /tmp/Rtmpusyr8Y/fileb75769cfb9bf/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpusyr8Y/fileb75769cfb9bf/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmpusyr8Y/fileb75769cfb9bf/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmpusyr8Y/fileb75769cfb9bf/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmpusyr8Y/fileb75769cfb9bf/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/Rtmpusyr8Y/fileb75769cfb9bf/sampleA_matched_reads_dedup.fastq.gz -> /tmp/Rtmpusyr8Y/fileb75769cfb9bf/sampleA_realign2transcript.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample /tmp/Rtmpusyr8Y/fileb75769cfb9bf/sample1_matched_reads_dedup.fastq.gz -> /tmp/Rtmpusyr8Y/fileb75769cfb9bf/sample1_realign2transcript.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample /tmp/Rtmpusyr8Y/fileb75769cfb9bf/sample2_matched_reads_dedup.fastq.gz -> /tmp/Rtmpusyr8Y/fileb75769cfb9bf/sample2_realign2transcript.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample /tmp/Rtmpusyr8Y/fileb75769cfb9bf/sample3_matched_reads_dedup.fastq.gz -> /tmp/Rtmpusyr8Y/fileb75769cfb9bf/sample3_realign2transcript.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Fri Dec 19 04:47:56 2025 ----------
04:47:56 Fri Dec 19 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/Rtmpusyr8Y/fileb75769cfb9bf/sample3_realign2transcript.bam...
parsing /tmp/Rtmpusyr8Y/fileb75769cfb9bf/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpusyr8Y/fileb75769cfb9bf/sample3_realign2transcript.bamdone
parsing /tmp/Rtmpusyr8Y/fileb75769cfb9bf/sampleA_realign2transcript.bam...
parsing /tmp/Rtmpusyr8Y/fileb75769cfb9bf/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpusyr8Y/fileb75769cfb9bf/sampleA_realign2transcript.bamdone
parsing /tmp/Rtmpusyr8Y/fileb75769cfb9bf/sample2_realign2transcript.bam...
parsing /tmp/Rtmpusyr8Y/fileb75769cfb9bf/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpusyr8Y/fileb75769cfb9bf/sample2_realign2transcript.bamdone
parsing /tmp/Rtmpusyr8Y/fileb75769cfb9bf/sample1_realign2transcript.bam...
parsing /tmp/Rtmpusyr8Y/fileb75769cfb9bf/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpusyr8Y/fileb75769cfb9bf/sample1_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpusyr8Y/fileb7576852223b/config_file_46935.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Fri Dec 19 04:48:04 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpusyr8Y/fileb7576852223b/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpusyr8Y/fileb7576852223b/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpusyr8Y/fileb7576852223b/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpusyr8Y/fileb7576852223b/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpusyr8Y/fileb7576852223b/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpusyr8Y/fileb7576852223b/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 92
Number of reads with exactly one barcode match: 91
Number of chimera reads: 1
All done!
Reads	Barcodes
4	1
3	9
2	9
1	44
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpusyr8Y/fileb7576852223b/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpusyr8Y/fileb7576852223b/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 95
Number of reads with exactly one barcode match: 94
Number of chimera reads: 0
All done!
Reads	Barcodes
4	2
3	3
2	16
1	47
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpusyr8Y/fileb7576852223b/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpusyr8Y/fileb7576852223b/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Fri Dec 19 04:48:06 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmpusyr8Y/fileb7576852223b/sampleA_matched_reads.fastq.gz ->/tmp/Rtmpusyr8Y/fileb7576852223b/sampleA_align2genome.bam
/tmp/Rtmpusyr8Y/fileb7576852223b/sample1_matched_reads.fastq.gz ->/tmp/Rtmpusyr8Y/fileb7576852223b/sample1_align2genome.bam
/tmp/Rtmpusyr8Y/fileb7576852223b/sample2_matched_reads.fastq.gz ->/tmp/Rtmpusyr8Y/fileb7576852223b/sample2_align2genome.bam
/tmp/Rtmpusyr8Y/fileb7576852223b/sample3_matched_reads.fastq.gz ->/tmp/Rtmpusyr8Y/fileb7576852223b/sample3_align2genome.bam
-- Running step: gene_quantification @ Fri Dec 19 04:48:47 2025 ----------------
04:48:47 Fri Dec 19 2025 quantify genes 
Using BAM(s): '/tmp/Rtmpusyr8Y/fileb7576852223b/sampleA_align2genome.bam',
'/tmp/Rtmpusyr8Y/fileb7576852223b/sample1_align2genome.bam',
'/tmp/Rtmpusyr8Y/fileb7576852223b/sample2_align2genome.bam', and
'/tmp/Rtmpusyr8Y/fileb7576852223b/sample3_align2genome.bam'
	Counter({'counted_reads': 176})
	Counter({'counted_reads': 358})
	Counter({'counted_reads': 95})
	Counter({'counted_reads': 91})
parsing /tmp/Rtmpusyr8Y/fileb7576852223b/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  6.63gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  6.61gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 132367.55Read/s]
parsing /tmp/Rtmpusyr8Y/fileb7576852223b/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 15.47gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 698678.04Read/s]
parsing /tmp/Rtmpusyr8Y/fileb7576852223b/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 17.06gene_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, 612700.71Read/s]
parsing /tmp/Rtmpusyr8Y/fileb7576852223b/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 12.11gene_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, 266277.97Read/s]
-- Running step: isoform_identification @ Fri Dec 19 04:48:49 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri Dec 19 04:48:50 2025 -------------------
Checking for fastq file(s) /tmp/Rtmpusyr8Y/fileb7576852223b/fastq, /tmp/Rtmpusyr8Y/fileb7576852223b/fastq/sample1.fq.gz, /tmp/Rtmpusyr8Y/fileb7576852223b/fastq/sample2.fq.gz, /tmp/Rtmpusyr8Y/fileb7576852223b/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpusyr8Y/fileb7576852223b/sampleA_matched_reads.fastq.gz, /tmp/Rtmpusyr8Y/fileb7576852223b/sample1_matched_reads.fastq.gz, /tmp/Rtmpusyr8Y/fileb7576852223b/sample2_matched_reads.fastq.gz, /tmp/Rtmpusyr8Y/fileb7576852223b/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpusyr8Y/fileb7576852223b/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmpusyr8Y/fileb7576852223b/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmpusyr8Y/fileb7576852223b/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmpusyr8Y/fileb7576852223b/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/Rtmpusyr8Y/fileb7576852223b/sampleA_matched_reads_dedup.fastq.gz ->/tmp/Rtmpusyr8Y/fileb7576852223b/sampleA_realign2transcript.bam
/tmp/Rtmpusyr8Y/fileb7576852223b/sample1_matched_reads_dedup.fastq.gz ->/tmp/Rtmpusyr8Y/fileb7576852223b/sample1_realign2transcript.bam
/tmp/Rtmpusyr8Y/fileb7576852223b/sample2_matched_reads_dedup.fastq.gz ->/tmp/Rtmpusyr8Y/fileb7576852223b/sample2_realign2transcript.bam
/tmp/Rtmpusyr8Y/fileb7576852223b/sample3_matched_reads_dedup.fastq.gz ->/tmp/Rtmpusyr8Y/fileb7576852223b/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Fri Dec 19 04:49:31 2025 ----------
04:49:31 Fri Dec 19 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/Rtmpusyr8Y/fileb7576852223b/sample3_realign2transcript.bam...
parsing /tmp/Rtmpusyr8Y/fileb7576852223b/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpusyr8Y/fileb7576852223b/sample3_realign2transcript.bamdone
parsing /tmp/Rtmpusyr8Y/fileb7576852223b/sampleA_realign2transcript.bam...
parsing /tmp/Rtmpusyr8Y/fileb7576852223b/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpusyr8Y/fileb7576852223b/sampleA_realign2transcript.bamdone
parsing /tmp/Rtmpusyr8Y/fileb7576852223b/sample2_realign2transcript.bam...
parsing /tmp/Rtmpusyr8Y/fileb7576852223b/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpusyr8Y/fileb7576852223b/sample2_realign2transcript.bamdone
parsing /tmp/Rtmpusyr8Y/fileb7576852223b/sample1_realign2transcript.bam...
parsing /tmp/Rtmpusyr8Y/fileb7576852223b/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpusyr8Y/fileb7576852223b/sample1_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
[ FAIL 12 | WARN 106 | SKIP 0 | PASS 47 ]

══ Failed tests ════════════════════════════════════════════════════════════════
── Failure ('test-run_FLAMES.R:29:11'): run_FLAMES() completes and returns experiment for all pipeline types and option combinations ──
Expected `is.null(experiment(result))` to be FALSE.
Differences:
`actual`:   TRUE 
`expected`: FALSE

experiment is NULL for run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
── Failure ('test-run_FLAMES.R:29:11'): run_FLAMES() completes and returns experiment for all pipeline types and option combinations ──
Expected `is.null(experiment(result))` to be FALSE.
Differences:
`actual`:   TRUE 
`expected`: FALSE

experiment is NULL for run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
── Failure ('test-run_FLAMES.R:29:11'): run_FLAMES() completes and returns experiment for all pipeline types and option combinations ──
Expected `is.null(experiment(result))` to be FALSE.
Differences:
`actual`:   TRUE 
`expected`: FALSE

experiment is NULL for run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
── Failure ('test-run_FLAMES.R:29:11'): run_FLAMES() completes and returns experiment for all pipeline types and option combinations ──
Expected `is.null(experiment(result))` to be FALSE.
Differences:
`actual`:   TRUE 
`expected`: FALSE

experiment is NULL for run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
── Failure ('test-run_FLAMES.R:29:11'): run_FLAMES() completes and returns experiment for all pipeline types and option combinations ──
Expected `is.null(experiment(result))` to be FALSE.
Differences:
`actual`:   TRUE 
`expected`: FALSE

experiment is NULL for run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
── Failure ('test-run_FLAMES.R:29:11'): run_FLAMES() completes and returns experiment for all pipeline types and option combinations ──
Expected `is.null(experiment(result))` to be FALSE.
Differences:
`actual`:   TRUE 
`expected`: FALSE

experiment is NULL for run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
── Failure ('test-run_FLAMES.R:29:11'): run_FLAMES() completes and returns experiment for all pipeline types and option combinations ──
Expected `is.null(experiment(result))` to be FALSE.
Differences:
`actual`:   TRUE 
`expected`: FALSE

experiment is NULL for run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
── Failure ('test-run_FLAMES.R:29:11'): run_FLAMES() completes and returns experiment for all pipeline types and option combinations ──
Expected `is.null(experiment(result))` to be FALSE.
Differences:
`actual`:   TRUE 
`expected`: FALSE

experiment is NULL for run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
── Failure ('test-run_FLAMES.R:29:11'): run_FLAMES() completes and returns experiment for all pipeline types and option combinations ──
Expected `is.null(experiment(result))` to be FALSE.
Differences:
`actual`:   TRUE 
`expected`: FALSE

experiment is NULL for run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
── Failure ('test-run_FLAMES.R:29:11'): run_FLAMES() completes and returns experiment for all pipeline types and option combinations ──
Expected `is.null(experiment(result))` to be FALSE.
Differences:
`actual`:   TRUE 
`expected`: FALSE

experiment is NULL for run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
── Failure ('test-run_FLAMES.R:29:11'): run_FLAMES() completes and returns experiment for all pipeline types and option combinations ──
Expected `is.null(experiment(result))` to be FALSE.
Differences:
`actual`:   TRUE 
`expected`: FALSE

experiment is NULL for run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
── Failure ('test-run_FLAMES.R:29:11'): run_FLAMES() completes and returns experiment for all pipeline types and option combinations ──
Expected `is.null(experiment(result))` to be FALSE.
Differences:
`actual`:   TRUE 
`expected`: FALSE

experiment is NULL for run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)

[ FAIL 12 | WARN 106 | SKIP 0 | PASS 47 ]
Error:
! Test failures.
Execution halted
/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 '