Back to Multiple platform build/check report for BioC 3.22:   simplified   long
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This page was generated on 2025-10-25 12:04 -0400 (Sat, 25 Oct 2025).

HostnameOSArch (*)R versionInstalled pkgs
nebbiolo2Linux (Ubuntu 24.04.3 LTS)x86_644.5.1 Patched (2025-08-23 r88802) -- "Great Square Root" 4901
lconwaymacOS 12.7.6 Montereyx86_644.5.1 Patched (2025-09-10 r88807) -- "Great Square Root" 4691
kjohnson3macOS 13.7.7 Venturaarm644.5.1 Patched (2025-09-10 r88807) -- "Great Square Root" 4637
taishanLinux (openEuler 24.03 LTS)aarch644.5.0 (2025-04-11) -- "How About a Twenty-Six" 4658
Click on any hostname to see more info about the system (e.g. compilers)      (*) as reported by 'uname -p', except on Windows and Mac OS X

Package 744/2361HostnameOS / ArchINSTALLBUILDCHECKBUILD BIN
FLAMES 2.3.5  (landing page)
Changqing Wang
Snapshot Date: 2025-10-24 13:45 -0400 (Fri, 24 Oct 2025)
git_url: https://git.bioconductor.org/packages/FLAMES
git_branch: devel
git_last_commit: 5771592
git_last_commit_date: 2025-09-12 02:43:50 -0400 (Fri, 12 Sep 2025)
nebbiolo2Linux (Ubuntu 24.04.3 LTS) / x86_64  OK    OK    OK  UNNEEDED, same version is already published
lconwaymacOS 12.7.6 Monterey / x86_64  OK    OK    OK    OK  UNNEEDED, same version is already published
kjohnson3macOS 13.7.7 Ventura / arm64  OK    OK    OK    OK  UNNEEDED, same version is already published
taishanLinux (openEuler 24.03 LTS) / aarch64  ERROR    NA    NA  


CHECK results for FLAMES on lconway

To the developers/maintainers of the FLAMES package:
- Allow up to 24 hours (and sometimes 48 hours) for your latest push to git@git.bioconductor.org:packages/FLAMES.git to reflect on this report. See Troubleshooting Build Report for more information.
- Use the following Renviron settings to reproduce errors and warnings.
- If 'R CMD check' started to fail recently on the Linux builder(s) over a missing dependency, add the missing dependency to 'Suggests:' in your DESCRIPTION file. See Renviron.bioc for more information.

raw results


Summary

Package: FLAMES
Version: 2.3.5
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_2.3.5.tar.gz
StartedAt: 2025-10-24 21:15:19 -0400 (Fri, 24 Oct 2025)
EndedAt: 2025-10-24 21:40:41 -0400 (Fri, 24 Oct 2025)
EllapsedTime: 1522.2 seconds
RetCode: 0
Status:   OK  
CheckDir: FLAMES.Rcheck
Warnings: 0

Command output

##############################################################################
##############################################################################
###
### 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_2.3.5.tar.gz
###
##############################################################################
##############################################################################


* using log directory ‘/Users/biocbuild/bbs-3.22-bioc/meat/FLAMES.Rcheck’
* using R version 4.5.1 Patched (2025-09-10 r88807)
* 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 ‘2.3.5’
* package encoding: UTF-8
* checking package namespace information ... OK
* checking package dependencies ... INFO
Imports includes 47 non-default packages.
Importing from so many packages makes the package vulnerable to any of
them becoming unavailable.  Move as many as possible to Suggests and
use conditionally.
* checking if this is a source package ... OK
* checking if there is a namespace ... OK
* checking for hidden files and directories ... 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     28.672  0.446  29.387
find_variants                25.764  0.429  25.664
sc_long_multisample_pipeline 16.313  2.967  16.363
MultiSampleSCPipeline        13.175  1.964  16.920
sc_plot_genotype             11.920  0.339  11.405
sc_DTU_analysis              10.241  1.260  10.222
plot_isoform_heatmap         10.226  0.594  10.971
blaze                         8.345  1.178  10.491
create_sce_from_dir           7.402  1.425   7.492
sc_long_pipeline              5.319  0.807   4.819
bulk_long_pipeline            4.650  1.043   4.567
sc_genotype                   4.030  1.122   4.025
BulkPipeline                  4.360  0.616   5.387
* checking for unstated dependencies in ‘tests’ ... OK
* checking tests ...
  Running ‘testthat.R’
 OK
* checking for unstated dependencies in vignettes ... OK
* checking package vignettes ... OK
* checking running R code from vignettes ... SKIPPED
* checking re-building of vignette outputs ... SKIPPED
* checking PDF version of manual ... OK
* DONE

Status: 5 NOTEs
See
  ‘/Users/biocbuild/bbs-3.22-bioc/meat/FLAMES.Rcheck/00check.log’
for details.


Installation output

FLAMES.Rcheck/00install.out

##############################################################################
##############################################################################
###
### 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 ‘2.3.5’
** 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)

Tests output

FLAMES.Rcheck/tests/testthat.Rout


R version 4.5.1 Patched (2025-09-10 r88807) -- "Great Square Root"
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/Rtmpar2j2K/file4c5573081fc/config_file_1221.json 
Writing configuration parameters to:  /tmp/Rtmpar2j2K/file4c5573081fc/config_file_1221.json 
Writing configuration parameters to:  /tmp/Rtmpar2j2K/file4c5573081fc/config_file_1221.json 
Writing configuration parameters to:  /tmp/Rtmpar2j2K/file4c54f596659/config_file_1221.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/Rtmpar2j2K/file4c57091f213/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/Rtmpar2j2K/file4c57f476f35/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/Rtmpar2j2K/file4c57f476f35/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpar2j2K/file4c5587da6d5/musc_rps24_1.fastq
Searching for barcodes...
Processing file: /tmp/Rtmpar2j2K/file4c5587da6d5/musc_rps24_2.fastq
Searching for barcodes...
Processing file: /tmp/Rtmpar2j2K/file4c5587da6d5/musc_rps24_3.fastq
Searching for barcodes...
Processing file: /tmp/Rtmpar2j2K/file4c5587da6d5/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/Rtmpar2j2K/file4c55590ae48/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/Rtmpar2j2K/file4c55ea26738/config_file_1221.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 Oct 24 21:25:59 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/Rtmpar2j2K/file4c55ea26738/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/Rtmpar2j2K/file4c55ea26738/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/Rtmpar2j2K/file4c55ea26738/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Fri Oct 24 21:26:00 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

  |                                                                            
  |                                                                      |   0%[21:26:08] WARNING: src/learner.cc:553: 
  If you are loading a serialized model (like pickle in Python, RDS in R) generated by
  older XGBoost, please export the model by calling `Booster.save_model` from that version
  first, then load it back in current version. See:

    https://xgboost.readthedocs.io/en/latest/tutorials/saving_model.html

  for more details about differences between saving model and serializing.

[21:26:08] WARNING: src/learner.cc:553: 
  If you are loading a serialized model (like pickle in Python, RDS in R) generated by
  older XGBoost, please export the model by calling `Booster.save_model` from that version
  first, then load it back in current version. See:

    https://xgboost.readthedocs.io/en/latest/tutorials/saving_model.html

  for more details about differences between saving model and serializing.

[21:26:08] WARNING: src/learner.cc:553: 
  If you are loading a serialized model (like pickle in Python, RDS in R) generated by
  older XGBoost, please export the model by calling `Booster.save_model` from that version
  first, then load it back in current version. See:

    https://xgboost.readthedocs.io/en/latest/tutorials/saving_model.html

  for more details about differences between saving model and serializing.

[21:26:08] WARNING: src/learner.cc:553: 
  If you are loading a serialized model (like pickle in Python, RDS in R) generated by
  older XGBoost, please export the model by calling `Booster.save_model` from that version
  first, then load it back in current version. See:

    https://xgboost.readthedocs.io/en/latest/tutorials/saving_model.html

  for more details about differences between saving model and serializing.

[21:26:10] WARNING: src/learner.cc:553: 
  If you are loading a serialized model (like pickle in Python, RDS in R) generated by
  older XGBoost, please export the model by calling `Booster.save_model` from that version
  first, then load it back in current version. See:

    https://xgboost.readthedocs.io/en/latest/tutorials/saving_model.html

  for more details about differences between saving model and serializing.

[21:26:10] WARNING: src/learner.cc:553: 
  If you are loading a serialized model (like pickle in Python, RDS in R) generated by
  older XGBoost, please export the model by calling `Booster.save_model` from that version
  first, then load it back in current version. See:

    https://xgboost.readthedocs.io/en/latest/tutorials/saving_model.html

  for more details about differences between saving model and serializing.


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

Detected 4 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Fri Oct 24 21:26:31 2025 -------------------
Realigning sample sample1 -> /tmp/Rtmpar2j2K/file4c55ea26738/sample1_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample2 -> /tmp/Rtmpar2j2K/file4c55ea26738/sample2_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample3 -> /tmp/Rtmpar2j2K/file4c55ea26738/sample3_realign2transcript.bam
Skipped sorting BAM files.

-- Running step: transcript_quantification @ Fri Oct 24 21:26:31 2025 ----------
2025-10-25T01:26:32.051715Z  INFO oarfish: setting user-provided filter parameters.
2025-10-25T01:26:32.052345Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpar2j2K/file4c55ea26738/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-10-25T01:26:32.052386Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-25T01:26:32.052401Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-25T01:26:32.052506Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-25T01:26:32.052522Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-25T01:26:32.054674Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-25T01:26:32.054978Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 253   │
│ aligned fraction too low        │ 4     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 1     │
│ reads with valid best alignment │ 96    │
╰─────────────────────────────────┴───────╯

2025-10-25T01:26:32.055038Z  INFO oarfish::bulk: Total number of alignment records : 116
2025-10-25T01:26:32.055080Z  INFO oarfish::bulk: number of aligned reads : 96
2025-10-25T01:26:32.055089Z  INFO oarfish::bulk: number of unique alignments : 86
2025-10-25T01:26:32.060933Z  INFO oarfish: oarfish completed successfully.
2025-10-25T01:26:32.116218Z  INFO oarfish: setting user-provided filter parameters.
2025-10-25T01:26:32.116999Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpar2j2K/file4c55ea26738/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-10-25T01:26:32.117048Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-25T01:26:32.117077Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-25T01:26:32.117249Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-25T01:26:32.117270Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-25T01:26:32.119855Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-25T01:26:32.120174Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 251   │
│ aligned fraction too low        │ 5     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 2     │
│ reads with valid best alignment │ 95    │
╰─────────────────────────────────┴───────╯

2025-10-25T01:26:32.120248Z  INFO oarfish::bulk: Total number of alignment records : 114
2025-10-25T01:26:32.120258Z  INFO oarfish::bulk: number of aligned reads : 95
2025-10-25T01:26:32.120264Z  INFO oarfish::bulk: number of unique alignments : 82
2025-10-25T01:26:32.125496Z  INFO oarfish: oarfish completed successfully.
2025-10-25T01:26:32.175110Z  INFO oarfish: setting user-provided filter parameters.
2025-10-25T01:26:32.175741Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpar2j2K/file4c55ea26738/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-10-25T01:26:32.175783Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-25T01:26:32.175796Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-25T01:26:32.175926Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-25T01:26:32.175944Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-25T01:26:32.180460Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2025-10-25T01:26:32.180897Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 398   │
│ aligned fraction too low        │ 12    │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 5     │
│ reads with valid best alignment │ 179   │
╰─────────────────────────────────┴───────╯

2025-10-25T01:26:32.180981Z  INFO oarfish::bulk: Total number of alignment records : 239
2025-10-25T01:26:32.180991Z  INFO oarfish::bulk: number of aligned reads : 179
2025-10-25T01:26:32.180998Z  INFO oarfish::bulk: number of unique alignments : 143
2025-10-25T01:26:32.185436Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpar2j2K/file4c53aa92d22/config_file_1221.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 Oct 24 21:26:33 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/Rtmpar2j2K/file4c53aa92d22/sample1_align2genome.bam
sample2 ->/tmp/Rtmpar2j2K/file4c53aa92d22/sample2_align2genome.bam
sample3 ->/tmp/Rtmpar2j2K/file4c53aa92d22/sample3_align2genome.bam
-- Running step: isoform_identification @ Fri Oct 24 21:26:55 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 4 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Fri Oct 24 21:27:18 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/Rtmpar2j2K/file4c53aa92d22/sample1_realign2transcript.bam
sample2 ->/tmp/Rtmpar2j2K/file4c53aa92d22/sample2_realign2transcript.bam
sample3 ->/tmp/Rtmpar2j2K/file4c53aa92d22/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Fri Oct 24 21:27:40 2025 ----------
2025-10-25T01:27:40.234569Z  INFO oarfish: setting user-provided filter parameters.
2025-10-25T01:27:40.235508Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpar2j2K/file4c53aa92d22/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-10-25T01:27:40.235584Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-25T01:27:40.235598Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-25T01:27:40.235738Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-25T01:27:40.235775Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-25T01:27:40.238718Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-25T01:27:40.239118Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 253   │
│ aligned fraction too low        │ 4     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 1     │
│ reads with valid best alignment │ 96    │
╰─────────────────────────────────┴───────╯

2025-10-25T01:27:40.239203Z  INFO oarfish::bulk: Total number of alignment records : 116
2025-10-25T01:27:40.239216Z  INFO oarfish::bulk: number of aligned reads : 96
2025-10-25T01:27:40.239225Z  INFO oarfish::bulk: number of unique alignments : 86
2025-10-25T01:27:40.246235Z  INFO oarfish: oarfish completed successfully.
2025-10-25T01:27:40.323057Z  INFO oarfish: setting user-provided filter parameters.
2025-10-25T01:27:40.323788Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpar2j2K/file4c53aa92d22/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-10-25T01:27:40.323834Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-25T01:27:40.323851Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-25T01:27:40.323986Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-25T01:27:40.324009Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-25T01:27:40.326971Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-25T01:27:40.327400Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 251   │
│ aligned fraction too low        │ 5     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 2     │
│ reads with valid best alignment │ 95    │
╰─────────────────────────────────┴───────╯

2025-10-25T01:27:40.327480Z  INFO oarfish::bulk: Total number of alignment records : 114
2025-10-25T01:27:40.327553Z  INFO oarfish::bulk: number of aligned reads : 95
2025-10-25T01:27:40.327565Z  INFO oarfish::bulk: number of unique alignments : 82
2025-10-25T01:27:40.337595Z  INFO oarfish: oarfish completed successfully.
2025-10-25T01:27:40.399106Z  INFO oarfish: setting user-provided filter parameters.
2025-10-25T01:27:40.399838Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpar2j2K/file4c53aa92d22/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-10-25T01:27:40.399880Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-25T01:27:40.399896Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-25T01:27:40.400123Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-25T01:27:40.400153Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-25T01:27:40.405005Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2025-10-25T01:27:40.405392Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 398   │
│ aligned fraction too low        │ 12    │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 5     │
│ reads with valid best alignment │ 179   │
╰─────────────────────────────────┴───────╯

2025-10-25T01:27:40.405458Z  INFO oarfish::bulk: Total number of alignment records : 239
2025-10-25T01:27:40.405474Z  INFO oarfish::bulk: number of aligned reads : 179
2025-10-25T01:27:40.405487Z  INFO oarfish::bulk: number of unique alignments : 143
2025-10-25T01:27:40.413981Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpar2j2K/file4c523c9d363/config_file_1221.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 Oct 24 21:27:41 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/Rtmpar2j2K/file4c523c9d363/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/Rtmpar2j2K/file4c523c9d363/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/Rtmpar2j2K/file4c523c9d363/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Fri Oct 24 21:27:42 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 4 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Fri Oct 24 21:28:05 2025 -------------------
Realigning sample sample1 -> /tmp/Rtmpar2j2K/file4c523c9d363/sample1_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample2 -> /tmp/Rtmpar2j2K/file4c523c9d363/sample2_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample3 -> /tmp/Rtmpar2j2K/file4c523c9d363/sample3_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Fri Oct 24 21:28:06 2025 ----------
21:28:06 Fri Oct 24 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpar2j2K/file4c53352cd48/config_file_1221.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 Oct 24 21:28:08 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/Rtmpar2j2K/file4c53352cd48/sample1_align2genome.bam
sample2 ->/tmp/Rtmpar2j2K/file4c53352cd48/sample2_align2genome.bam
sample3 ->/tmp/Rtmpar2j2K/file4c53352cd48/sample3_align2genome.bam
-- Running step: isoform_identification @ Fri Oct 24 21:28:31 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

  |                                                                            
  |                                                                      |   0%
  |                                                                            
  |=======================                                               |  33%
  |                                                                            
  |===============================================                       |  67%
  |                                                                            
  |======================================================================| 100%

Detected 4 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Fri Oct 24 21:28:53 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/Rtmpar2j2K/file4c53352cd48/sample1_realign2transcript.bam
sample2 ->/tmp/Rtmpar2j2K/file4c53352cd48/sample2_realign2transcript.bam
sample3 ->/tmp/Rtmpar2j2K/file4c53352cd48/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Fri Oct 24 21:29:14 2025 ----------
21:29:14 Fri Oct 24 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Inputs:  ['/tmp/Rtmpar2j2K/file4c523c9d363/sample3_realign2transcript.bam', '/tmp/Rtmpar2j2K/file4c523c9d363/sample2_realign2transcript.bam', '/tmp/Rtmpar2j2K/file4c523c9d363/sample1_realign2transcript.bam'] /tmp/Rtmpar2j2K/file4c523c9d363/transcript_assembly.fa.fai 5 0.4 0.4
	Counter({'counted_reads': 375, 'not_enough_coverage': 16, 'unmapped': 2})
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpar2j2K/file4c5718536f8/config_file_1221.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 Oct 24 21:29:16 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/Rtmpar2j2K/file4c5718536f8/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/Rtmpar2j2K/file4c5718536f8/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/Rtmpar2j2K/file4c5718536f8/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Fri Oct 24 21:29:17 2025 -------------
Inputs:  ['/tmp/Rtmpar2j2K/file4c53352cd48/sample3_realign2transcript.bam', '/tmp/Rtmpar2j2K/file4c53352cd48/sample2_realign2transcript.bam', '/tmp/Rtmpar2j2K/file4c53352cd48/sample1_realign2transcript.bam'] /tmp/Rtmpar2j2K/file4c53352cd48/transcript_assembly.fa.fai 5 0.4 0.4
	Counter({'counted_reads': 375, 'not_enough_coverage': 16, 'unmapped': 2})
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri Oct 24 21:29:17 2025 -------------------
Realigning sample sample1 -> /tmp/Rtmpar2j2K/file4c5718536f8/sample1_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample2 -> /tmp/Rtmpar2j2K/file4c5718536f8/sample2_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample3 -> /tmp/Rtmpar2j2K/file4c5718536f8/sample3_realign2transcript.bam
Skipped sorting BAM files.

-- Running step: transcript_quantification @ Fri Oct 24 21:29:18 2025 ----------
2025-10-25T01:29:18.862922Z  INFO oarfish: setting user-provided filter parameters.
2025-10-25T01:29:18.863433Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpar2j2K/file4c5718536f8/sample1_realign2transcript.bam, contains 10 reference sequences.
2025-10-25T01:29:18.863467Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-25T01:29:18.863479Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-25T01:29:18.863621Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-25T01:29:18.863647Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-25T01:29:18.868423Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-25T01:29:18.868729Z  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    │
╰─────────────────────────────────┴───────╯

2025-10-25T01:29:18.868774Z  INFO oarfish::bulk: Total number of alignment records : 125
2025-10-25T01:29:18.868782Z  INFO oarfish::bulk: number of aligned reads : 98
2025-10-25T01:29:18.868788Z  INFO oarfish::bulk: number of unique alignments : 86
2025-10-25T01:29:18.873523Z  INFO oarfish: oarfish completed successfully.
2025-10-25T01:29:18.925503Z  INFO oarfish: setting user-provided filter parameters.
2025-10-25T01:29:18.926140Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpar2j2K/file4c5718536f8/sample2_realign2transcript.bam, contains 10 reference sequences.
2025-10-25T01:29:18.926172Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-25T01:29:18.926184Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-25T01:29:18.926298Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-25T01:29:18.926311Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-25T01:29:18.931227Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-25T01:29:18.931519Z  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    │
╰─────────────────────────────────┴───────╯

2025-10-25T01:29:18.931593Z  INFO oarfish::bulk: Total number of alignment records : 136
2025-10-25T01:29:18.931606Z  INFO oarfish::bulk: number of aligned reads : 97
2025-10-25T01:29:18.931615Z  INFO oarfish::bulk: number of unique alignments : 79
2025-10-25T01:29:18.936849Z  INFO oarfish: oarfish completed successfully.
2025-10-25T01:29:18.997363Z  INFO oarfish: setting user-provided filter parameters.
2025-10-25T01:29:18.998096Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpar2j2K/file4c5718536f8/sample3_realign2transcript.bam, contains 10 reference sequences.
2025-10-25T01:29:18.998163Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-25T01:29:18.998175Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-25T01:29:18.998291Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-25T01:29:18.998304Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-25T01:29:19.005533Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-25T01:29:19.005910Z  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   │
╰─────────────────────────────────┴───────╯

2025-10-25T01:29:19.005966Z  INFO oarfish::bulk: Total number of alignment records : 272
2025-10-25T01:29:19.005974Z  INFO oarfish::bulk: number of aligned reads : 187
2025-10-25T01:29:19.005980Z  INFO oarfish::bulk: number of unique alignments : 140
2025-10-25T01:29:19.009787Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpar2j2K/file4c5ef40c17/config_file_1221.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 Oct 24 21:29:19 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/Rtmpar2j2K/file4c5ef40c17/sample1_align2genome.bam
sample2 ->/tmp/Rtmpar2j2K/file4c5ef40c17/sample2_align2genome.bam
sample3 ->/tmp/Rtmpar2j2K/file4c5ef40c17/sample3_align2genome.bam
-- Running step: isoform_identification @ Fri Oct 24 21:29:41 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri Oct 24 21:29:41 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/Rtmpar2j2K/file4c5ef40c17/sample1_realign2transcript.bam
sample2 ->/tmp/Rtmpar2j2K/file4c5ef40c17/sample2_realign2transcript.bam
sample3 ->/tmp/Rtmpar2j2K/file4c5ef40c17/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Fri Oct 24 21:30:02 2025 ----------
2025-10-25T01:30:02.541744Z  INFO oarfish: setting user-provided filter parameters.
2025-10-25T01:30:02.542501Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpar2j2K/file4c5ef40c17/sample1_realign2transcript.bam, contains 10 reference sequences.
2025-10-25T01:30:02.542559Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-25T01:30:02.542579Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-25T01:30:02.542767Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-25T01:30:02.542793Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-25T01:30:02.546452Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-25T01:30:02.546951Z  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    │
╰─────────────────────────────────┴───────╯

2025-10-25T01:30:02.547060Z  INFO oarfish::bulk: Total number of alignment records : 125
2025-10-25T01:30:02.547072Z  INFO oarfish::bulk: number of aligned reads : 98
2025-10-25T01:30:02.547078Z  INFO oarfish::bulk: number of unique alignments : 86
2025-10-25T01:30:02.550715Z  INFO oarfish: oarfish completed successfully.
2025-10-25T01:30:02.602593Z  INFO oarfish: setting user-provided filter parameters.
2025-10-25T01:30:02.603238Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpar2j2K/file4c5ef40c17/sample2_realign2transcript.bam, contains 10 reference sequences.
2025-10-25T01:30:02.603297Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-25T01:30:02.603308Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-25T01:30:02.603477Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-25T01:30:02.603490Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-25T01:30:02.608117Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-25T01:30:02.608509Z  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    │
╰─────────────────────────────────┴───────╯

2025-10-25T01:30:02.608572Z  INFO oarfish::bulk: Total number of alignment records : 136
2025-10-25T01:30:02.608581Z  INFO oarfish::bulk: number of aligned reads : 97
2025-10-25T01:30:02.608589Z  INFO oarfish::bulk: number of unique alignments : 79
2025-10-25T01:30:02.612903Z  INFO oarfish: oarfish completed successfully.
2025-10-25T01:30:02.668665Z  INFO oarfish: setting user-provided filter parameters.
2025-10-25T01:30:02.669430Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpar2j2K/file4c5ef40c17/sample3_realign2transcript.bam, contains 10 reference sequences.
2025-10-25T01:30:02.669470Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-25T01:30:02.669480Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-25T01:30:02.669596Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-25T01:30:02.669609Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-25T01:30:02.675942Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-25T01:30:02.676312Z  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   │
╰─────────────────────────────────┴───────╯

2025-10-25T01:30:02.676366Z  INFO oarfish::bulk: Total number of alignment records : 272
2025-10-25T01:30:02.676374Z  INFO oarfish::bulk: number of aligned reads : 187
2025-10-25T01:30:02.676380Z  INFO oarfish::bulk: number of unique alignments : 140
2025-10-25T01:30:02.680728Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpar2j2K/file4c54d4e0b58/config_file_1221.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 Oct 24 21:30:03 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/Rtmpar2j2K/file4c54d4e0b58/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/Rtmpar2j2K/file4c54d4e0b58/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/Rtmpar2j2K/file4c54d4e0b58/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Fri Oct 24 21:30:04 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri Oct 24 21:30:05 2025 -------------------
Realigning sample sample1 -> /tmp/Rtmpar2j2K/file4c54d4e0b58/sample1_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample2 -> /tmp/Rtmpar2j2K/file4c54d4e0b58/sample2_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample3 -> /tmp/Rtmpar2j2K/file4c54d4e0b58/sample3_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Fri Oct 24 21:30:05 2025 ----------
21:30:05 Fri Oct 24 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/Rtmpar2j2K/file4c54a4bdf32/config_file_1221.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 Oct 24 21:30:07 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/Rtmpar2j2K/file4c54a4bdf32/sample1_align2genome.bam
sample2 ->/tmp/Rtmpar2j2K/file4c54a4bdf32/sample2_align2genome.bam
sample3 ->/tmp/Rtmpar2j2K/file4c54a4bdf32/sample3_align2genome.bam
-- Running step: isoform_identification @ Fri Oct 24 21:30:29 2025 -------------
Inputs:  ['/tmp/Rtmpar2j2K/file4c54d4e0b58/sample3_realign2transcript.bam', '/tmp/Rtmpar2j2K/file4c54d4e0b58/sample2_realign2transcript.bam', '/tmp/Rtmpar2j2K/file4c54d4e0b58/sample1_realign2transcript.bam'] /tmp/Rtmpar2j2K/file4c54d4e0b58/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 Oct 24 21:30:29 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/Rtmpar2j2K/file4c54a4bdf32/sample1_realign2transcript.bam
sample2 ->/tmp/Rtmpar2j2K/file4c54a4bdf32/sample2_realign2transcript.bam
sample3 ->/tmp/Rtmpar2j2K/file4c54a4bdf32/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Fri Oct 24 21:30:51 2025 ----------
21:30:51 Fri Oct 24 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Inputs:  ['/tmp/Rtmpar2j2K/file4c54a4bdf32/sample3_realign2transcript.bam', '/tmp/Rtmpar2j2K/file4c54a4bdf32/sample2_realign2transcript.bam', '/tmp/Rtmpar2j2K/file4c54a4bdf32/sample1_realign2transcript.bam'] /tmp/Rtmpar2j2K/file4c54a4bdf32/transcript_assembly.fa.fai 5 0.4 0.4
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpar2j2K/file4c57c8a740/config_file_1221.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 Oct 24 21:30:52 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/Rtmpar2j2K/file4c57c8a740/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 Oct 24 21:30:53 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmpar2j2K/file4c57c8a740/matched_reads.fastq.gz -> /tmp/Rtmpar2j2K/file4c57c8a740/align2genome.bam
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: isoform_identification @ Fri Oct 24 21:30:53 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---
Detected 2 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Fri Oct 24 21:31:04 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/Rtmpar2j2K/file4c57c8a740/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpar2j2K/file4c57c8a740/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/Rtmpar2j2K/file4c57c8a740/matched_reads.fastq.gz -> /tmp/Rtmpar2j2K/file4c57c8a740/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 Oct 24 21:31:05 2025 ----------
2025-10-25T01:31:05.388074Z  INFO oarfish: setting user-provided filter parameters.
2025-10-25T01:31:05.388920Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpar2j2K/file4c57c8a740/realign2transcript.bam, contains 5 reference sequences.
2025-10-25T01:31:05.388960Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-25T01:31:05.388970Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-25T01:31:05.389076Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-25T01:31:05.389089Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-25T01:31:05.397781Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpar2j2K/file4c5fc35b3b/config_file_1221.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 Oct 24 21:31:06 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/Rtmpar2j2K/file4c5fc35b3b/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 Oct 24 21:31:06 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmpar2j2K/file4c5fc35b3b/matched_reads.fastq.gz ->/tmp/Rtmpar2j2K/file4c5fc35b3b/align2genome.bam
-- Running step: isoform_identification @ Fri Oct 24 21:31:27 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---
Detected 2 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Fri Oct 24 21:31:39 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/Rtmpar2j2K/file4c5fc35b3b/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpar2j2K/file4c5fc35b3b/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/Rtmpar2j2K/file4c5fc35b3b/matched_reads.fastq.gz ->/tmp/Rtmpar2j2K/file4c5fc35b3b/realign2transcript.bam
-- Running step: transcript_quantification @ Fri Oct 24 21:31:59 2025 ----------
2025-10-25T01:31:59.161714Z  INFO oarfish: setting user-provided filter parameters.
2025-10-25T01:31:59.162502Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpar2j2K/file4c5fc35b3b/realign2transcript.bam, contains 5 reference sequences.
2025-10-25T01:31:59.162548Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-25T01:31:59.162569Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-25T01:31:59.162704Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-25T01:31:59.162732Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-25T01:31:59.170287Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpar2j2K/file4c574561a89/config_file_1221.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 Oct 24 21:31:59 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/Rtmpar2j2K/file4c574561a89/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 Oct 24 21:32:00 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmpar2j2K/file4c574561a89/matched_reads.fastq.gz -> /tmp/Rtmpar2j2K/file4c574561a89/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 Oct 24 21:32:00 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---
Detected 2 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Fri Oct 24 21:32:12 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/Rtmpar2j2K/file4c574561a89/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpar2j2K/file4c574561a89/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/Rtmpar2j2K/file4c574561a89/matched_reads.fastq.gz -> /tmp/Rtmpar2j2K/file4c574561a89/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 Oct 24 21:32:12 2025 ----------
21:32:12 Fri Oct 24 2025 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
	Counter({'counted_reads': 391, 'not_enough_coverage': 2})
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpar2j2K/file4c51b3a4f57/config_file_1221.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 Oct 24 21:32:13 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/Rtmpar2j2K/file4c51b3a4f57/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 Oct 24 21:32:14 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmpar2j2K/file4c51b3a4f57/matched_reads.fastq.gz ->/tmp/Rtmpar2j2K/file4c51b3a4f57/align2genome.bam
-- Running step: isoform_identification @ Fri Oct 24 21:32:34 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---
Detected 2 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Fri Oct 24 21:32:46 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/Rtmpar2j2K/file4c51b3a4f57/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpar2j2K/file4c51b3a4f57/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/Rtmpar2j2K/file4c51b3a4f57/matched_reads.fastq.gz ->/tmp/Rtmpar2j2K/file4c51b3a4f57/realign2transcript.bam
-- Running step: transcript_quantification @ Fri Oct 24 21:33:07 2025 ----------
21:33:07 Fri Oct 24 2025 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
	Counter({'counted_reads': 354, 'not_enough_coverage': 12, 'unmapped': 6})
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpar2j2K/file4c536de046a/config_file_1221.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 Oct 24 21:33:08 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 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpar2j2K/file4c536de046a/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 Oct 24 21:33:09 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmpar2j2K/file4c536de046a/matched_reads.fastq.gz -> /tmp/Rtmpar2j2K/file4c536de046a/align2genome.bam
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: isoform_identification @ Fri Oct 24 21:33:09 2025 -------------
	Counter({'counted_reads': 354, 'not_enough_coverage': 12, 'unmapped': 6})
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri Oct 24 21:33:10 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/Rtmpar2j2K/file4c536de046a/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpar2j2K/file4c536de046a/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/Rtmpar2j2K/file4c536de046a/matched_reads.fastq.gz -> /tmp/Rtmpar2j2K/file4c536de046a/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 Oct 24 21:33:10 2025 ----------
2025-10-25T01:33:10.767456Z  INFO oarfish: setting user-provided filter parameters.
2025-10-25T01:33:10.768237Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpar2j2K/file4c536de046a/realign2transcript.bam, contains 10 reference sequences.
2025-10-25T01:33:10.768288Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-25T01:33:10.768306Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-25T01:33:10.768586Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-25T01:33:10.768632Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-25T01:33:10.781290Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpar2j2K/file4c56416d36c/config_file_1221.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 Oct 24 21:33:11 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/Rtmpar2j2K/file4c56416d36c/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 Oct 24 21:33:12 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmpar2j2K/file4c56416d36c/matched_reads.fastq.gz ->/tmp/Rtmpar2j2K/file4c56416d36c/align2genome.bam
-- Running step: isoform_identification @ Fri Oct 24 21:33:32 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri Oct 24 21:33:33 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/Rtmpar2j2K/file4c56416d36c/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpar2j2K/file4c56416d36c/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/Rtmpar2j2K/file4c56416d36c/matched_reads.fastq.gz ->/tmp/Rtmpar2j2K/file4c56416d36c/realign2transcript.bam
-- Running step: transcript_quantification @ Fri Oct 24 21:33:53 2025 ----------
2025-10-25T01:33:53.944759Z  INFO oarfish: setting user-provided filter parameters.
2025-10-25T01:33:53.945798Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpar2j2K/file4c56416d36c/realign2transcript.bam, contains 10 reference sequences.
2025-10-25T01:33:53.945864Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-25T01:33:53.945895Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-25T01:33:53.946042Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-25T01:33:53.946069Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-25T01:33:53.957560Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpar2j2K/file4c5191b16/config_file_1221.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 Oct 24 21:33:55 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/Rtmpar2j2K/file4c5191b16/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 Oct 24 21:33:55 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmpar2j2K/file4c5191b16/matched_reads.fastq.gz -> /tmp/Rtmpar2j2K/file4c5191b16/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 Oct 24 21:33:55 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri Oct 24 21:33:56 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/Rtmpar2j2K/file4c5191b16/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpar2j2K/file4c5191b16/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/Rtmpar2j2K/file4c5191b16/matched_reads.fastq.gz -> /tmp/Rtmpar2j2K/file4c5191b16/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 Oct 24 21:33:56 2025 ----------
21:33:56 Fri Oct 24 2025 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpar2j2K/file4c56735774e/config_file_1221.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 Oct 24 21:33:57 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/Rtmpar2j2K/file4c56735774e/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 Oct 24 21:33:58 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmpar2j2K/file4c56735774e/matched_reads.fastq.gz ->/tmp/Rtmpar2j2K/file4c56735774e/align2genome.bam
-- Running step: isoform_identification @ Fri Oct 24 21:34:18 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 Oct 24 21:34:19 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/Rtmpar2j2K/file4c56735774e/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpar2j2K/file4c56735774e/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/Rtmpar2j2K/file4c56735774e/matched_reads.fastq.gz ->/tmp/Rtmpar2j2K/file4c56735774e/realign2transcript.bam
-- Running step: transcript_quantification @ Fri Oct 24 21:34:39 2025 ----------
21:34:39 Fri Oct 24 2025 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpar2j2K/file4c57d358c70/config_file_1221.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 Oct 24 21:34:41 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 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpar2j2K/file4c57d358c70/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpar2j2K/file4c57d358c70/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpar2j2K/file4c57d358c70/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpar2j2K/file4c57d358c70/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/Rtmpar2j2K/file4c57d358c70/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpar2j2K/file4c57d358c70/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/Rtmpar2j2K/file4c57d358c70/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpar2j2K/file4c57d358c70/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/Rtmpar2j2K/file4c57d358c70/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpar2j2K/file4c57d358c70/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 Oct 24 21:34:43 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmpar2j2K/file4c57d358c70/sampleA_matched_reads.fastq.gz -> /tmp/Rtmpar2j2K/file4c57d358c70/sampleA_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmpar2j2K/file4c57d358c70/sample1_matched_reads.fastq.gz -> /tmp/Rtmpar2j2K/file4c57d358c70/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmpar2j2K/file4c57d358c70/sample2_matched_reads.fastq.gz -> /tmp/Rtmpar2j2K/file4c57d358c70/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmpar2j2K/file4c57d358c70/sample3_matched_reads.fastq.gz -> /tmp/Rtmpar2j2K/file4c57d358c70/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Fri Oct 24 21:34:45 2025 ----------------
21:34:45 Fri Oct 24 2025 quantify genes 
Using BAM(s): '/tmp/Rtmpar2j2K/file4c57d358c70/sampleA_align2genome.bam',
'/tmp/Rtmpar2j2K/file4c57d358c70/sample1_align2genome.bam',
'/tmp/Rtmpar2j2K/file4c57d358c70/sample2_align2genome.bam', and
'/tmp/Rtmpar2j2K/file4c57d358c70/sample3_align2genome.bam'
	Counter({'counted_reads': 368, 'unmapped': 4})
parsing /tmp/Rtmpar2j2K/file4c57d358c70/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 19.08gene_group/s]
2025-10-24 21:34:46.728 R[1221:639523667] XType: com.apple.fonts is not accessible.
2025-10-24 21:34:46.729 R[1221:639523667] 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, 400954.42Read/s]
parsing /tmp/Rtmpar2j2K/file4c57d358c70/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 34.12gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1004479.36Read/s]
parsing /tmp/Rtmpar2j2K/file4c57d358c70/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 37.70gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1298706.96Read/s]
parsing /tmp/Rtmpar2j2K/file4c57d358c70/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 32.15gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 800500.80Read/s]
-- Running step: isoform_identification @ Fri Oct 24 21:34:47 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 6 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Fri Oct 24 21:35:13 2025 -------------------
Checking for fastq file(s) /tmp/Rtmpar2j2K/file4c57d358c70/fastq, /tmp/Rtmpar2j2K/file4c57d358c70/fastq/sample1.fq.gz, /tmp/Rtmpar2j2K/file4c57d358c70/fastq/sample2.fq.gz, /tmp/Rtmpar2j2K/file4c57d358c70/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpar2j2K/file4c57d358c70/sampleA_matched_reads.fastq.gz, /tmp/Rtmpar2j2K/file4c57d358c70/sample1_matched_reads.fastq.gz, /tmp/Rtmpar2j2K/file4c57d358c70/sample2_matched_reads.fastq.gz, /tmp/Rtmpar2j2K/file4c57d358c70/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpar2j2K/file4c57d358c70/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmpar2j2K/file4c57d358c70/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmpar2j2K/file4c57d358c70/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmpar2j2K/file4c57d358c70/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/Rtmpar2j2K/file4c57d358c70/sampleA_matched_reads_dedup.fastq.gz -> /tmp/Rtmpar2j2K/file4c57d358c70/sampleA_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/Rtmpar2j2K/file4c57d358c70/sample1_matched_reads_dedup.fastq.gz -> /tmp/Rtmpar2j2K/file4c57d358c70/sample1_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/Rtmpar2j2K/file4c57d358c70/sample2_matched_reads_dedup.fastq.gz -> /tmp/Rtmpar2j2K/file4c57d358c70/sample2_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/Rtmpar2j2K/file4c57d358c70/sample3_matched_reads_dedup.fastq.gz -> /tmp/Rtmpar2j2K/file4c57d358c70/sample3_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

-- Running step: transcript_quantification @ Fri Oct 24 21:35:14 2025 ----------
2025-10-25T01:35:14.792544Z  INFO oarfish: setting user-provided filter parameters.
2025-10-25T01:35:14.793248Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpar2j2K/file4c57d358c70/sampleA_realign2transcript.bam, contains 5 reference sequences.
2025-10-25T01:35:14.793306Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-25T01:35:14.793328Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-25T01:35:14.793462Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-25T01:35:14.793490Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-25T01:35:14.801605Z  INFO oarfish::single_cell: Processed 100 cells.
2025-10-25T01:35:15.191026Z  INFO oarfish: setting user-provided filter parameters.
2025-10-25T01:35:15.191688Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpar2j2K/file4c57d358c70/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-10-25T01:35:15.191733Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-25T01:35:15.191746Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-25T01:35:15.191849Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-25T01:35:15.191864Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-25T01:35:15.601122Z  INFO oarfish: setting user-provided filter parameters.
2025-10-25T01:35:15.601816Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpar2j2K/file4c57d358c70/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-10-25T01:35:15.601866Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-25T01:35:15.601887Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-25T01:35:15.602059Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-25T01:35:15.602090Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-25T01:35:15.975805Z  INFO oarfish: setting user-provided filter parameters.
2025-10-25T01:35:15.976428Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpar2j2K/file4c57d358c70/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-10-25T01:35:15.976481Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-25T01:35:15.976500Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-25T01:35:15.976889Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-25T01:35:15.976948Z  INFO oarfish: parsed reference information for 5 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpar2j2K/file4c5184fc403/config_file_1221.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 Oct 24 21:35:16 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/Rtmpar2j2K/file4c5184fc403/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpar2j2K/file4c5184fc403/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpar2j2K/file4c5184fc403/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpar2j2K/file4c5184fc403/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/Rtmpar2j2K/file4c5184fc403/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpar2j2K/file4c5184fc403/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/Rtmpar2j2K/file4c5184fc403/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpar2j2K/file4c5184fc403/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/Rtmpar2j2K/file4c5184fc403/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpar2j2K/file4c5184fc403/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 Oct 24 21:35:18 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmpar2j2K/file4c5184fc403/sampleA_matched_reads.fastq.gz ->/tmp/Rtmpar2j2K/file4c5184fc403/sampleA_align2genome.bam
/tmp/Rtmpar2j2K/file4c5184fc403/sample1_matched_reads.fastq.gz ->/tmp/Rtmpar2j2K/file4c5184fc403/sample1_align2genome.bam
/tmp/Rtmpar2j2K/file4c5184fc403/sample2_matched_reads.fastq.gz ->/tmp/Rtmpar2j2K/file4c5184fc403/sample2_align2genome.bam
/tmp/Rtmpar2j2K/file4c5184fc403/sample3_matched_reads.fastq.gz ->/tmp/Rtmpar2j2K/file4c5184fc403/sample3_align2genome.bam
-- Running step: gene_quantification @ Fri Oct 24 21:35:40 2025 ----------------
21:35:40 Fri Oct 24 2025 quantify genes 
Using BAM(s): '/tmp/Rtmpar2j2K/file4c5184fc403/sampleA_align2genome.bam',
'/tmp/Rtmpar2j2K/file4c5184fc403/sample1_align2genome.bam',
'/tmp/Rtmpar2j2K/file4c5184fc403/sample2_align2genome.bam', and
'/tmp/Rtmpar2j2K/file4c5184fc403/sample3_align2genome.bam'
parsing /tmp/Rtmpar2j2K/file4c5184fc403/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 15.75gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 364569.92Read/s]
parsing /tmp/Rtmpar2j2K/file4c5184fc403/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 37.32gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1252031.04Read/s]
parsing /tmp/Rtmpar2j2K/file4c5184fc403/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 34.39gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1040357.18Read/s]
parsing /tmp/Rtmpar2j2K/file4c5184fc403/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 25.47gene_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, 778568.46Read/s]
-- Running step: isoform_identification @ Fri Oct 24 21:35:41 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 6 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Fri Oct 24 21:36:08 2025 -------------------
Checking for fastq file(s) /tmp/Rtmpar2j2K/file4c5184fc403/fastq, /tmp/Rtmpar2j2K/file4c5184fc403/fastq/sample1.fq.gz, /tmp/Rtmpar2j2K/file4c5184fc403/fastq/sample2.fq.gz, /tmp/Rtmpar2j2K/file4c5184fc403/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpar2j2K/file4c5184fc403/sampleA_matched_reads.fastq.gz, /tmp/Rtmpar2j2K/file4c5184fc403/sample1_matched_reads.fastq.gz, /tmp/Rtmpar2j2K/file4c5184fc403/sample2_matched_reads.fastq.gz, /tmp/Rtmpar2j2K/file4c5184fc403/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpar2j2K/file4c5184fc403/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmpar2j2K/file4c5184fc403/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmpar2j2K/file4c5184fc403/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmpar2j2K/file4c5184fc403/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/Rtmpar2j2K/file4c5184fc403/sampleA_matched_reads_dedup.fastq.gz ->/tmp/Rtmpar2j2K/file4c5184fc403/sampleA_realign2transcript.bam
/tmp/Rtmpar2j2K/file4c5184fc403/sample1_matched_reads_dedup.fastq.gz ->/tmp/Rtmpar2j2K/file4c5184fc403/sample1_realign2transcript.bam
/tmp/Rtmpar2j2K/file4c5184fc403/sample2_matched_reads_dedup.fastq.gz ->/tmp/Rtmpar2j2K/file4c5184fc403/sample2_realign2transcript.bam
/tmp/Rtmpar2j2K/file4c5184fc403/sample3_matched_reads_dedup.fastq.gz ->/tmp/Rtmpar2j2K/file4c5184fc403/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Fri Oct 24 21:36:29 2025 ----------
2025-10-25T01:36:29.743679Z  INFO oarfish: setting user-provided filter parameters.
2025-10-25T01:36:29.744448Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpar2j2K/file4c5184fc403/sampleA_realign2transcript.bam, contains 5 reference sequences.
2025-10-25T01:36:29.744501Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-25T01:36:29.744518Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-25T01:36:29.744657Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-25T01:36:29.744679Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-25T01:36:29.754066Z  INFO oarfish::single_cell: Processed 100 cells.
2025-10-25T01:36:30.234877Z  INFO oarfish: setting user-provided filter parameters.
2025-10-25T01:36:30.235575Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpar2j2K/file4c5184fc403/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-10-25T01:36:30.235641Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-25T01:36:30.235682Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-25T01:36:30.236036Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-25T01:36:30.236068Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-25T01:36:30.714435Z  INFO oarfish: setting user-provided filter parameters.
2025-10-25T01:36:30.715281Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpar2j2K/file4c5184fc403/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-10-25T01:36:30.715322Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-25T01:36:30.715335Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-25T01:36:30.715442Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-25T01:36:30.715458Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-25T01:36:31.174372Z  INFO oarfish: setting user-provided filter parameters.
2025-10-25T01:36:31.175098Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpar2j2K/file4c5184fc403/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-10-25T01:36:31.175138Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-25T01:36:31.175151Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-25T01:36:31.175383Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-25T01:36:31.175428Z  INFO oarfish: parsed reference information for 5 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpar2j2K/file4c524e346a6/config_file_1221.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 Oct 24 21:36:32 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/Rtmpar2j2K/file4c524e346a6/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpar2j2K/file4c524e346a6/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpar2j2K/file4c524e346a6/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpar2j2K/file4c524e346a6/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/Rtmpar2j2K/file4c524e346a6/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpar2j2K/file4c524e346a6/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/Rtmpar2j2K/file4c524e346a6/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpar2j2K/file4c524e346a6/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/Rtmpar2j2K/file4c524e346a6/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpar2j2K/file4c524e346a6/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 Oct 24 21:36:34 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmpar2j2K/file4c524e346a6/sampleA_matched_reads.fastq.gz -> /tmp/Rtmpar2j2K/file4c524e346a6/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/Rtmpar2j2K/file4c524e346a6/sample1_matched_reads.fastq.gz -> /tmp/Rtmpar2j2K/file4c524e346a6/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/Rtmpar2j2K/file4c524e346a6/sample2_matched_reads.fastq.gz -> /tmp/Rtmpar2j2K/file4c524e346a6/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/Rtmpar2j2K/file4c524e346a6/sample3_matched_reads.fastq.gz -> /tmp/Rtmpar2j2K/file4c524e346a6/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 Oct 24 21:36:35 2025 ----------------
21:36:35 Fri Oct 24 2025 quantify genes 
Using BAM(s): '/tmp/Rtmpar2j2K/file4c524e346a6/sampleA_align2genome.bam',
'/tmp/Rtmpar2j2K/file4c524e346a6/sample1_align2genome.bam',
'/tmp/Rtmpar2j2K/file4c524e346a6/sample2_align2genome.bam', and
'/tmp/Rtmpar2j2K/file4c524e346a6/sample3_align2genome.bam'
parsing /tmp/Rtmpar2j2K/file4c524e346a6/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 19.85gene_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, 376630.15Read/s]
parsing /tmp/Rtmpar2j2K/file4c524e346a6/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 38.56gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1118958.49Read/s]
parsing /tmp/Rtmpar2j2K/file4c524e346a6/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 38.75gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1155328.34Read/s]
parsing /tmp/Rtmpar2j2K/file4c524e346a6/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 28.23gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 835918.37Read/s]
-- Running step: isoform_identification @ Fri Oct 24 21:36:37 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 6 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Fri Oct 24 21:37:06 2025 -------------------
Checking for fastq file(s) /tmp/Rtmpar2j2K/file4c524e346a6/fastq, /tmp/Rtmpar2j2K/file4c524e346a6/fastq/sample1.fq.gz, /tmp/Rtmpar2j2K/file4c524e346a6/fastq/sample2.fq.gz, /tmp/Rtmpar2j2K/file4c524e346a6/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpar2j2K/file4c524e346a6/sampleA_matched_reads.fastq.gz, /tmp/Rtmpar2j2K/file4c524e346a6/sample1_matched_reads.fastq.gz, /tmp/Rtmpar2j2K/file4c524e346a6/sample2_matched_reads.fastq.gz, /tmp/Rtmpar2j2K/file4c524e346a6/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpar2j2K/file4c524e346a6/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmpar2j2K/file4c524e346a6/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmpar2j2K/file4c524e346a6/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmpar2j2K/file4c524e346a6/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/Rtmpar2j2K/file4c524e346a6/sampleA_matched_reads_dedup.fastq.gz -> /tmp/Rtmpar2j2K/file4c524e346a6/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/Rtmpar2j2K/file4c524e346a6/sample1_matched_reads_dedup.fastq.gz -> /tmp/Rtmpar2j2K/file4c524e346a6/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/Rtmpar2j2K/file4c524e346a6/sample2_matched_reads_dedup.fastq.gz -> /tmp/Rtmpar2j2K/file4c524e346a6/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/Rtmpar2j2K/file4c524e346a6/sample3_matched_reads_dedup.fastq.gz -> /tmp/Rtmpar2j2K/file4c524e346a6/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 Oct 24 21:37:07 2025 ----------
21:37:07 Fri Oct 24 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/Rtmpar2j2K/file4c524e346a6/sample3_realign2transcript.bam...
parsing /tmp/Rtmpar2j2K/file4c524e346a6/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpar2j2K/file4c524e346a6/sample3_realign2transcript.bamdone
parsing /tmp/Rtmpar2j2K/file4c524e346a6/sampleA_realign2transcript.bam...
parsing /tmp/Rtmpar2j2K/file4c524e346a6/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpar2j2K/file4c524e346a6/sampleA_realign2transcript.bamdone
parsing /tmp/Rtmpar2j2K/file4c524e346a6/sample2_realign2transcript.bam...
parsing /tmp/Rtmpar2j2K/file4c524e346a6/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpar2j2K/file4c524e346a6/sample2_realign2transcript.bamdone
parsing /tmp/Rtmpar2j2K/file4c524e346a6/sample1_realign2transcript.bam...
parsing /tmp/Rtmpar2j2K/file4c524e346a6/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpar2j2K/file4c524e346a6/sample1_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpar2j2K/file4c538ddd3fd/config_file_1221.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 Oct 24 21:37:10 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/Rtmpar2j2K/file4c538ddd3fd/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpar2j2K/file4c538ddd3fd/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpar2j2K/file4c538ddd3fd/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpar2j2K/file4c538ddd3fd/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/Rtmpar2j2K/file4c538ddd3fd/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpar2j2K/file4c538ddd3fd/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/Rtmpar2j2K/file4c538ddd3fd/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpar2j2K/file4c538ddd3fd/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/Rtmpar2j2K/file4c538ddd3fd/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpar2j2K/file4c538ddd3fd/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 Oct 24 21:37:11 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmpar2j2K/file4c538ddd3fd/sampleA_matched_reads.fastq.gz ->/tmp/Rtmpar2j2K/file4c538ddd3fd/sampleA_align2genome.bam
/tmp/Rtmpar2j2K/file4c538ddd3fd/sample1_matched_reads.fastq.gz ->/tmp/Rtmpar2j2K/file4c538ddd3fd/sample1_align2genome.bam
/tmp/Rtmpar2j2K/file4c538ddd3fd/sample2_matched_reads.fastq.gz ->/tmp/Rtmpar2j2K/file4c538ddd3fd/sample2_align2genome.bam
/tmp/Rtmpar2j2K/file4c538ddd3fd/sample3_matched_reads.fastq.gz ->/tmp/Rtmpar2j2K/file4c538ddd3fd/sample3_align2genome.bam
-- Running step: gene_quantification @ Fri Oct 24 21:37:33 2025 ----------------
21:37:33 Fri Oct 24 2025 quantify genes 
Using BAM(s): '/tmp/Rtmpar2j2K/file4c538ddd3fd/sampleA_align2genome.bam',
'/tmp/Rtmpar2j2K/file4c538ddd3fd/sample1_align2genome.bam',
'/tmp/Rtmpar2j2K/file4c538ddd3fd/sample2_align2genome.bam', and
'/tmp/Rtmpar2j2K/file4c538ddd3fd/sample3_align2genome.bam'
	Counter({'counted_reads': 168, 'not_enough_coverage': 6, 'unmapped': 2})
	Counter({'counted_reads': 347, 'not_enough_coverage': 9, 'unmapped': 2})
	Counter({'counted_reads': 92, 'not_enough_coverage': 3})
	Counter({'counted_reads': 89, 'not_enough_coverage': 2})
parsing /tmp/Rtmpar2j2K/file4c538ddd3fd/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 14.60gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 343547.61Read/s]
parsing /tmp/Rtmpar2j2K/file4c538ddd3fd/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 40.20gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1370687.58Read/s]
parsing /tmp/Rtmpar2j2K/file4c538ddd3fd/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 36.27gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1261368.94Read/s]
parsing /tmp/Rtmpar2j2K/file4c538ddd3fd/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 28.43gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 475911.59Read/s]
-- Running step: isoform_identification @ Fri Oct 24 21:37:35 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 6 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Fri Oct 24 21:38:02 2025 -------------------
Checking for fastq file(s) /tmp/Rtmpar2j2K/file4c538ddd3fd/fastq, /tmp/Rtmpar2j2K/file4c538ddd3fd/fastq/sample1.fq.gz, /tmp/Rtmpar2j2K/file4c538ddd3fd/fastq/sample2.fq.gz, /tmp/Rtmpar2j2K/file4c538ddd3fd/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpar2j2K/file4c538ddd3fd/sampleA_matched_reads.fastq.gz, /tmp/Rtmpar2j2K/file4c538ddd3fd/sample1_matched_reads.fastq.gz, /tmp/Rtmpar2j2K/file4c538ddd3fd/sample2_matched_reads.fastq.gz, /tmp/Rtmpar2j2K/file4c538ddd3fd/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpar2j2K/file4c538ddd3fd/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmpar2j2K/file4c538ddd3fd/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmpar2j2K/file4c538ddd3fd/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmpar2j2K/file4c538ddd3fd/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/Rtmpar2j2K/file4c538ddd3fd/sampleA_matched_reads_dedup.fastq.gz ->/tmp/Rtmpar2j2K/file4c538ddd3fd/sampleA_realign2transcript.bam
/tmp/Rtmpar2j2K/file4c538ddd3fd/sample1_matched_reads_dedup.fastq.gz ->/tmp/Rtmpar2j2K/file4c538ddd3fd/sample1_realign2transcript.bam
/tmp/Rtmpar2j2K/file4c538ddd3fd/sample2_matched_reads_dedup.fastq.gz ->/tmp/Rtmpar2j2K/file4c538ddd3fd/sample2_realign2transcript.bam
/tmp/Rtmpar2j2K/file4c538ddd3fd/sample3_matched_reads_dedup.fastq.gz ->/tmp/Rtmpar2j2K/file4c538ddd3fd/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Fri Oct 24 21:38:23 2025 ----------
21:38:23 Fri Oct 24 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/Rtmpar2j2K/file4c538ddd3fd/sample3_realign2transcript.bam...
parsing /tmp/Rtmpar2j2K/file4c538ddd3fd/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpar2j2K/file4c538ddd3fd/sample3_realign2transcript.bamdone
parsing /tmp/Rtmpar2j2K/file4c538ddd3fd/sampleA_realign2transcript.bam...
parsing /tmp/Rtmpar2j2K/file4c538ddd3fd/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpar2j2K/file4c538ddd3fd/sampleA_realign2transcript.bamdone
parsing /tmp/Rtmpar2j2K/file4c538ddd3fd/sample2_realign2transcript.bam...
parsing /tmp/Rtmpar2j2K/file4c538ddd3fd/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpar2j2K/file4c538ddd3fd/sample2_realign2transcript.bamdone
parsing /tmp/Rtmpar2j2K/file4c538ddd3fd/sample1_realign2transcript.bam...
parsing /tmp/Rtmpar2j2K/file4c538ddd3fd/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpar2j2K/file4c538ddd3fd/sample1_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpar2j2K/file4c53e10a561/config_file_1221.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 Oct 24 21:38:26 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/Rtmpar2j2K/file4c53e10a561/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpar2j2K/file4c53e10a561/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpar2j2K/file4c53e10a561/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpar2j2K/file4c53e10a561/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/Rtmpar2j2K/file4c53e10a561/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpar2j2K/file4c53e10a561/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/Rtmpar2j2K/file4c53e10a561/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpar2j2K/file4c53e10a561/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/Rtmpar2j2K/file4c53e10a561/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpar2j2K/file4c53e10a561/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 Oct 24 21:38:28 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmpar2j2K/file4c53e10a561/sampleA_matched_reads.fastq.gz -> /tmp/Rtmpar2j2K/file4c53e10a561/sampleA_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmpar2j2K/file4c53e10a561/sample1_matched_reads.fastq.gz -> /tmp/Rtmpar2j2K/file4c53e10a561/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmpar2j2K/file4c53e10a561/sample2_matched_reads.fastq.gz -> /tmp/Rtmpar2j2K/file4c53e10a561/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmpar2j2K/file4c53e10a561/sample3_matched_reads.fastq.gz -> /tmp/Rtmpar2j2K/file4c53e10a561/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Fri Oct 24 21:38:29 2025 ----------------
21:38:29 Fri Oct 24 2025 quantify genes 
Using BAM(s): '/tmp/Rtmpar2j2K/file4c53e10a561/sampleA_align2genome.bam',
'/tmp/Rtmpar2j2K/file4c53e10a561/sample1_align2genome.bam',
'/tmp/Rtmpar2j2K/file4c53e10a561/sample2_align2genome.bam', and
'/tmp/Rtmpar2j2K/file4c53e10a561/sample3_align2genome.bam'
	Counter({'counted_reads': 168, 'not_enough_coverage': 6, 'unmapped': 2})
	Counter({'counted_reads': 347, 'not_enough_coverage': 9, 'unmapped': 2})
	Counter({'counted_reads': 92, 'not_enough_coverage': 3})
	Counter({'counted_reads': 89, 'not_enough_coverage': 2})
parsing /tmp/Rtmpar2j2K/file4c53e10a561/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 19.18gene_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, 379520.07Read/s]
parsing /tmp/Rtmpar2j2K/file4c53e10a561/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 40.16gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1318631.79Read/s]
parsing /tmp/Rtmpar2j2K/file4c53e10a561/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 35.96gene_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, 1490725.05Read/s]
parsing /tmp/Rtmpar2j2K/file4c53e10a561/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 25.47gene_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, 627363.89Read/s]
-- Running step: isoform_identification @ Fri Oct 24 21:38:31 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri Oct 24 21:38:31 2025 -------------------
Checking for fastq file(s) /tmp/Rtmpar2j2K/file4c53e10a561/fastq, /tmp/Rtmpar2j2K/file4c53e10a561/fastq/sample1.fq.gz, /tmp/Rtmpar2j2K/file4c53e10a561/fastq/sample2.fq.gz, /tmp/Rtmpar2j2K/file4c53e10a561/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpar2j2K/file4c53e10a561/sampleA_matched_reads.fastq.gz, /tmp/Rtmpar2j2K/file4c53e10a561/sample1_matched_reads.fastq.gz, /tmp/Rtmpar2j2K/file4c53e10a561/sample2_matched_reads.fastq.gz, /tmp/Rtmpar2j2K/file4c53e10a561/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpar2j2K/file4c53e10a561/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmpar2j2K/file4c53e10a561/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmpar2j2K/file4c53e10a561/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmpar2j2K/file4c53e10a561/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/Rtmpar2j2K/file4c53e10a561/sampleA_matched_reads_dedup.fastq.gz -> /tmp/Rtmpar2j2K/file4c53e10a561/sampleA_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/Rtmpar2j2K/file4c53e10a561/sample1_matched_reads_dedup.fastq.gz -> /tmp/Rtmpar2j2K/file4c53e10a561/sample1_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/Rtmpar2j2K/file4c53e10a561/sample2_matched_reads_dedup.fastq.gz -> /tmp/Rtmpar2j2K/file4c53e10a561/sample2_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/Rtmpar2j2K/file4c53e10a561/sample3_matched_reads_dedup.fastq.gz -> /tmp/Rtmpar2j2K/file4c53e10a561/sample3_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

-- Running step: transcript_quantification @ Fri Oct 24 21:38:33 2025 ----------
2025-10-25T01:38:33.759121Z  INFO oarfish: setting user-provided filter parameters.
2025-10-25T01:38:33.760104Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpar2j2K/file4c53e10a561/sampleA_realign2transcript.bam, contains 14 reference sequences.
2025-10-25T01:38:33.760144Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-25T01:38:33.760158Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-25T01:38:33.760777Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-25T01:38:33.760821Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-10-25T01:38:33.777776Z  INFO oarfish::single_cell: Processed 100 cells.
2025-10-25T01:38:34.526337Z  INFO oarfish: setting user-provided filter parameters.
2025-10-25T01:38:34.526923Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpar2j2K/file4c53e10a561/sample1_realign2transcript.bam, contains 14 reference sequences.
2025-10-25T01:38:34.526957Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-25T01:38:34.526966Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-25T01:38:34.527095Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-25T01:38:34.527110Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-10-25T01:38:35.247013Z  INFO oarfish: setting user-provided filter parameters.
2025-10-25T01:38:35.247717Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpar2j2K/file4c53e10a561/sample2_realign2transcript.bam, contains 14 reference sequences.
2025-10-25T01:38:35.247747Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-25T01:38:35.247756Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-25T01:38:35.247976Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-25T01:38:35.248000Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-10-25T01:38:35.984906Z  INFO oarfish: setting user-provided filter parameters.
2025-10-25T01:38:35.985676Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpar2j2K/file4c53e10a561/sample3_realign2transcript.bam, contains 14 reference sequences.
2025-10-25T01:38:35.985724Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-25T01:38:35.985735Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-25T01:38:35.985936Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-25T01:38:35.985958Z  INFO oarfish: parsed reference information for 14 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpar2j2K/file4c57972d1bd/config_file_1221.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 Oct 24 21:38:37 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 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpar2j2K/file4c57972d1bd/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpar2j2K/file4c57972d1bd/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpar2j2K/file4c57972d1bd/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpar2j2K/file4c57972d1bd/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/Rtmpar2j2K/file4c57972d1bd/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpar2j2K/file4c57972d1bd/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/Rtmpar2j2K/file4c57972d1bd/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpar2j2K/file4c57972d1bd/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/Rtmpar2j2K/file4c57972d1bd/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpar2j2K/file4c57972d1bd/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 Oct 24 21:38:38 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmpar2j2K/file4c57972d1bd/sampleA_matched_reads.fastq.gz ->/tmp/Rtmpar2j2K/file4c57972d1bd/sampleA_align2genome.bam
/tmp/Rtmpar2j2K/file4c57972d1bd/sample1_matched_reads.fastq.gz ->/tmp/Rtmpar2j2K/file4c57972d1bd/sample1_align2genome.bam
/tmp/Rtmpar2j2K/file4c57972d1bd/sample2_matched_reads.fastq.gz ->/tmp/Rtmpar2j2K/file4c57972d1bd/sample2_align2genome.bam
/tmp/Rtmpar2j2K/file4c57972d1bd/sample3_matched_reads.fastq.gz ->/tmp/Rtmpar2j2K/file4c57972d1bd/sample3_align2genome.bam
-- Running step: gene_quantification @ Fri Oct 24 21:38:59 2025 ----------------
21:38:59 Fri Oct 24 2025 quantify genes 
Using BAM(s): '/tmp/Rtmpar2j2K/file4c57972d1bd/sampleA_align2genome.bam',
'/tmp/Rtmpar2j2K/file4c57972d1bd/sample1_align2genome.bam',
'/tmp/Rtmpar2j2K/file4c57972d1bd/sample2_align2genome.bam', and
'/tmp/Rtmpar2j2K/file4c57972d1bd/sample3_align2genome.bam'
parsing /tmp/Rtmpar2j2K/file4c57972d1bd/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 16.28gene_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, 379822.51Read/s]
parsing /tmp/Rtmpar2j2K/file4c57972d1bd/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 42.29gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 895682.92Read/s]
parsing /tmp/Rtmpar2j2K/file4c57972d1bd/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 45.20gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 817539.37Read/s]
parsing /tmp/Rtmpar2j2K/file4c57972d1bd/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 29.85gene_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, 647668.93Read/s]
-- Running step: isoform_identification @ Fri Oct 24 21:39:00 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri Oct 24 21:39:01 2025 -------------------
Checking for fastq file(s) /tmp/Rtmpar2j2K/file4c57972d1bd/fastq, /tmp/Rtmpar2j2K/file4c57972d1bd/fastq/sample1.fq.gz, /tmp/Rtmpar2j2K/file4c57972d1bd/fastq/sample2.fq.gz, /tmp/Rtmpar2j2K/file4c57972d1bd/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpar2j2K/file4c57972d1bd/sampleA_matched_reads.fastq.gz, /tmp/Rtmpar2j2K/file4c57972d1bd/sample1_matched_reads.fastq.gz, /tmp/Rtmpar2j2K/file4c57972d1bd/sample2_matched_reads.fastq.gz, /tmp/Rtmpar2j2K/file4c57972d1bd/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpar2j2K/file4c57972d1bd/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmpar2j2K/file4c57972d1bd/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmpar2j2K/file4c57972d1bd/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmpar2j2K/file4c57972d1bd/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/Rtmpar2j2K/file4c57972d1bd/sampleA_matched_reads_dedup.fastq.gz ->/tmp/Rtmpar2j2K/file4c57972d1bd/sampleA_realign2transcript.bam
/tmp/Rtmpar2j2K/file4c57972d1bd/sample1_matched_reads_dedup.fastq.gz ->/tmp/Rtmpar2j2K/file4c57972d1bd/sample1_realign2transcript.bam
/tmp/Rtmpar2j2K/file4c57972d1bd/sample2_matched_reads_dedup.fastq.gz ->/tmp/Rtmpar2j2K/file4c57972d1bd/sample2_realign2transcript.bam
/tmp/Rtmpar2j2K/file4c57972d1bd/sample3_matched_reads_dedup.fastq.gz ->/tmp/Rtmpar2j2K/file4c57972d1bd/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Fri Oct 24 21:39:24 2025 ----------
2025-10-25T01:39:24.121244Z  INFO oarfish: setting user-provided filter parameters.
2025-10-25T01:39:24.122304Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpar2j2K/file4c57972d1bd/sampleA_realign2transcript.bam, contains 14 reference sequences.
2025-10-25T01:39:24.122357Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-25T01:39:24.122375Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-25T01:39:24.122568Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-25T01:39:24.122599Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-10-25T01:39:24.138447Z  INFO oarfish::single_cell: Processed 100 cells.
2025-10-25T01:39:24.991552Z  INFO oarfish: setting user-provided filter parameters.
2025-10-25T01:39:24.992183Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpar2j2K/file4c57972d1bd/sample1_realign2transcript.bam, contains 14 reference sequences.
2025-10-25T01:39:24.992238Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-25T01:39:24.992257Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-25T01:39:24.992456Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-25T01:39:24.992483Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-10-25T01:39:25.764901Z  INFO oarfish: setting user-provided filter parameters.
2025-10-25T01:39:25.765605Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpar2j2K/file4c57972d1bd/sample2_realign2transcript.bam, contains 14 reference sequences.
2025-10-25T01:39:25.765658Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-25T01:39:25.765677Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-25T01:39:25.765865Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-25T01:39:25.765893Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-10-25T01:39:26.424765Z  INFO oarfish: setting user-provided filter parameters.
2025-10-25T01:39:26.425450Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpar2j2K/file4c57972d1bd/sample3_realign2transcript.bam, contains 14 reference sequences.
2025-10-25T01:39:26.425502Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-25T01:39:26.425521Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-25T01:39:26.425714Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-25T01:39:26.425743Z  INFO oarfish: parsed reference information for 14 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpar2j2K/file4c542872d5/config_file_1221.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 Oct 24 21:39:27 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/Rtmpar2j2K/file4c542872d5/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpar2j2K/file4c542872d5/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpar2j2K/file4c542872d5/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpar2j2K/file4c542872d5/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/Rtmpar2j2K/file4c542872d5/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpar2j2K/file4c542872d5/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/Rtmpar2j2K/file4c542872d5/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpar2j2K/file4c542872d5/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/Rtmpar2j2K/file4c542872d5/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpar2j2K/file4c542872d5/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 Oct 24 21:39:29 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmpar2j2K/file4c542872d5/sampleA_matched_reads.fastq.gz -> /tmp/Rtmpar2j2K/file4c542872d5/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/Rtmpar2j2K/file4c542872d5/sample1_matched_reads.fastq.gz -> /tmp/Rtmpar2j2K/file4c542872d5/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/Rtmpar2j2K/file4c542872d5/sample2_matched_reads.fastq.gz -> /tmp/Rtmpar2j2K/file4c542872d5/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/Rtmpar2j2K/file4c542872d5/sample3_matched_reads.fastq.gz -> /tmp/Rtmpar2j2K/file4c542872d5/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 Oct 24 21:39:31 2025 ----------------
21:39:31 Fri Oct 24 2025 quantify genes 
Using BAM(s): '/tmp/Rtmpar2j2K/file4c542872d5/sampleA_align2genome.bam',
'/tmp/Rtmpar2j2K/file4c542872d5/sample1_align2genome.bam',
'/tmp/Rtmpar2j2K/file4c542872d5/sample2_align2genome.bam', and
'/tmp/Rtmpar2j2K/file4c542872d5/sample3_align2genome.bam'
parsing /tmp/Rtmpar2j2K/file4c542872d5/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 17.62gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 367007.11Read/s]
parsing /tmp/Rtmpar2j2K/file4c542872d5/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 46.82gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1155201.06Read/s]
parsing /tmp/Rtmpar2j2K/file4c542872d5/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 36.31gene_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, 1250687.02Read/s]
parsing /tmp/Rtmpar2j2K/file4c542872d5/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 27.33gene_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, 614100.15Read/s]
-- Running step: isoform_identification @ Fri Oct 24 21:39:32 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri Oct 24 21:39:33 2025 -------------------
Checking for fastq file(s) /tmp/Rtmpar2j2K/file4c542872d5/fastq, /tmp/Rtmpar2j2K/file4c542872d5/fastq/sample1.fq.gz, /tmp/Rtmpar2j2K/file4c542872d5/fastq/sample2.fq.gz, /tmp/Rtmpar2j2K/file4c542872d5/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpar2j2K/file4c542872d5/sampleA_matched_reads.fastq.gz, /tmp/Rtmpar2j2K/file4c542872d5/sample1_matched_reads.fastq.gz, /tmp/Rtmpar2j2K/file4c542872d5/sample2_matched_reads.fastq.gz, /tmp/Rtmpar2j2K/file4c542872d5/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpar2j2K/file4c542872d5/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmpar2j2K/file4c542872d5/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmpar2j2K/file4c542872d5/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmpar2j2K/file4c542872d5/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/Rtmpar2j2K/file4c542872d5/sampleA_matched_reads_dedup.fastq.gz -> /tmp/Rtmpar2j2K/file4c542872d5/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/Rtmpar2j2K/file4c542872d5/sample1_matched_reads_dedup.fastq.gz -> /tmp/Rtmpar2j2K/file4c542872d5/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/Rtmpar2j2K/file4c542872d5/sample2_matched_reads_dedup.fastq.gz -> /tmp/Rtmpar2j2K/file4c542872d5/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/Rtmpar2j2K/file4c542872d5/sample3_matched_reads_dedup.fastq.gz -> /tmp/Rtmpar2j2K/file4c542872d5/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 Oct 24 21:39:34 2025 ----------
21:39:34 Fri Oct 24 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/Rtmpar2j2K/file4c542872d5/sample3_realign2transcript.bam...
parsing /tmp/Rtmpar2j2K/file4c542872d5/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpar2j2K/file4c542872d5/sample3_realign2transcript.bamdone
parsing /tmp/Rtmpar2j2K/file4c542872d5/sampleA_realign2transcript.bam...
parsing /tmp/Rtmpar2j2K/file4c542872d5/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpar2j2K/file4c542872d5/sampleA_realign2transcript.bamdone
parsing /tmp/Rtmpar2j2K/file4c542872d5/sample2_realign2transcript.bam...
parsing /tmp/Rtmpar2j2K/file4c542872d5/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpar2j2K/file4c542872d5/sample2_realign2transcript.bamdone
parsing /tmp/Rtmpar2j2K/file4c542872d5/sample1_realign2transcript.bam...
parsing /tmp/Rtmpar2j2K/file4c542872d5/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpar2j2K/file4c542872d5/sample1_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpar2j2K/file4c55edf4bcf/config_file_1221.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 Oct 24 21:39:38 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/Rtmpar2j2K/file4c55edf4bcf/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpar2j2K/file4c55edf4bcf/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpar2j2K/file4c55edf4bcf/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpar2j2K/file4c55edf4bcf/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/Rtmpar2j2K/file4c55edf4bcf/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpar2j2K/file4c55edf4bcf/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/Rtmpar2j2K/file4c55edf4bcf/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpar2j2K/file4c55edf4bcf/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/Rtmpar2j2K/file4c55edf4bcf/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpar2j2K/file4c55edf4bcf/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 Oct 24 21:39:40 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmpar2j2K/file4c55edf4bcf/sampleA_matched_reads.fastq.gz ->/tmp/Rtmpar2j2K/file4c55edf4bcf/sampleA_align2genome.bam
/tmp/Rtmpar2j2K/file4c55edf4bcf/sample1_matched_reads.fastq.gz ->/tmp/Rtmpar2j2K/file4c55edf4bcf/sample1_align2genome.bam
/tmp/Rtmpar2j2K/file4c55edf4bcf/sample2_matched_reads.fastq.gz ->/tmp/Rtmpar2j2K/file4c55edf4bcf/sample2_align2genome.bam
/tmp/Rtmpar2j2K/file4c55edf4bcf/sample3_matched_reads.fastq.gz ->/tmp/Rtmpar2j2K/file4c55edf4bcf/sample3_align2genome.bam
-- Running step: gene_quantification @ Fri Oct 24 21:40:03 2025 ----------------
21:40:03 Fri Oct 24 2025 quantify genes 
Using BAM(s): '/tmp/Rtmpar2j2K/file4c55edf4bcf/sampleA_align2genome.bam',
'/tmp/Rtmpar2j2K/file4c55edf4bcf/sample1_align2genome.bam',
'/tmp/Rtmpar2j2K/file4c55edf4bcf/sample2_align2genome.bam', and
'/tmp/Rtmpar2j2K/file4c55edf4bcf/sample3_align2genome.bam'
	Counter({'counted_reads': 176})
	Counter({'counted_reads': 358})
	Counter({'counted_reads': 95})
	Counter({'counted_reads': 91})
parsing /tmp/Rtmpar2j2K/file4c55edf4bcf/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 17.31gene_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, 360038.46Read/s]
parsing /tmp/Rtmpar2j2K/file4c55edf4bcf/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 38.30gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1067578.90Read/s]
parsing /tmp/Rtmpar2j2K/file4c55edf4bcf/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 40.80gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1060667.61Read/s]
parsing /tmp/Rtmpar2j2K/file4c55edf4bcf/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 30.34gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 687410.52Read/s]
-- Running step: isoform_identification @ Fri Oct 24 21:40:04 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri Oct 24 21:40:04 2025 -------------------
Checking for fastq file(s) /tmp/Rtmpar2j2K/file4c55edf4bcf/fastq, /tmp/Rtmpar2j2K/file4c55edf4bcf/fastq/sample1.fq.gz, /tmp/Rtmpar2j2K/file4c55edf4bcf/fastq/sample2.fq.gz, /tmp/Rtmpar2j2K/file4c55edf4bcf/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpar2j2K/file4c55edf4bcf/sampleA_matched_reads.fastq.gz, /tmp/Rtmpar2j2K/file4c55edf4bcf/sample1_matched_reads.fastq.gz, /tmp/Rtmpar2j2K/file4c55edf4bcf/sample2_matched_reads.fastq.gz, /tmp/Rtmpar2j2K/file4c55edf4bcf/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpar2j2K/file4c55edf4bcf/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmpar2j2K/file4c55edf4bcf/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmpar2j2K/file4c55edf4bcf/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmpar2j2K/file4c55edf4bcf/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/Rtmpar2j2K/file4c55edf4bcf/sampleA_matched_reads_dedup.fastq.gz ->/tmp/Rtmpar2j2K/file4c55edf4bcf/sampleA_realign2transcript.bam
/tmp/Rtmpar2j2K/file4c55edf4bcf/sample1_matched_reads_dedup.fastq.gz ->/tmp/Rtmpar2j2K/file4c55edf4bcf/sample1_realign2transcript.bam
/tmp/Rtmpar2j2K/file4c55edf4bcf/sample2_matched_reads_dedup.fastq.gz ->/tmp/Rtmpar2j2K/file4c55edf4bcf/sample2_realign2transcript.bam
/tmp/Rtmpar2j2K/file4c55edf4bcf/sample3_matched_reads_dedup.fastq.gz ->/tmp/Rtmpar2j2K/file4c55edf4bcf/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Fri Oct 24 21:40:25 2025 ----------
21:40:25 Fri Oct 24 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/Rtmpar2j2K/file4c55edf4bcf/sample3_realign2transcript.bam...
parsing /tmp/Rtmpar2j2K/file4c55edf4bcf/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpar2j2K/file4c55edf4bcf/sample3_realign2transcript.bamdone
parsing /tmp/Rtmpar2j2K/file4c55edf4bcf/sampleA_realign2transcript.bam...
parsing /tmp/Rtmpar2j2K/file4c55edf4bcf/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpar2j2K/file4c55edf4bcf/sampleA_realign2transcript.bamdone
parsing /tmp/Rtmpar2j2K/file4c55edf4bcf/sample2_realign2transcript.bam...
parsing /tmp/Rtmpar2j2K/file4c55edf4bcf/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpar2j2K/file4c55edf4bcf/sample2_realign2transcript.bamdone
parsing /tmp/Rtmpar2j2K/file4c55edf4bcf/sample1_realign2transcript.bam...
parsing /tmp/Rtmpar2j2K/file4c55edf4bcf/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpar2j2K/file4c55edf4bcf/sample1_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
[ FAIL 0 | WARN 183 | SKIP 0 | PASS 59 ]

[ FAIL 0 | WARN 183 | SKIP 0 | PASS 59 ]
	Counter({'counted_reads': 176})
	Counter({'counted_reads': 358})
	Counter({'counted_reads': 95})
	Counter({'counted_reads': 91})
> 
> proc.time()
   user  system elapsed 
829.632  57.050 893.366 
/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 '

Example timings

FLAMES.Rcheck/FLAMES-Ex.timings

nameusersystemelapsed
BulkPipeline4.3600.6165.387
MultiSampleSCPipeline13.175 1.96416.920
SingleCellPipeline4.0130.3183.260
add_gene_counts0.4000.0100.414
annotation_to_fasta0.2580.0070.267
blaze 8.345 1.17810.491
bulk_long_pipeline4.6501.0434.567
combine_sce1.0100.0861.110
config-set0.3160.1510.503
config0.3040.1710.545
controllers-set0.5240.0900.650
controllers0.3940.1660.588
convolution_filter0.0010.0010.001
create_config0.0130.0030.017
create_sce_from_dir7.4021.4257.492
create_se_from_dir3.2480.6694.283
cutadapt0.1720.0630.255
example_pipeline0.4450.0470.535
experiment3.0210.4303.815
filter_annotation0.0590.0060.065
filter_coverage1.4230.2501.815
find_barcode1.8370.2822.278
find_bin0.0060.0090.060
find_variants25.764 0.42925.664
get_coverage1.2180.2091.535
index_genome0.2760.1440.467
mutation_positions1.5440.0241.576
plot_coverage3.1970.2763.597
plot_demultiplex3.7230.5004.896
plot_demultiplex_raw2.2020.1222.425
plot_durations3.3610.4554.195
plot_isoform_heatmap10.226 0.59410.971
plot_isoform_reduced_dim28.672 0.44629.387
plot_isoforms4.1650.0484.256
resume_FLAMES2.8860.4203.666
run_FLAMES2.7330.4263.675
run_step1.4950.2562.000
sc_DTU_analysis10.241 1.26010.222
sc_gene_entropy1.6660.0582.047
sc_genotype4.0301.1224.025
sc_impute_transcript0.8560.0180.891
sc_long_multisample_pipeline16.313 2.96716.363
sc_long_pipeline5.3190.8074.819
sc_mutations3.3100.4313.230
sc_plot_genotype11.920 0.33911.405
show-FLAMESPipeline0.4000.0390.557
steps-set0.5850.0490.666
steps0.2020.0420.301
weight_transcripts0.0310.0170.048