Back to Multiple platform build/check report for BioC 3.22:   simplified   long
ABCDE[F]GHIJKLMNOPQRSTUVWXYZ

This page was generated on 2025-10-11 12:04 -0400 (Sat, 11 Oct 2025).

HostnameOSArch (*)R versionInstalled pkgs
nebbiolo2Linux (Ubuntu 24.04.3 LTS)x86_644.5.1 Patched (2025-08-23 r88802) -- "Great Square Root" 4864
lconwaymacOS 12.7.1 Montereyx86_644.5.1 Patched (2025-09-10 r88807) -- "Great Square Root" 4652
kjohnson3macOS 13.7.7 Venturaarm644.5.1 Patched (2025-09-10 r88807) -- "Great Square Root" 4597
taishanLinux (openEuler 24.03 LTS)aarch644.5.0 (2025-04-11) -- "How About a Twenty-Six" 4586
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 737/2346HostnameOS / ArchINSTALLBUILDCHECKBUILD BIN
FLAMES 2.3.5  (landing page)
Changqing Wang
Snapshot Date: 2025-10-10 13:45 -0400 (Fri, 10 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 12.7.1 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    ERROR  skipped


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: 2025-10-10 21:52:42 -0400 (Fri, 10 Oct 2025)
EndedAt: 2025-10-10 22:24:01 -0400 (Fri, 10 Oct 2025)
EllapsedTime: 1878.2 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     27.379  0.690  30.003
find_variants                26.542  0.423  26.553
sc_long_multisample_pipeline 17.179  3.839  20.393
MultiSampleSCPipeline        13.357  3.677  27.575
sc_plot_genotype             11.339  0.404  11.031
sc_DTU_analysis               9.851  1.435  10.335
plot_isoform_heatmap          9.923  0.591  10.700
create_sce_from_dir           7.893  1.743   8.487
blaze                         7.650  1.390  10.112
sc_long_pipeline              5.328  1.235   8.512
bulk_long_pipeline            4.608  1.306   4.951
BulkPipeline                  4.533  0.836   8.905
sc_genotype                   4.079  1.158   4.184
create_se_from_dir            3.833  0.931   5.133
* 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/RtmpEpOS1m/file3a8f60904761/config_file_14991.json 
Writing configuration parameters to:  /tmp/RtmpEpOS1m/file3a8f60904761/config_file_14991.json 
Writing configuration parameters to:  /tmp/RtmpEpOS1m/file3a8f60904761/config_file_14991.json 
Writing configuration parameters to:  /tmp/RtmpEpOS1m/file3a8f8cc8f29/config_file_14991.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/RtmpEpOS1m/file3a8fbb72682/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/RtmpEpOS1m/file3a8f23d463c0/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/RtmpEpOS1m/file3a8f23d463c0/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpEpOS1m/file3a8f40c1ebce/musc_rps24_1.fastq
Searching for barcodes...
Processing file: /tmp/RtmpEpOS1m/file3a8f40c1ebce/musc_rps24_2.fastq
Searching for barcodes...
Processing file: /tmp/RtmpEpOS1m/file3a8f40c1ebce/musc_rps24_3.fastq
Searching for barcodes...
Processing file: /tmp/RtmpEpOS1m/file3a8f40c1ebce/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/RtmpEpOS1m/file3a8f45108817/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/RtmpEpOS1m/file3a8f1e5af34c/config_file_14991.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 10 22:05:49 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpEpOS1m/file3a8f1e5af34c/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpEpOS1m/file3a8f1e5af34c/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpEpOS1m/file3a8f1e5af34c/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Fri Oct 10 22:05:52 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

  |                                                                            
  |                                                                      |   0%[22:05:59] 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.

[22:05:59] 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.

[22:05:59] 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.

[22:05:59] 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.

[22:06:02] 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.

[22:06:02] 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 10 22:06:22 2025 -------------------
Realigning sample sample1 -> /tmp/RtmpEpOS1m/file3a8f1e5af34c/sample1_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample2 -> /tmp/RtmpEpOS1m/file3a8f1e5af34c/sample2_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample3 -> /tmp/RtmpEpOS1m/file3a8f1e5af34c/sample3_realign2transcript.bam
Skipped sorting BAM files.

-- Running step: transcript_quantification @ Fri Oct 10 22:06:24 2025 ----------
2025-10-11T02:06:24.170745Z  INFO oarfish: setting user-provided filter parameters.
2025-10-11T02:06:24.182098Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpEpOS1m/file3a8f1e5af34c/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-10-11T02:06:24.182463Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-11T02:06:24.182506Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-11T02:06:24.187767Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-11T02:06:24.188236Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-11T02:06:24.198520Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-11T02:06:24.200156Z  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    │
╰─────────────────────────────────┴───────╯

2025-10-11T02:06:24.200354Z  INFO oarfish::bulk: Total number of alignment records : 116
2025-10-11T02:06:24.200377Z  INFO oarfish::bulk: number of aligned reads : 96
2025-10-11T02:06:24.200386Z  INFO oarfish::bulk: number of unique alignments : 86
2025-10-11T02:06:24.206011Z  INFO oarfish: oarfish completed successfully.
2025-10-11T02:06:24.318109Z  INFO oarfish: setting user-provided filter parameters.
2025-10-11T02:06:24.318735Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpEpOS1m/file3a8f1e5af34c/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-10-11T02:06:24.318775Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-11T02:06:24.318789Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-11T02:06:24.318917Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-11T02:06:24.318936Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-11T02:06:24.322063Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-11T02:06:24.322479Z  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    │
╰─────────────────────────────────┴───────╯

2025-10-11T02:06:24.322540Z  INFO oarfish::bulk: Total number of alignment records : 114
2025-10-11T02:06:24.322549Z  INFO oarfish::bulk: number of aligned reads : 95
2025-10-11T02:06:24.322555Z  INFO oarfish::bulk: number of unique alignments : 82
2025-10-11T02:06:24.329141Z  INFO oarfish: oarfish completed successfully.
2025-10-11T02:06:24.426552Z  INFO oarfish: setting user-provided filter parameters.
2025-10-11T02:06:24.427611Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpEpOS1m/file3a8f1e5af34c/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-10-11T02:06:24.427695Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-11T02:06:24.427722Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-11T02:06:24.427909Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-11T02:06:24.427933Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-11T02:06:24.433041Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2025-10-11T02:06:24.433369Z  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   │
╰─────────────────────────────────┴───────╯

2025-10-11T02:06:24.433430Z  INFO oarfish::bulk: Total number of alignment records : 239
2025-10-11T02:06:24.433439Z  INFO oarfish::bulk: number of aligned reads : 179
2025-10-11T02:06:24.433445Z  INFO oarfish::bulk: number of unique alignments : 143
2025-10-11T02:06:24.440310Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpEpOS1m/file3a8f2b2e5c4d/config_file_14991.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 10 22:06:25 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpEpOS1m/file3a8f2b2e5c4d/sample1_align2genome.bam
sample2 ->/tmp/RtmpEpOS1m/file3a8f2b2e5c4d/sample2_align2genome.bam
sample3 ->/tmp/RtmpEpOS1m/file3a8f2b2e5c4d/sample3_align2genome.bam
-- Running step: isoform_identification @ Fri Oct 10 22:06:49 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 10 22:07:11 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpEpOS1m/file3a8f2b2e5c4d/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpEpOS1m/file3a8f2b2e5c4d/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpEpOS1m/file3a8f2b2e5c4d/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Fri Oct 10 22:07:57 2025 ----------
2025-10-11T02:07:58.891436Z  INFO oarfish: setting user-provided filter parameters.
2025-10-11T02:07:59.085997Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpEpOS1m/file3a8f2b2e5c4d/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-10-11T02:07:59.625835Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-11T02:07:59.625882Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-11T02:07:59.709972Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-11T02:07:59.710026Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-11T02:07:59.772451Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-11T02:07:59.847777Z  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    │
╰─────────────────────────────────┴───────╯

2025-10-11T02:07:59.861952Z  INFO oarfish::bulk: Total number of alignment records : 116
2025-10-11T02:07:59.861972Z  INFO oarfish::bulk: number of aligned reads : 96
2025-10-11T02:07:59.861982Z  INFO oarfish::bulk: number of unique alignments : 86
2025-10-11T02:07:59.890498Z  INFO oarfish: oarfish completed successfully.
2025-10-11T02:08:00.024129Z  INFO oarfish: setting user-provided filter parameters.
2025-10-11T02:08:00.028086Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpEpOS1m/file3a8f2b2e5c4d/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-10-11T02:08:00.028224Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-11T02:08:00.028664Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-11T02:08:00.029439Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-11T02:08:00.030155Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-11T02:08:00.040974Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-11T02:08:00.043215Z  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    │
╰─────────────────────────────────┴───────╯

2025-10-11T02:08:00.043919Z  INFO oarfish::bulk: Total number of alignment records : 114
2025-10-11T02:08:00.043962Z  INFO oarfish::bulk: number of aligned reads : 95
2025-10-11T02:08:00.043968Z  INFO oarfish::bulk: number of unique alignments : 82
2025-10-11T02:08:00.053101Z  INFO oarfish: oarfish completed successfully.
2025-10-11T02:08:00.360179Z  INFO oarfish: setting user-provided filter parameters.
2025-10-11T02:08:00.361464Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpEpOS1m/file3a8f2b2e5c4d/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-10-11T02:08:00.361506Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-11T02:08:00.361518Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-11T02:08:00.361637Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-11T02:08:00.361654Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-11T02:08:00.367972Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2025-10-11T02:08:00.368503Z  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   │
╰─────────────────────────────────┴───────╯

2025-10-11T02:08:00.368579Z  INFO oarfish::bulk: Total number of alignment records : 239
2025-10-11T02:08:00.368592Z  INFO oarfish::bulk: number of aligned reads : 179
2025-10-11T02:08:00.368601Z  INFO oarfish::bulk: number of unique alignments : 143
2025-10-11T02:08:00.377909Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpEpOS1m/file3a8f6db2ae7c/config_file_14991.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 10 22:08:02 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpEpOS1m/file3a8f6db2ae7c/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpEpOS1m/file3a8f6db2ae7c/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpEpOS1m/file3a8f6db2ae7c/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Fri Oct 10 22:08:03 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 10 22:08:28 2025 -------------------
Realigning sample sample1 -> /tmp/RtmpEpOS1m/file3a8f6db2ae7c/sample1_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample2 -> /tmp/RtmpEpOS1m/file3a8f6db2ae7c/sample2_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample3 -> /tmp/RtmpEpOS1m/file3a8f6db2ae7c/sample3_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Fri Oct 10 22:08:31 2025 ----------
22:08:31 Fri Oct 10 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Inputs:  ['/tmp/RtmpEpOS1m/file3a8f6db2ae7c/sample3_realign2transcript.bam', '/tmp/RtmpEpOS1m/file3a8f6db2ae7c/sample2_realign2transcript.bam', '/tmp/RtmpEpOS1m/file3a8f6db2ae7c/sample1_realign2transcript.bam'] /tmp/RtmpEpOS1m/file3a8f6db2ae7c/transcript_assembly.fa.fai 5 0.4 0.4
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpEpOS1m/file3a8f4e4b924/config_file_14991.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 10 22:08:57 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpEpOS1m/file3a8f4e4b924/sample1_align2genome.bam
sample2 ->/tmp/RtmpEpOS1m/file3a8f4e4b924/sample2_align2genome.bam
sample3 ->/tmp/RtmpEpOS1m/file3a8f4e4b924/sample3_align2genome.bam
-- Running step: isoform_identification @ Fri Oct 10 22:09:37 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 10 22:10:04 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpEpOS1m/file3a8f4e4b924/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpEpOS1m/file3a8f4e4b924/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpEpOS1m/file3a8f4e4b924/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Fri Oct 10 22:10:37 2025 ----------
22:10:37 Fri Oct 10 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	Counter({'counted_reads': 375, 'not_enough_coverage': 16, 'unmapped': 2})
Inputs:  ['/tmp/RtmpEpOS1m/file3a8f4e4b924/sample3_realign2transcript.bam', '/tmp/RtmpEpOS1m/file3a8f4e4b924/sample2_realign2transcript.bam', '/tmp/RtmpEpOS1m/file3a8f4e4b924/sample1_realign2transcript.bam'] /tmp/RtmpEpOS1m/file3a8f4e4b924/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/RtmpEpOS1m/file3a8f703ebfe5/config_file_14991.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 10 22:10:39 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpEpOS1m/file3a8f703ebfe5/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpEpOS1m/file3a8f703ebfe5/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpEpOS1m/file3a8f703ebfe5/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Fri Oct 10 22:10:41 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri Oct 10 22:10:42 2025 -------------------
Realigning sample sample1 -> /tmp/RtmpEpOS1m/file3a8f703ebfe5/sample1_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample2 -> /tmp/RtmpEpOS1m/file3a8f703ebfe5/sample2_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample3 -> /tmp/RtmpEpOS1m/file3a8f703ebfe5/sample3_realign2transcript.bam
Skipped sorting BAM files.

-- Running step: transcript_quantification @ Fri Oct 10 22:10:43 2025 ----------
2025-10-11T02:10:43.876534Z  INFO oarfish: setting user-provided filter parameters.
2025-10-11T02:10:43.934554Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpEpOS1m/file3a8f703ebfe5/sample1_realign2transcript.bam, contains 10 reference sequences.
2025-10-11T02:10:43.934631Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-11T02:10:43.934650Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-11T02:10:43.953318Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-11T02:10:43.953379Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-11T02:10:43.971048Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-11T02:10:43.989057Z  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    │
╰─────────────────────────────────┴───────╯

2025-10-11T02:10:44.003452Z  INFO oarfish::bulk: Total number of alignment records : 125
2025-10-11T02:10:44.003474Z  INFO oarfish::bulk: number of aligned reads : 98
2025-10-11T02:10:44.003483Z  INFO oarfish::bulk: number of unique alignments : 86
2025-10-11T02:10:44.009007Z  INFO oarfish: oarfish completed successfully.
2025-10-11T02:10:44.074890Z  INFO oarfish: setting user-provided filter parameters.
2025-10-11T02:10:44.076071Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpEpOS1m/file3a8f703ebfe5/sample2_realign2transcript.bam, contains 10 reference sequences.
2025-10-11T02:10:44.076112Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-11T02:10:44.076122Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-11T02:10:44.076307Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-11T02:10:44.076329Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-11T02:10:44.081933Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-11T02:10:44.082216Z  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    │
╰─────────────────────────────────┴───────╯

2025-10-11T02:10:44.082263Z  INFO oarfish::bulk: Total number of alignment records : 136
2025-10-11T02:10:44.082273Z  INFO oarfish::bulk: number of aligned reads : 97
2025-10-11T02:10:44.082279Z  INFO oarfish::bulk: number of unique alignments : 79
2025-10-11T02:10:44.086996Z  INFO oarfish: oarfish completed successfully.
2025-10-11T02:10:44.148212Z  INFO oarfish: setting user-provided filter parameters.
2025-10-11T02:10:44.149323Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpEpOS1m/file3a8f703ebfe5/sample3_realign2transcript.bam, contains 10 reference sequences.
2025-10-11T02:10:44.149391Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-11T02:10:44.149410Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-11T02:10:44.149672Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-11T02:10:44.149706Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-11T02:10:44.157690Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-11T02:10:44.158246Z  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   │
╰─────────────────────────────────┴───────╯

2025-10-11T02:10:44.158316Z  INFO oarfish::bulk: Total number of alignment records : 272
2025-10-11T02:10:44.158329Z  INFO oarfish::bulk: number of aligned reads : 187
2025-10-11T02:10:44.158341Z  INFO oarfish::bulk: number of unique alignments : 140
2025-10-11T02:10:44.162809Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpEpOS1m/file3a8f64db3983/config_file_14991.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 10 22:10:44 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpEpOS1m/file3a8f64db3983/sample1_align2genome.bam
sample2 ->/tmp/RtmpEpOS1m/file3a8f64db3983/sample2_align2genome.bam
sample3 ->/tmp/RtmpEpOS1m/file3a8f64db3983/sample3_align2genome.bam
-- Running step: isoform_identification @ Fri Oct 10 22:11:12 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri Oct 10 22:11:12 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpEpOS1m/file3a8f64db3983/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpEpOS1m/file3a8f64db3983/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpEpOS1m/file3a8f64db3983/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Fri Oct 10 22:11:36 2025 ----------
2025-10-11T02:11:37.101371Z  INFO oarfish: setting user-provided filter parameters.
2025-10-11T02:11:37.106327Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpEpOS1m/file3a8f64db3983/sample1_realign2transcript.bam, contains 10 reference sequences.
2025-10-11T02:11:37.106400Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-11T02:11:37.106439Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-11T02:11:37.107898Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-11T02:11:37.107958Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-11T02:11:37.115460Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-11T02:11:37.117705Z  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    │
╰─────────────────────────────────┴───────╯

2025-10-11T02:11:37.117836Z  INFO oarfish::bulk: Total number of alignment records : 125
2025-10-11T02:11:37.117851Z  INFO oarfish::bulk: number of aligned reads : 98
2025-10-11T02:11:37.117861Z  INFO oarfish::bulk: number of unique alignments : 86
2025-10-11T02:11:37.123152Z  INFO oarfish: oarfish completed successfully.
2025-10-11T02:11:37.185101Z  INFO oarfish: setting user-provided filter parameters.
2025-10-11T02:11:37.185817Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpEpOS1m/file3a8f64db3983/sample2_realign2transcript.bam, contains 10 reference sequences.
2025-10-11T02:11:37.185859Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-11T02:11:37.185872Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-11T02:11:37.186022Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-11T02:11:37.186043Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-11T02:11:37.189504Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-11T02:11:37.189864Z  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    │
╰─────────────────────────────────┴───────╯

2025-10-11T02:11:37.189928Z  INFO oarfish::bulk: Total number of alignment records : 136
2025-10-11T02:11:37.189941Z  INFO oarfish::bulk: number of aligned reads : 97
2025-10-11T02:11:37.189950Z  INFO oarfish::bulk: number of unique alignments : 79
2025-10-11T02:11:37.194738Z  INFO oarfish: oarfish completed successfully.
2025-10-11T02:11:37.259938Z  INFO oarfish: setting user-provided filter parameters.
2025-10-11T02:11:37.260719Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpEpOS1m/file3a8f64db3983/sample3_realign2transcript.bam, contains 10 reference sequences.
2025-10-11T02:11:37.260760Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-11T02:11:37.260773Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-11T02:11:37.260959Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-11T02:11:37.260983Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-11T02:11:37.268651Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-11T02:11:37.269203Z  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   │
╰─────────────────────────────────┴───────╯

2025-10-11T02:11:37.269286Z  INFO oarfish::bulk: Total number of alignment records : 272
2025-10-11T02:11:37.269301Z  INFO oarfish::bulk: number of aligned reads : 187
2025-10-11T02:11:37.269311Z  INFO oarfish::bulk: number of unique alignments : 140
2025-10-11T02:11:37.274594Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpEpOS1m/file3a8f5e076424/config_file_14991.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 10 22:11:38 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpEpOS1m/file3a8f5e076424/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpEpOS1m/file3a8f5e076424/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpEpOS1m/file3a8f5e076424/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Fri Oct 10 22:11:39 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri Oct 10 22:11:40 2025 -------------------
Realigning sample sample1 -> /tmp/RtmpEpOS1m/file3a8f5e076424/sample1_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample2 -> /tmp/RtmpEpOS1m/file3a8f5e076424/sample2_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample3 -> /tmp/RtmpEpOS1m/file3a8f5e076424/sample3_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Fri Oct 10 22:11:41 2025 ----------
22:11:41 Fri Oct 10 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Inputs:  ['/tmp/RtmpEpOS1m/file3a8f5e076424/sample3_realign2transcript.bam', '/tmp/RtmpEpOS1m/file3a8f5e076424/sample2_realign2transcript.bam', '/tmp/RtmpEpOS1m/file3a8f5e076424/sample1_realign2transcript.bam'] /tmp/RtmpEpOS1m/file3a8f5e076424/transcript_assembly.fa.fai 5 0.4 0.4
	Counter({'counted_reads': 391, 'not_enough_coverage': 2})
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpEpOS1m/file3a8f7b8c730d/config_file_14991.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 10 22:11:43 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpEpOS1m/file3a8f7b8c730d/sample1_align2genome.bam
sample2 ->/tmp/RtmpEpOS1m/file3a8f7b8c730d/sample2_align2genome.bam
sample3 ->/tmp/RtmpEpOS1m/file3a8f7b8c730d/sample3_align2genome.bam
-- Running step: isoform_identification @ Fri Oct 10 22:12:06 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri Oct 10 22:12:07 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpEpOS1m/file3a8f7b8c730d/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpEpOS1m/file3a8f7b8c730d/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpEpOS1m/file3a8f7b8c730d/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Fri Oct 10 22:12:38 2025 ----------
22:12:38 Fri Oct 10 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Inputs:  ['/tmp/RtmpEpOS1m/file3a8f7b8c730d/sample3_realign2transcript.bam', '/tmp/RtmpEpOS1m/file3a8f7b8c730d/sample2_realign2transcript.bam', '/tmp/RtmpEpOS1m/file3a8f7b8c730d/sample1_realign2transcript.bam'] /tmp/RtmpEpOS1m/file3a8f7b8c730d/transcript_assembly.fa.fai 5 0.4 0.4
	Counter({'counted_reads': 391, 'not_enough_coverage': 2})
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpEpOS1m/file3a8fdd9b1fa/config_file_14991.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 10 22:12:43 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/RtmpEpOS1m/file3a8fdd9b1fa/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 10 22:12:44 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpEpOS1m/file3a8fdd9b1fa/matched_reads.fastq.gz -> /tmp/RtmpEpOS1m/file3a8fdd9b1fa/align2genome.bam
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: isoform_identification @ Fri Oct 10 22:12:45 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 10 22:13:00 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/RtmpEpOS1m/file3a8fdd9b1fa/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpEpOS1m/file3a8fdd9b1fa/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/RtmpEpOS1m/file3a8fdd9b1fa/matched_reads.fastq.gz -> /tmp/RtmpEpOS1m/file3a8fdd9b1fa/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 10 22:13:01 2025 ----------
2025-10-11T02:13:01.955562Z  INFO oarfish: setting user-provided filter parameters.
2025-10-11T02:13:01.964499Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpEpOS1m/file3a8fdd9b1fa/realign2transcript.bam, contains 5 reference sequences.
2025-10-11T02:13:01.964611Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-11T02:13:01.964644Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-11T02:13:01.965777Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-11T02:13:01.965822Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-11T02:13:01.983600Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpEpOS1m/file3a8f466ce5cd/config_file_14991.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 10 22:13:03 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpEpOS1m/file3a8f466ce5cd/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 10 22:13:04 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpEpOS1m/file3a8f466ce5cd/matched_reads.fastq.gz ->/tmp/RtmpEpOS1m/file3a8f466ce5cd/align2genome.bam
-- Running step: isoform_identification @ Fri Oct 10 22:13:34 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 10 22:13:46 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/RtmpEpOS1m/file3a8f466ce5cd/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpEpOS1m/file3a8f466ce5cd/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/RtmpEpOS1m/file3a8f466ce5cd/matched_reads.fastq.gz ->/tmp/RtmpEpOS1m/file3a8f466ce5cd/realign2transcript.bam
-- Running step: transcript_quantification @ Fri Oct 10 22:14:13 2025 ----------
2025-10-11T02:14:13.762808Z  INFO oarfish: setting user-provided filter parameters.
2025-10-11T02:14:13.786130Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpEpOS1m/file3a8f466ce5cd/realign2transcript.bam, contains 5 reference sequences.
2025-10-11T02:14:13.786483Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-11T02:14:13.786755Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-11T02:14:13.792152Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-11T02:14:13.792443Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-11T02:14:13.883999Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpEpOS1m/file3a8f9eb19ce/config_file_14991.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 10 22:14:14 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpEpOS1m/file3a8f9eb19ce/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 10 22:14:15 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpEpOS1m/file3a8f9eb19ce/matched_reads.fastq.gz -> /tmp/RtmpEpOS1m/file3a8f9eb19ce/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 10 22:14:16 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 10 22:14:28 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/RtmpEpOS1m/file3a8f9eb19ce/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpEpOS1m/file3a8f9eb19ce/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/RtmpEpOS1m/file3a8f9eb19ce/matched_reads.fastq.gz -> /tmp/RtmpEpOS1m/file3a8f9eb19ce/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 10 22:14:29 2025 ----------
22:14:29 Fri Oct 10 2025 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpEpOS1m/file3a8f117219ad/config_file_14991.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 10 22:14:30 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/RtmpEpOS1m/file3a8f117219ad/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 10 22:14:30 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpEpOS1m/file3a8f117219ad/matched_reads.fastq.gz ->/tmp/RtmpEpOS1m/file3a8f117219ad/align2genome.bam
-- Running step: isoform_identification @ Fri Oct 10 22:14:55 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 10 22:15:07 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/RtmpEpOS1m/file3a8f117219ad/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpEpOS1m/file3a8f117219ad/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/RtmpEpOS1m/file3a8f117219ad/matched_reads.fastq.gz ->/tmp/RtmpEpOS1m/file3a8f117219ad/realign2transcript.bam
-- Running step: transcript_quantification @ Fri Oct 10 22:15:30 2025 ----------
22:15:30 Fri Oct 10 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/RtmpEpOS1m/file3a8f678702fd/config_file_14991.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 10 22:15:31 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/RtmpEpOS1m/file3a8f678702fd/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 10 22:15:32 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpEpOS1m/file3a8f678702fd/matched_reads.fastq.gz -> /tmp/RtmpEpOS1m/file3a8f678702fd/align2genome.bam
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: isoform_identification @ Fri Oct 10 22:15:33 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 10 22:15:33 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/RtmpEpOS1m/file3a8f678702fd/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpEpOS1m/file3a8f678702fd/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/RtmpEpOS1m/file3a8f678702fd/matched_reads.fastq.gz -> /tmp/RtmpEpOS1m/file3a8f678702fd/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 10 22:15:34 2025 ----------
2025-10-11T02:15:34.181001Z  INFO oarfish: setting user-provided filter parameters.
2025-10-11T02:15:34.186102Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpEpOS1m/file3a8f678702fd/realign2transcript.bam, contains 10 reference sequences.
2025-10-11T02:15:34.186215Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-11T02:15:34.186241Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-11T02:15:34.187503Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-11T02:15:34.187548Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-11T02:15:34.211424Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpEpOS1m/file3a8f1ce9289a/config_file_14991.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 10 22:15:35 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/RtmpEpOS1m/file3a8f1ce9289a/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 10 22:15:36 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpEpOS1m/file3a8f1ce9289a/matched_reads.fastq.gz ->/tmp/RtmpEpOS1m/file3a8f1ce9289a/align2genome.bam
-- Running step: isoform_identification @ Fri Oct 10 22:16:01 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri Oct 10 22:16:01 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/RtmpEpOS1m/file3a8f1ce9289a/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpEpOS1m/file3a8f1ce9289a/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/RtmpEpOS1m/file3a8f1ce9289a/matched_reads.fastq.gz ->/tmp/RtmpEpOS1m/file3a8f1ce9289a/realign2transcript.bam
-- Running step: transcript_quantification @ Fri Oct 10 22:16:26 2025 ----------
2025-10-11T02:16:26.687403Z  INFO oarfish: setting user-provided filter parameters.
2025-10-11T02:16:26.698875Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpEpOS1m/file3a8f1ce9289a/realign2transcript.bam, contains 10 reference sequences.
2025-10-11T02:16:26.699418Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-11T02:16:26.699508Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-11T02:16:26.703877Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-11T02:16:26.703926Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-11T02:16:26.733803Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpEpOS1m/file3a8f3889942/config_file_14991.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 10 22:16:31 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/RtmpEpOS1m/file3a8f3889942/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 10 22:16:32 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpEpOS1m/file3a8f3889942/matched_reads.fastq.gz -> /tmp/RtmpEpOS1m/file3a8f3889942/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 10 22:16:32 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri Oct 10 22:16:33 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/RtmpEpOS1m/file3a8f3889942/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpEpOS1m/file3a8f3889942/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/RtmpEpOS1m/file3a8f3889942/matched_reads.fastq.gz -> /tmp/RtmpEpOS1m/file3a8f3889942/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 10 22:16:33 2025 ----------
22:16:33 Fri Oct 10 2025 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpEpOS1m/file3a8f29191b88/config_file_14991.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 10 22:16:35 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/RtmpEpOS1m/file3a8f29191b88/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 10 22:16:35 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpEpOS1m/file3a8f29191b88/matched_reads.fastq.gz ->/tmp/RtmpEpOS1m/file3a8f29191b88/align2genome.bam
-- Running step: isoform_identification @ Fri Oct 10 22:16:59 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 10 22:16:59 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/RtmpEpOS1m/file3a8f29191b88/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpEpOS1m/file3a8f29191b88/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/RtmpEpOS1m/file3a8f29191b88/matched_reads.fastq.gz ->/tmp/RtmpEpOS1m/file3a8f29191b88/realign2transcript.bam
-- Running step: transcript_quantification @ Fri Oct 10 22:17:23 2025 ----------
22:17:23 Fri Oct 10 2025 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpEpOS1m/file3a8f58c559d9/config_file_14991.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 10 22:17:26 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/RtmpEpOS1m/file3a8f58c559d9/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpEpOS1m/file3a8f58c559d9/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpEpOS1m/file3a8f58c559d9/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpEpOS1m/file3a8f58c559d9/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/RtmpEpOS1m/file3a8f58c559d9/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpEpOS1m/file3a8f58c559d9/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/RtmpEpOS1m/file3a8f58c559d9/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpEpOS1m/file3a8f58c559d9/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/RtmpEpOS1m/file3a8f58c559d9/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpEpOS1m/file3a8f58c559d9/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 10 22:17:28 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpEpOS1m/file3a8f58c559d9/sampleA_matched_reads.fastq.gz -> /tmp/RtmpEpOS1m/file3a8f58c559d9/sampleA_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpEpOS1m/file3a8f58c559d9/sample1_matched_reads.fastq.gz -> /tmp/RtmpEpOS1m/file3a8f58c559d9/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpEpOS1m/file3a8f58c559d9/sample2_matched_reads.fastq.gz -> /tmp/RtmpEpOS1m/file3a8f58c559d9/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpEpOS1m/file3a8f58c559d9/sample3_matched_reads.fastq.gz -> /tmp/RtmpEpOS1m/file3a8f58c559d9/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Fri Oct 10 22:17:31 2025 ----------------
22:17:31 Fri Oct 10 2025 quantify genes 
Using BAM(s): '/tmp/RtmpEpOS1m/file3a8f58c559d9/sampleA_align2genome.bam',
'/tmp/RtmpEpOS1m/file3a8f58c559d9/sample1_align2genome.bam',
'/tmp/RtmpEpOS1m/file3a8f58c559d9/sample2_align2genome.bam', and
'/tmp/RtmpEpOS1m/file3a8f58c559d9/sample3_align2genome.bam'
	Counter({'counted_reads': 368, 'unmapped': 4})
parsing /tmp/RtmpEpOS1m/file3a8f58c559d9/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  8.52gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  8.47gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
2025-10-10 22:17:36.503 R[14991:512483663] XType: com.apple.fonts is not accessible.
2025-10-10 22:17:36.503 R[14991:512483663] XType: XTFontStaticRegistry is enabled.
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 56925.64Read/s]
parsing /tmp/RtmpEpOS1m/file3a8f58c559d9/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 33.38gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 346751.32Read/s]
parsing /tmp/RtmpEpOS1m/file3a8f58c559d9/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 23.52gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 810900.94Read/s]
parsing /tmp/RtmpEpOS1m/file3a8f58c559d9/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 26.56gene_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, 230735.17Read/s]
-- Running step: isoform_identification @ Fri Oct 10 22:17:38 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 10 22:18:06 2025 -------------------
Checking for fastq file(s) /tmp/RtmpEpOS1m/file3a8f58c559d9/fastq, /tmp/RtmpEpOS1m/file3a8f58c559d9/fastq/sample1.fq.gz, /tmp/RtmpEpOS1m/file3a8f58c559d9/fastq/sample2.fq.gz, /tmp/RtmpEpOS1m/file3a8f58c559d9/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpEpOS1m/file3a8f58c559d9/sampleA_matched_reads.fastq.gz, /tmp/RtmpEpOS1m/file3a8f58c559d9/sample1_matched_reads.fastq.gz, /tmp/RtmpEpOS1m/file3a8f58c559d9/sample2_matched_reads.fastq.gz, /tmp/RtmpEpOS1m/file3a8f58c559d9/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpEpOS1m/file3a8f58c559d9/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpEpOS1m/file3a8f58c559d9/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpEpOS1m/file3a8f58c559d9/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpEpOS1m/file3a8f58c559d9/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpEpOS1m/file3a8f58c559d9/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpEpOS1m/file3a8f58c559d9/sampleA_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpEpOS1m/file3a8f58c559d9/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpEpOS1m/file3a8f58c559d9/sample1_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpEpOS1m/file3a8f58c559d9/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpEpOS1m/file3a8f58c559d9/sample2_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpEpOS1m/file3a8f58c559d9/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpEpOS1m/file3a8f58c559d9/sample3_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

-- Running step: transcript_quantification @ Fri Oct 10 22:18:07 2025 ----------
2025-10-11T02:18:07.365993Z  INFO oarfish: setting user-provided filter parameters.
2025-10-11T02:18:07.371135Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpEpOS1m/file3a8f58c559d9/sampleA_realign2transcript.bam, contains 5 reference sequences.
2025-10-11T02:18:07.371303Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-11T02:18:07.371348Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-11T02:18:07.373233Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-11T02:18:07.373276Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-11T02:18:07.393879Z  INFO oarfish::single_cell: Processed 100 cells.
2025-10-11T02:18:07.790803Z  INFO oarfish: setting user-provided filter parameters.
2025-10-11T02:18:07.791577Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpEpOS1m/file3a8f58c559d9/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-10-11T02:18:07.791630Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-11T02:18:07.791649Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-11T02:18:07.791877Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-11T02:18:07.791947Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-11T02:18:08.213471Z  INFO oarfish: setting user-provided filter parameters.
2025-10-11T02:18:08.214252Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpEpOS1m/file3a8f58c559d9/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-10-11T02:18:08.214312Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-11T02:18:08.214331Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-11T02:18:08.214478Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-11T02:18:08.214505Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-11T02:18:08.623939Z  INFO oarfish: setting user-provided filter parameters.
2025-10-11T02:18:08.624647Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpEpOS1m/file3a8f58c559d9/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-10-11T02:18:08.624710Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-11T02:18:08.624730Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-11T02:18:08.624949Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-11T02:18:08.624994Z  INFO oarfish: parsed reference information for 5 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpEpOS1m/file3a8f1d24b596/config_file_14991.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 10 22:18:09 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/RtmpEpOS1m/file3a8f1d24b596/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpEpOS1m/file3a8f1d24b596/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpEpOS1m/file3a8f1d24b596/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpEpOS1m/file3a8f1d24b596/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/RtmpEpOS1m/file3a8f1d24b596/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpEpOS1m/file3a8f1d24b596/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/RtmpEpOS1m/file3a8f1d24b596/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpEpOS1m/file3a8f1d24b596/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/RtmpEpOS1m/file3a8f1d24b596/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpEpOS1m/file3a8f1d24b596/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 10 22:18:11 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpEpOS1m/file3a8f1d24b596/sampleA_matched_reads.fastq.gz ->/tmp/RtmpEpOS1m/file3a8f1d24b596/sampleA_align2genome.bam
/tmp/RtmpEpOS1m/file3a8f1d24b596/sample1_matched_reads.fastq.gz ->/tmp/RtmpEpOS1m/file3a8f1d24b596/sample1_align2genome.bam
/tmp/RtmpEpOS1m/file3a8f1d24b596/sample2_matched_reads.fastq.gz ->/tmp/RtmpEpOS1m/file3a8f1d24b596/sample2_align2genome.bam
/tmp/RtmpEpOS1m/file3a8f1d24b596/sample3_matched_reads.fastq.gz ->/tmp/RtmpEpOS1m/file3a8f1d24b596/sample3_align2genome.bam
-- Running step: gene_quantification @ Fri Oct 10 22:18:39 2025 ----------------
22:18:39 Fri Oct 10 2025 quantify genes 
Using BAM(s): '/tmp/RtmpEpOS1m/file3a8f1d24b596/sampleA_align2genome.bam',
'/tmp/RtmpEpOS1m/file3a8f1d24b596/sample1_align2genome.bam',
'/tmp/RtmpEpOS1m/file3a8f1d24b596/sample2_align2genome.bam', and
'/tmp/RtmpEpOS1m/file3a8f1d24b596/sample3_align2genome.bam'
parsing /tmp/RtmpEpOS1m/file3a8f1d24b596/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  4.98gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  4.97gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 359310.56Read/s]
parsing /tmp/RtmpEpOS1m/file3a8f1d24b596/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 33.55gene_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, 1003134.03Read/s]
parsing /tmp/RtmpEpOS1m/file3a8f1d24b596/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 32.59gene_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, 620495.89Read/s]
parsing /tmp/RtmpEpOS1m/file3a8f1d24b596/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 21.64gene_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, 521057.44Read/s]
-- Running step: isoform_identification @ Fri Oct 10 22:18:41 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 10 22:19:09 2025 -------------------
Checking for fastq file(s) /tmp/RtmpEpOS1m/file3a8f1d24b596/fastq, /tmp/RtmpEpOS1m/file3a8f1d24b596/fastq/sample1.fq.gz, /tmp/RtmpEpOS1m/file3a8f1d24b596/fastq/sample2.fq.gz, /tmp/RtmpEpOS1m/file3a8f1d24b596/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpEpOS1m/file3a8f1d24b596/sampleA_matched_reads.fastq.gz, /tmp/RtmpEpOS1m/file3a8f1d24b596/sample1_matched_reads.fastq.gz, /tmp/RtmpEpOS1m/file3a8f1d24b596/sample2_matched_reads.fastq.gz, /tmp/RtmpEpOS1m/file3a8f1d24b596/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpEpOS1m/file3a8f1d24b596/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpEpOS1m/file3a8f1d24b596/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpEpOS1m/file3a8f1d24b596/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpEpOS1m/file3a8f1d24b596/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpEpOS1m/file3a8f1d24b596/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpEpOS1m/file3a8f1d24b596/sampleA_realign2transcript.bam
/tmp/RtmpEpOS1m/file3a8f1d24b596/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpEpOS1m/file3a8f1d24b596/sample1_realign2transcript.bam
/tmp/RtmpEpOS1m/file3a8f1d24b596/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpEpOS1m/file3a8f1d24b596/sample2_realign2transcript.bam
/tmp/RtmpEpOS1m/file3a8f1d24b596/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpEpOS1m/file3a8f1d24b596/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Fri Oct 10 22:19:33 2025 ----------
2025-10-11T02:19:33.086073Z  INFO oarfish: setting user-provided filter parameters.
2025-10-11T02:19:33.092884Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpEpOS1m/file3a8f1d24b596/sampleA_realign2transcript.bam, contains 5 reference sequences.
2025-10-11T02:19:33.093185Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-11T02:19:33.093228Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-11T02:19:33.095493Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-11T02:19:33.095687Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-11T02:19:33.117038Z  INFO oarfish::single_cell: Processed 100 cells.
2025-10-11T02:19:33.628505Z  INFO oarfish: setting user-provided filter parameters.
2025-10-11T02:19:33.629321Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpEpOS1m/file3a8f1d24b596/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-10-11T02:19:33.629397Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-11T02:19:33.629430Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-11T02:19:33.629562Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-11T02:19:33.629581Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-11T02:19:34.117239Z  INFO oarfish: setting user-provided filter parameters.
2025-10-11T02:19:34.118019Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpEpOS1m/file3a8f1d24b596/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-10-11T02:19:34.118073Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-11T02:19:34.118090Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-11T02:19:34.118265Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-11T02:19:34.118288Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-11T02:19:34.656951Z  INFO oarfish: setting user-provided filter parameters.
2025-10-11T02:19:34.657788Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpEpOS1m/file3a8f1d24b596/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-10-11T02:19:34.657831Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-11T02:19:34.657926Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-11T02:19:34.658070Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-11T02:19:34.658127Z  INFO oarfish: parsed reference information for 5 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpEpOS1m/file3a8f5b7db548/config_file_14991.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 10 22:19:36 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/RtmpEpOS1m/file3a8f5b7db548/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpEpOS1m/file3a8f5b7db548/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpEpOS1m/file3a8f5b7db548/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpEpOS1m/file3a8f5b7db548/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/RtmpEpOS1m/file3a8f5b7db548/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpEpOS1m/file3a8f5b7db548/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/RtmpEpOS1m/file3a8f5b7db548/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpEpOS1m/file3a8f5b7db548/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/RtmpEpOS1m/file3a8f5b7db548/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpEpOS1m/file3a8f5b7db548/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 10 22:19:37 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpEpOS1m/file3a8f5b7db548/sampleA_matched_reads.fastq.gz -> /tmp/RtmpEpOS1m/file3a8f5b7db548/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/RtmpEpOS1m/file3a8f5b7db548/sample1_matched_reads.fastq.gz -> /tmp/RtmpEpOS1m/file3a8f5b7db548/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/RtmpEpOS1m/file3a8f5b7db548/sample2_matched_reads.fastq.gz -> /tmp/RtmpEpOS1m/file3a8f5b7db548/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/RtmpEpOS1m/file3a8f5b7db548/sample3_matched_reads.fastq.gz -> /tmp/RtmpEpOS1m/file3a8f5b7db548/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 10 22:19:39 2025 ----------------
22:19:39 Fri Oct 10 2025 quantify genes 
Using BAM(s): '/tmp/RtmpEpOS1m/file3a8f5b7db548/sampleA_align2genome.bam',
'/tmp/RtmpEpOS1m/file3a8f5b7db548/sample1_align2genome.bam',
'/tmp/RtmpEpOS1m/file3a8f5b7db548/sample2_align2genome.bam', and
'/tmp/RtmpEpOS1m/file3a8f5b7db548/sample3_align2genome.bam'
parsing /tmp/RtmpEpOS1m/file3a8f5b7db548/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 18.25gene_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, 307356.08Read/s]
parsing /tmp/RtmpEpOS1m/file3a8f5b7db548/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 40.22gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1387923.23Read/s]
parsing /tmp/RtmpEpOS1m/file3a8f5b7db548/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 34.40gene_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, 1111603.94Read/s]
parsing /tmp/RtmpEpOS1m/file3a8f5b7db548/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 26.65gene_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, 339950.07Read/s]
-- Running step: isoform_identification @ Fri Oct 10 22:19:41 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 10 22:20:12 2025 -------------------
Checking for fastq file(s) /tmp/RtmpEpOS1m/file3a8f5b7db548/fastq, /tmp/RtmpEpOS1m/file3a8f5b7db548/fastq/sample1.fq.gz, /tmp/RtmpEpOS1m/file3a8f5b7db548/fastq/sample2.fq.gz, /tmp/RtmpEpOS1m/file3a8f5b7db548/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpEpOS1m/file3a8f5b7db548/sampleA_matched_reads.fastq.gz, /tmp/RtmpEpOS1m/file3a8f5b7db548/sample1_matched_reads.fastq.gz, /tmp/RtmpEpOS1m/file3a8f5b7db548/sample2_matched_reads.fastq.gz, /tmp/RtmpEpOS1m/file3a8f5b7db548/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpEpOS1m/file3a8f5b7db548/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpEpOS1m/file3a8f5b7db548/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpEpOS1m/file3a8f5b7db548/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpEpOS1m/file3a8f5b7db548/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpEpOS1m/file3a8f5b7db548/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpEpOS1m/file3a8f5b7db548/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/RtmpEpOS1m/file3a8f5b7db548/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpEpOS1m/file3a8f5b7db548/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/RtmpEpOS1m/file3a8f5b7db548/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpEpOS1m/file3a8f5b7db548/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/RtmpEpOS1m/file3a8f5b7db548/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpEpOS1m/file3a8f5b7db548/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 10 22:20:13 2025 ----------
22:20:13 Fri Oct 10 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpEpOS1m/file3a8f5b7db548/sample3_realign2transcript.bam...
parsing /tmp/RtmpEpOS1m/file3a8f5b7db548/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpEpOS1m/file3a8f5b7db548/sample3_realign2transcript.bamdone
parsing /tmp/RtmpEpOS1m/file3a8f5b7db548/sampleA_realign2transcript.bam...
parsing /tmp/RtmpEpOS1m/file3a8f5b7db548/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpEpOS1m/file3a8f5b7db548/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpEpOS1m/file3a8f5b7db548/sample2_realign2transcript.bam...
parsing /tmp/RtmpEpOS1m/file3a8f5b7db548/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpEpOS1m/file3a8f5b7db548/sample2_realign2transcript.bamdone
parsing /tmp/RtmpEpOS1m/file3a8f5b7db548/sample1_realign2transcript.bam...
parsing /tmp/RtmpEpOS1m/file3a8f5b7db548/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpEpOS1m/file3a8f5b7db548/sample1_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpEpOS1m/file3a8f1f642489/config_file_14991.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 10 22:20:16 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/RtmpEpOS1m/file3a8f1f642489/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpEpOS1m/file3a8f1f642489/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpEpOS1m/file3a8f1f642489/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpEpOS1m/file3a8f1f642489/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/RtmpEpOS1m/file3a8f1f642489/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpEpOS1m/file3a8f1f642489/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/RtmpEpOS1m/file3a8f1f642489/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpEpOS1m/file3a8f1f642489/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/RtmpEpOS1m/file3a8f1f642489/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpEpOS1m/file3a8f1f642489/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 10 22:20:18 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpEpOS1m/file3a8f1f642489/sampleA_matched_reads.fastq.gz ->/tmp/RtmpEpOS1m/file3a8f1f642489/sampleA_align2genome.bam
/tmp/RtmpEpOS1m/file3a8f1f642489/sample1_matched_reads.fastq.gz ->/tmp/RtmpEpOS1m/file3a8f1f642489/sample1_align2genome.bam
/tmp/RtmpEpOS1m/file3a8f1f642489/sample2_matched_reads.fastq.gz ->/tmp/RtmpEpOS1m/file3a8f1f642489/sample2_align2genome.bam
/tmp/RtmpEpOS1m/file3a8f1f642489/sample3_matched_reads.fastq.gz ->/tmp/RtmpEpOS1m/file3a8f1f642489/sample3_align2genome.bam
-- Running step: gene_quantification @ Fri Oct 10 22:20:44 2025 ----------------
22:20:44 Fri Oct 10 2025 quantify genes 
Using BAM(s): '/tmp/RtmpEpOS1m/file3a8f1f642489/sampleA_align2genome.bam',
'/tmp/RtmpEpOS1m/file3a8f1f642489/sample1_align2genome.bam',
'/tmp/RtmpEpOS1m/file3a8f1f642489/sample2_align2genome.bam', and
'/tmp/RtmpEpOS1m/file3a8f1f642489/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/RtmpEpOS1m/file3a8f1f642489/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 15.01gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 226244.63Read/s]
parsing /tmp/RtmpEpOS1m/file3a8f1f642489/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 35.43gene_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, 798003.04Read/s]
parsing /tmp/RtmpEpOS1m/file3a8f1f642489/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 35.42gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 777875.37Read/s]
parsing /tmp/RtmpEpOS1m/file3a8f1f642489/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 24.35gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 657414.42Read/s]
-- Running step: isoform_identification @ Fri Oct 10 22:20:46 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 10 22:21:13 2025 -------------------
Checking for fastq file(s) /tmp/RtmpEpOS1m/file3a8f1f642489/fastq, /tmp/RtmpEpOS1m/file3a8f1f642489/fastq/sample1.fq.gz, /tmp/RtmpEpOS1m/file3a8f1f642489/fastq/sample2.fq.gz, /tmp/RtmpEpOS1m/file3a8f1f642489/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpEpOS1m/file3a8f1f642489/sampleA_matched_reads.fastq.gz, /tmp/RtmpEpOS1m/file3a8f1f642489/sample1_matched_reads.fastq.gz, /tmp/RtmpEpOS1m/file3a8f1f642489/sample2_matched_reads.fastq.gz, /tmp/RtmpEpOS1m/file3a8f1f642489/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpEpOS1m/file3a8f1f642489/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpEpOS1m/file3a8f1f642489/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpEpOS1m/file3a8f1f642489/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpEpOS1m/file3a8f1f642489/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpEpOS1m/file3a8f1f642489/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpEpOS1m/file3a8f1f642489/sampleA_realign2transcript.bam
/tmp/RtmpEpOS1m/file3a8f1f642489/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpEpOS1m/file3a8f1f642489/sample1_realign2transcript.bam
/tmp/RtmpEpOS1m/file3a8f1f642489/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpEpOS1m/file3a8f1f642489/sample2_realign2transcript.bam
/tmp/RtmpEpOS1m/file3a8f1f642489/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpEpOS1m/file3a8f1f642489/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Fri Oct 10 22:21:36 2025 ----------
22:21:36 Fri Oct 10 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpEpOS1m/file3a8f1f642489/sample3_realign2transcript.bam...
parsing /tmp/RtmpEpOS1m/file3a8f1f642489/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpEpOS1m/file3a8f1f642489/sample3_realign2transcript.bamdone
parsing /tmp/RtmpEpOS1m/file3a8f1f642489/sampleA_realign2transcript.bam...
parsing /tmp/RtmpEpOS1m/file3a8f1f642489/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpEpOS1m/file3a8f1f642489/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpEpOS1m/file3a8f1f642489/sample2_realign2transcript.bam...
parsing /tmp/RtmpEpOS1m/file3a8f1f642489/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpEpOS1m/file3a8f1f642489/sample2_realign2transcript.bamdone
parsing /tmp/RtmpEpOS1m/file3a8f1f642489/sample1_realign2transcript.bam...
parsing /tmp/RtmpEpOS1m/file3a8f1f642489/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpEpOS1m/file3a8f1f642489/sample1_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpEpOS1m/file3a8f35d83883/config_file_14991.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 10 22:21:39 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpEpOS1m/file3a8f35d83883/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpEpOS1m/file3a8f35d83883/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpEpOS1m/file3a8f35d83883/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpEpOS1m/file3a8f35d83883/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/RtmpEpOS1m/file3a8f35d83883/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpEpOS1m/file3a8f35d83883/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/RtmpEpOS1m/file3a8f35d83883/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpEpOS1m/file3a8f35d83883/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/RtmpEpOS1m/file3a8f35d83883/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpEpOS1m/file3a8f35d83883/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 10 22:21:41 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpEpOS1m/file3a8f35d83883/sampleA_matched_reads.fastq.gz -> /tmp/RtmpEpOS1m/file3a8f35d83883/sampleA_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpEpOS1m/file3a8f35d83883/sample1_matched_reads.fastq.gz -> /tmp/RtmpEpOS1m/file3a8f35d83883/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpEpOS1m/file3a8f35d83883/sample2_matched_reads.fastq.gz -> /tmp/RtmpEpOS1m/file3a8f35d83883/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpEpOS1m/file3a8f35d83883/sample3_matched_reads.fastq.gz -> /tmp/RtmpEpOS1m/file3a8f35d83883/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Fri Oct 10 22:21:43 2025 ----------------
22:21:43 Fri Oct 10 2025 quantify genes 
Using BAM(s): '/tmp/RtmpEpOS1m/file3a8f35d83883/sampleA_align2genome.bam',
'/tmp/RtmpEpOS1m/file3a8f35d83883/sample1_align2genome.bam',
'/tmp/RtmpEpOS1m/file3a8f35d83883/sample2_align2genome.bam', and
'/tmp/RtmpEpOS1m/file3a8f35d83883/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/RtmpEpOS1m/file3a8f35d83883/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 16.77gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 377865.23Read/s]
parsing /tmp/RtmpEpOS1m/file3a8f35d83883/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 43.19gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1285334.64Read/s]
parsing /tmp/RtmpEpOS1m/file3a8f35d83883/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 36.24gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1300640.04Read/s]
parsing /tmp/RtmpEpOS1m/file3a8f35d83883/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 33.06gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 390211.37Read/s]
-- Running step: isoform_identification @ Fri Oct 10 22:21:44 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri Oct 10 22:21:44 2025 -------------------
Checking for fastq file(s) /tmp/RtmpEpOS1m/file3a8f35d83883/fastq, /tmp/RtmpEpOS1m/file3a8f35d83883/fastq/sample1.fq.gz, /tmp/RtmpEpOS1m/file3a8f35d83883/fastq/sample2.fq.gz, /tmp/RtmpEpOS1m/file3a8f35d83883/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpEpOS1m/file3a8f35d83883/sampleA_matched_reads.fastq.gz, /tmp/RtmpEpOS1m/file3a8f35d83883/sample1_matched_reads.fastq.gz, /tmp/RtmpEpOS1m/file3a8f35d83883/sample2_matched_reads.fastq.gz, /tmp/RtmpEpOS1m/file3a8f35d83883/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpEpOS1m/file3a8f35d83883/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpEpOS1m/file3a8f35d83883/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpEpOS1m/file3a8f35d83883/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpEpOS1m/file3a8f35d83883/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpEpOS1m/file3a8f35d83883/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpEpOS1m/file3a8f35d83883/sampleA_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpEpOS1m/file3a8f35d83883/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpEpOS1m/file3a8f35d83883/sample1_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpEpOS1m/file3a8f35d83883/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpEpOS1m/file3a8f35d83883/sample2_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpEpOS1m/file3a8f35d83883/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpEpOS1m/file3a8f35d83883/sample3_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

-- Running step: transcript_quantification @ Fri Oct 10 22:21:47 2025 ----------
2025-10-11T02:21:47.078809Z  INFO oarfish: setting user-provided filter parameters.
2025-10-11T02:21:47.082477Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpEpOS1m/file3a8f35d83883/sampleA_realign2transcript.bam, contains 14 reference sequences.
2025-10-11T02:21:47.082540Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-11T02:21:47.082555Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-11T02:21:47.084390Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-11T02:21:47.084432Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-10-11T02:21:47.108188Z  INFO oarfish::single_cell: Processed 100 cells.
2025-10-11T02:21:47.877867Z  INFO oarfish: setting user-provided filter parameters.
2025-10-11T02:21:47.878705Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpEpOS1m/file3a8f35d83883/sample1_realign2transcript.bam, contains 14 reference sequences.
2025-10-11T02:21:47.878748Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-11T02:21:47.878762Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-11T02:21:47.879018Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-11T02:21:47.879058Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-10-11T02:21:48.656748Z  INFO oarfish: setting user-provided filter parameters.
2025-10-11T02:21:48.657427Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpEpOS1m/file3a8f35d83883/sample2_realign2transcript.bam, contains 14 reference sequences.
2025-10-11T02:21:48.657468Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-11T02:21:48.657480Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-11T02:21:48.657686Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-11T02:21:48.657703Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-10-11T02:21:49.403190Z  INFO oarfish: setting user-provided filter parameters.
2025-10-11T02:21:49.404058Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpEpOS1m/file3a8f35d83883/sample3_realign2transcript.bam, contains 14 reference sequences.
2025-10-11T02:21:49.404099Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-11T02:21:49.404114Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-11T02:21:49.404314Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-11T02:21:49.404352Z  INFO oarfish: parsed reference information for 14 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpEpOS1m/file3a8f3824842f/config_file_14991.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 10 22:21:50 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/RtmpEpOS1m/file3a8f3824842f/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpEpOS1m/file3a8f3824842f/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpEpOS1m/file3a8f3824842f/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpEpOS1m/file3a8f3824842f/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/RtmpEpOS1m/file3a8f3824842f/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpEpOS1m/file3a8f3824842f/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/RtmpEpOS1m/file3a8f3824842f/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpEpOS1m/file3a8f3824842f/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/RtmpEpOS1m/file3a8f3824842f/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpEpOS1m/file3a8f3824842f/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 10 22:21:52 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpEpOS1m/file3a8f3824842f/sampleA_matched_reads.fastq.gz ->/tmp/RtmpEpOS1m/file3a8f3824842f/sampleA_align2genome.bam
/tmp/RtmpEpOS1m/file3a8f3824842f/sample1_matched_reads.fastq.gz ->/tmp/RtmpEpOS1m/file3a8f3824842f/sample1_align2genome.bam
/tmp/RtmpEpOS1m/file3a8f3824842f/sample2_matched_reads.fastq.gz ->/tmp/RtmpEpOS1m/file3a8f3824842f/sample2_align2genome.bam
/tmp/RtmpEpOS1m/file3a8f3824842f/sample3_matched_reads.fastq.gz ->/tmp/RtmpEpOS1m/file3a8f3824842f/sample3_align2genome.bam
-- Running step: gene_quantification @ Fri Oct 10 22:22:15 2025 ----------------
22:22:15 Fri Oct 10 2025 quantify genes 
Using BAM(s): '/tmp/RtmpEpOS1m/file3a8f3824842f/sampleA_align2genome.bam',
'/tmp/RtmpEpOS1m/file3a8f3824842f/sample1_align2genome.bam',
'/tmp/RtmpEpOS1m/file3a8f3824842f/sample2_align2genome.bam', and
'/tmp/RtmpEpOS1m/file3a8f3824842f/sample3_align2genome.bam'
parsing /tmp/RtmpEpOS1m/file3a8f3824842f/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 14.82gene_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, 334399.34Read/s]
parsing /tmp/RtmpEpOS1m/file3a8f3824842f/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 36.64gene_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, 1113610.88Read/s]
parsing /tmp/RtmpEpOS1m/file3a8f3824842f/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 37.88gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1431894.03Read/s]
parsing /tmp/RtmpEpOS1m/file3a8f3824842f/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 23.47gene_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, 567749.20Read/s]
-- Running step: isoform_identification @ Fri Oct 10 22:22:16 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri Oct 10 22:22:17 2025 -------------------
Checking for fastq file(s) /tmp/RtmpEpOS1m/file3a8f3824842f/fastq, /tmp/RtmpEpOS1m/file3a8f3824842f/fastq/sample1.fq.gz, /tmp/RtmpEpOS1m/file3a8f3824842f/fastq/sample2.fq.gz, /tmp/RtmpEpOS1m/file3a8f3824842f/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpEpOS1m/file3a8f3824842f/sampleA_matched_reads.fastq.gz, /tmp/RtmpEpOS1m/file3a8f3824842f/sample1_matched_reads.fastq.gz, /tmp/RtmpEpOS1m/file3a8f3824842f/sample2_matched_reads.fastq.gz, /tmp/RtmpEpOS1m/file3a8f3824842f/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpEpOS1m/file3a8f3824842f/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpEpOS1m/file3a8f3824842f/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpEpOS1m/file3a8f3824842f/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpEpOS1m/file3a8f3824842f/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpEpOS1m/file3a8f3824842f/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpEpOS1m/file3a8f3824842f/sampleA_realign2transcript.bam
/tmp/RtmpEpOS1m/file3a8f3824842f/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpEpOS1m/file3a8f3824842f/sample1_realign2transcript.bam
/tmp/RtmpEpOS1m/file3a8f3824842f/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpEpOS1m/file3a8f3824842f/sample2_realign2transcript.bam
/tmp/RtmpEpOS1m/file3a8f3824842f/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpEpOS1m/file3a8f3824842f/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Fri Oct 10 22:22:40 2025 ----------
2025-10-11T02:22:40.892812Z  INFO oarfish: setting user-provided filter parameters.
2025-10-11T02:22:40.893686Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpEpOS1m/file3a8f3824842f/sampleA_realign2transcript.bam, contains 14 reference sequences.
2025-10-11T02:22:40.893725Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-11T02:22:40.893738Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-11T02:22:40.893893Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-11T02:22:40.893911Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-10-11T02:22:40.911962Z  INFO oarfish::single_cell: Processed 100 cells.
2025-10-11T02:22:41.862443Z  INFO oarfish: setting user-provided filter parameters.
2025-10-11T02:22:41.863137Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpEpOS1m/file3a8f3824842f/sample1_realign2transcript.bam, contains 14 reference sequences.
2025-10-11T02:22:41.863187Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-11T02:22:41.863220Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-11T02:22:41.863393Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-11T02:22:41.863413Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-10-11T02:22:42.664654Z  INFO oarfish: setting user-provided filter parameters.
2025-10-11T02:22:42.665487Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpEpOS1m/file3a8f3824842f/sample2_realign2transcript.bam, contains 14 reference sequences.
2025-10-11T02:22:42.665545Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-11T02:22:42.665565Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-11T02:22:42.665781Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-11T02:22:42.665825Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-10-11T02:22:43.407975Z  INFO oarfish: setting user-provided filter parameters.
2025-10-11T02:22:43.408638Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpEpOS1m/file3a8f3824842f/sample3_realign2transcript.bam, contains 14 reference sequences.
2025-10-11T02:22:43.408681Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-11T02:22:43.408694Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-11T02:22:43.408832Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-11T02:22:43.408850Z  INFO oarfish: parsed reference information for 14 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpEpOS1m/file3a8f5750992/config_file_14991.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 10 22:22:45 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/RtmpEpOS1m/file3a8f5750992/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpEpOS1m/file3a8f5750992/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpEpOS1m/file3a8f5750992/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpEpOS1m/file3a8f5750992/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/RtmpEpOS1m/file3a8f5750992/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpEpOS1m/file3a8f5750992/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/RtmpEpOS1m/file3a8f5750992/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpEpOS1m/file3a8f5750992/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/RtmpEpOS1m/file3a8f5750992/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpEpOS1m/file3a8f5750992/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 10 22:22:46 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpEpOS1m/file3a8f5750992/sampleA_matched_reads.fastq.gz -> /tmp/RtmpEpOS1m/file3a8f5750992/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/RtmpEpOS1m/file3a8f5750992/sample1_matched_reads.fastq.gz -> /tmp/RtmpEpOS1m/file3a8f5750992/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/RtmpEpOS1m/file3a8f5750992/sample2_matched_reads.fastq.gz -> /tmp/RtmpEpOS1m/file3a8f5750992/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/RtmpEpOS1m/file3a8f5750992/sample3_matched_reads.fastq.gz -> /tmp/RtmpEpOS1m/file3a8f5750992/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 10 22:22:48 2025 ----------------
22:22:48 Fri Oct 10 2025 quantify genes 
Using BAM(s): '/tmp/RtmpEpOS1m/file3a8f5750992/sampleA_align2genome.bam',
'/tmp/RtmpEpOS1m/file3a8f5750992/sample1_align2genome.bam',
'/tmp/RtmpEpOS1m/file3a8f5750992/sample2_align2genome.bam', and
'/tmp/RtmpEpOS1m/file3a8f5750992/sample3_align2genome.bam'
parsing /tmp/RtmpEpOS1m/file3a8f5750992/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 15.79gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 333113.92Read/s]
parsing /tmp/RtmpEpOS1m/file3a8f5750992/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 37.05gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1528982.21Read/s]
parsing /tmp/RtmpEpOS1m/file3a8f5750992/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, 1250090.61Read/s]
parsing /tmp/RtmpEpOS1m/file3a8f5750992/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  6.23gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  6.21gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 806721.03Read/s]
-- Running step: isoform_identification @ Fri Oct 10 22:22:50 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri Oct 10 22:22:50 2025 -------------------
Checking for fastq file(s) /tmp/RtmpEpOS1m/file3a8f5750992/fastq, /tmp/RtmpEpOS1m/file3a8f5750992/fastq/sample1.fq.gz, /tmp/RtmpEpOS1m/file3a8f5750992/fastq/sample2.fq.gz, /tmp/RtmpEpOS1m/file3a8f5750992/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpEpOS1m/file3a8f5750992/sampleA_matched_reads.fastq.gz, /tmp/RtmpEpOS1m/file3a8f5750992/sample1_matched_reads.fastq.gz, /tmp/RtmpEpOS1m/file3a8f5750992/sample2_matched_reads.fastq.gz, /tmp/RtmpEpOS1m/file3a8f5750992/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpEpOS1m/file3a8f5750992/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpEpOS1m/file3a8f5750992/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpEpOS1m/file3a8f5750992/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpEpOS1m/file3a8f5750992/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpEpOS1m/file3a8f5750992/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpEpOS1m/file3a8f5750992/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/RtmpEpOS1m/file3a8f5750992/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpEpOS1m/file3a8f5750992/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/RtmpEpOS1m/file3a8f5750992/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpEpOS1m/file3a8f5750992/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/RtmpEpOS1m/file3a8f5750992/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpEpOS1m/file3a8f5750992/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 10 22:22:51 2025 ----------
22:22:51 Fri Oct 10 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpEpOS1m/file3a8f5750992/sample3_realign2transcript.bam...
parsing /tmp/RtmpEpOS1m/file3a8f5750992/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpEpOS1m/file3a8f5750992/sample3_realign2transcript.bamdone
parsing /tmp/RtmpEpOS1m/file3a8f5750992/sampleA_realign2transcript.bam...
parsing /tmp/RtmpEpOS1m/file3a8f5750992/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpEpOS1m/file3a8f5750992/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpEpOS1m/file3a8f5750992/sample2_realign2transcript.bam...
parsing /tmp/RtmpEpOS1m/file3a8f5750992/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpEpOS1m/file3a8f5750992/sample2_realign2transcript.bamdone
parsing /tmp/RtmpEpOS1m/file3a8f5750992/sample1_realign2transcript.bam...
parsing /tmp/RtmpEpOS1m/file3a8f5750992/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpEpOS1m/file3a8f5750992/sample1_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpEpOS1m/file3a8f5b3f718f/config_file_14991.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 10 22:22:56 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/RtmpEpOS1m/file3a8f5b3f718f/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpEpOS1m/file3a8f5b3f718f/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpEpOS1m/file3a8f5b3f718f/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpEpOS1m/file3a8f5b3f718f/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/RtmpEpOS1m/file3a8f5b3f718f/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpEpOS1m/file3a8f5b3f718f/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/RtmpEpOS1m/file3a8f5b3f718f/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpEpOS1m/file3a8f5b3f718f/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/RtmpEpOS1m/file3a8f5b3f718f/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpEpOS1m/file3a8f5b3f718f/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 10 22:22:58 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpEpOS1m/file3a8f5b3f718f/sampleA_matched_reads.fastq.gz ->/tmp/RtmpEpOS1m/file3a8f5b3f718f/sampleA_align2genome.bam
/tmp/RtmpEpOS1m/file3a8f5b3f718f/sample1_matched_reads.fastq.gz ->/tmp/RtmpEpOS1m/file3a8f5b3f718f/sample1_align2genome.bam
/tmp/RtmpEpOS1m/file3a8f5b3f718f/sample2_matched_reads.fastq.gz ->/tmp/RtmpEpOS1m/file3a8f5b3f718f/sample2_align2genome.bam
/tmp/RtmpEpOS1m/file3a8f5b3f718f/sample3_matched_reads.fastq.gz ->/tmp/RtmpEpOS1m/file3a8f5b3f718f/sample3_align2genome.bam
-- Running step: gene_quantification @ Fri Oct 10 22:23:21 2025 ----------------
22:23:21 Fri Oct 10 2025 quantify genes 
Using BAM(s): '/tmp/RtmpEpOS1m/file3a8f5b3f718f/sampleA_align2genome.bam',
'/tmp/RtmpEpOS1m/file3a8f5b3f718f/sample1_align2genome.bam',
'/tmp/RtmpEpOS1m/file3a8f5b3f718f/sample2_align2genome.bam', and
'/tmp/RtmpEpOS1m/file3a8f5b3f718f/sample3_align2genome.bam'
	Counter({'counted_reads': 176})
	Counter({'counted_reads': 358})
	Counter({'counted_reads': 95})
	Counter({'counted_reads': 91})
parsing /tmp/RtmpEpOS1m/file3a8f5b3f718f/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 14.37gene_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, 286550.98Read/s]
parsing /tmp/RtmpEpOS1m/file3a8f5b3f718f/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 33.68gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1092039.16Read/s]
parsing /tmp/RtmpEpOS1m/file3a8f5b3f718f/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 27.29gene_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, 1088977.05Read/s]
parsing /tmp/RtmpEpOS1m/file3a8f5b3f718f/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 25.37gene_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, 540558.82Read/s]
-- Running step: isoform_identification @ Fri Oct 10 22:23:22 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri Oct 10 22:23:22 2025 -------------------
Checking for fastq file(s) /tmp/RtmpEpOS1m/file3a8f5b3f718f/fastq, /tmp/RtmpEpOS1m/file3a8f5b3f718f/fastq/sample1.fq.gz, /tmp/RtmpEpOS1m/file3a8f5b3f718f/fastq/sample2.fq.gz, /tmp/RtmpEpOS1m/file3a8f5b3f718f/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpEpOS1m/file3a8f5b3f718f/sampleA_matched_reads.fastq.gz, /tmp/RtmpEpOS1m/file3a8f5b3f718f/sample1_matched_reads.fastq.gz, /tmp/RtmpEpOS1m/file3a8f5b3f718f/sample2_matched_reads.fastq.gz, /tmp/RtmpEpOS1m/file3a8f5b3f718f/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpEpOS1m/file3a8f5b3f718f/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpEpOS1m/file3a8f5b3f718f/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpEpOS1m/file3a8f5b3f718f/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpEpOS1m/file3a8f5b3f718f/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpEpOS1m/file3a8f5b3f718f/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpEpOS1m/file3a8f5b3f718f/sampleA_realign2transcript.bam
/tmp/RtmpEpOS1m/file3a8f5b3f718f/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpEpOS1m/file3a8f5b3f718f/sample1_realign2transcript.bam
/tmp/RtmpEpOS1m/file3a8f5b3f718f/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpEpOS1m/file3a8f5b3f718f/sample2_realign2transcript.bam
/tmp/RtmpEpOS1m/file3a8f5b3f718f/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpEpOS1m/file3a8f5b3f718f/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Fri Oct 10 22:23:44 2025 ----------
22:23:44 Fri Oct 10 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpEpOS1m/file3a8f5b3f718f/sample3_realign2transcript.bam...
parsing /tmp/RtmpEpOS1m/file3a8f5b3f718f/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpEpOS1m/file3a8f5b3f718f/sample3_realign2transcript.bamdone
parsing /tmp/RtmpEpOS1m/file3a8f5b3f718f/sampleA_realign2transcript.bam...
parsing /tmp/RtmpEpOS1m/file3a8f5b3f718f/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpEpOS1m/file3a8f5b3f718f/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpEpOS1m/file3a8f5b3f718f/sample2_realign2transcript.bam...
parsing /tmp/RtmpEpOS1m/file3a8f5b3f718f/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpEpOS1m/file3a8f5b3f718f/sample2_realign2transcript.bamdone
parsing /tmp/RtmpEpOS1m/file3a8f5b3f718f/sample1_realign2transcript.bam...
parsing /tmp/RtmpEpOS1m/file3a8f5b3f718f/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpEpOS1m/file3a8f5b3f718f/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 
 841.984   82.517 1108.180 
/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
BulkPipeline4.5330.8368.905
MultiSampleSCPipeline13.357 3.67727.575
SingleCellPipeline3.7020.3983.135
add_gene_counts0.3470.0070.355
annotation_to_fasta0.2460.0070.263
blaze 7.650 1.39010.112
bulk_long_pipeline4.6081.3064.951
combine_sce1.0540.1031.176
config-set0.3220.2010.644
config0.3120.2220.600
controllers-set0.5350.0990.732
controllers0.4100.2090.674
convolution_filter0.0010.0000.001
create_config0.0150.0030.017
create_sce_from_dir7.8931.7438.487
create_se_from_dir3.8330.9315.133
cutadapt0.1820.0690.284
example_pipeline0.4740.0530.575
experiment3.0820.5614.059
filter_annotation0.0630.0040.068
filter_coverage1.4490.3361.912
find_barcode1.8900.2972.352
find_bin0.0070.0100.027
find_variants26.542 0.42326.553
get_coverage1.4340.3381.944
index_genome0.3100.2290.612
mutation_positions1.5060.0361.561
plot_coverage3.5540.4024.113
plot_demultiplex3.2710.5704.618
plot_demultiplex_raw1.8720.0912.126
plot_durations2.8360.4773.607
plot_isoform_heatmap 9.923 0.59110.700
plot_isoform_reduced_dim27.379 0.69030.003
plot_isoforms4.0350.0484.179
resume_FLAMES3.0500.8024.331
run_FLAMES2.8210.6304.148
run_step1.3960.2931.833
sc_DTU_analysis 9.851 1.43510.335
sc_gene_entropy1.7300.0632.143
sc_genotype4.0791.1584.184
sc_impute_transcript0.8570.0210.899
sc_long_multisample_pipeline17.179 3.83920.393
sc_long_pipeline5.3281.2358.512
sc_mutations3.2730.4993.444
sc_plot_genotype11.339 0.40411.031
show-FLAMESPipeline0.4310.0430.546
steps-set0.6100.0510.738
steps0.1940.0460.294
weight_transcripts0.0270.0250.053