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

This page was generated on 2025-10-13 12:04 -0400 (Mon, 13 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: 2025-10-12 13:45 -0400 (Sun, 12 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: 2025-10-12 21:54:55 -0400 (Sun, 12 Oct 2025)
EndedAt: 2025-10-12 22:26:31 -0400 (Sun, 12 Oct 2025)
EllapsedTime: 1895.1 seconds
RetCode: 0
Status:   OK  
CheckDir: FLAMES.Rcheck
Warnings: 0

Command output

##############################################################################
##############################################################################
###
### Running command:
###
###   /Library/Frameworks/R.framework/Resources/bin/R CMD check --install=check:FLAMES.install-out.txt --library=/Library/Frameworks/R.framework/Resources/library --no-vignettes --timings FLAMES_2.3.5.tar.gz
###
##############################################################################
##############################################################################


* using log directory ‘/Users/biocbuild/bbs-3.22-bioc/meat/FLAMES.Rcheck’
* using R version 4.5.1 Patched (2025-09-10 r88807)
* using platform: x86_64-apple-darwin20
* R was compiled by
    Apple clang version 14.0.0 (clang-1400.0.29.202)
    GNU Fortran (GCC) 14.2.0
* running under: macOS Monterey 12.7.6
* using session charset: UTF-8
* using option ‘--no-vignettes’
* checking for file ‘FLAMES/DESCRIPTION’ ... OK
* this is package ‘FLAMES’ version ‘2.3.5’
* package encoding: UTF-8
* checking package namespace information ... OK
* checking package dependencies ... INFO
Imports includes 47 non-default packages.
Importing from so many packages makes the package vulnerable to any of
them becoming unavailable.  Move as many as possible to Suggests and
use conditionally.
* checking if this is a source package ... OK
* checking if there is a namespace ... OK
* checking for hidden files and directories ... NOTE
Found the following hidden files and directories:
  .dockerignore
These were most likely included in error. See section ‘Package
structure’ in the ‘Writing R Extensions’ manual.
* checking for portable file names ... OK
* checking for sufficient/correct file permissions ... OK
* checking whether package ‘FLAMES’ can be installed ... NOTE
Found the following notes/warnings:
  Non-staged installation was used
See ‘/Users/biocbuild/bbs-3.22-bioc/meat/FLAMES.Rcheck/00install.out’ for details.
* used C++ compiler: ‘Apple clang version 14.0.0 (clang-1400.0.29.202)’
* used SDK: ‘MacOSX11.3.1.sdk’
* checking C++ specification ... OK
* checking installed package size ... INFO
  installed size is  5.5Mb
  sub-directories of 1Mb or more:
    data   1.8Mb
    libs   1.8Mb
* checking package directory ... OK
* checking ‘build’ directory ... OK
* checking DESCRIPTION meta-information ... OK
* checking top-level files ... OK
* checking for left-over files ... OK
* checking index information ... OK
* checking package subdirectories ... OK
* checking code files for non-ASCII characters ... OK
* checking R files for syntax errors ... OK
* checking whether the package can be loaded ... OK
* checking whether the package can be loaded with stated dependencies ... OK
* checking whether the package can be unloaded cleanly ... OK
* checking whether the namespace can be loaded with stated dependencies ... OK
* checking whether the namespace can be unloaded cleanly ... OK
* checking dependencies in R code ... NOTE
There are ::: calls to the package's namespace in its code. A package
  almost never needs to use ::: for its own objects:
  'blaze' 'find_barcode' 'gene_quantification' 'isoform_identification'
  'minimap2_align' 'transcript_quantification'
* checking S3 generic/method consistency ... OK
* checking replacement functions ... OK
* checking foreign function calls ... OK
* checking R code for possible problems ... NOTE
BulkPipeline: no visible global function definition for ‘new’
BulkPipeline: no visible global function definition for ‘setNames’
MultiSampleSCPipeline: no visible global function definition for ‘new’
MultiSampleSCPipeline: no visible global function definition for
  ‘setNames’
SingleCellPipeline: no visible global function definition for ‘new’
SingleCellPipeline: no visible global function definition for
  ‘setNames’
addRowRanges: no visible global function definition for ‘head’
addRowRanges: no visible global function definition for ‘as’
add_gene_counts: no visible global function definition for ‘as’
cache_dir: no visible global function definition for ‘packageVersion’
chisq_test_by_gene: no visible global function definition for
  ‘chisq.test’
create_sce_from_dir: no visible global function definition for
  ‘setNames’
create_spe: no visible binding for global variable ‘barcode’
create_spe: no visible binding for global variable ‘in_tissue’
download_oarfish: no visible global function definition for
  ‘download.file’
download_oarfish: no visible global function definition for ‘unzip’
filter_coverage: no visible global function definition for
  ‘starts_with’
filter_coverage: no visible binding for global variable ‘filter_res’
find_variants: no visible global function definition for ‘setNames’
find_variants_grange: no visible binding for global variable
  ‘which_label’
find_variants_grange: no visible binding for global variable
  ‘nucleotide’
find_variants_grange: no visible binding for global variable ‘pos’
find_variants_grange: no visible binding for global variable ‘count’
find_variants_grange: no visible binding for global variable
  ‘counts_no_ins’
find_variants_grange: no visible binding for global variable ‘ref’
generate_sc_sce: no visible binding for global variable ‘FSM_match’
get_coverage: no visible binding for global variable ‘Freq’
homopolymer_pct : <anonymous>: no visible binding for global variable
  ‘Freq’
homopolymer_pct : <anonymous>: no visible binding for global variable
  ‘pct’
plot_coverage: no visible binding for global variable ‘tr_length’
plot_coverage: no visible binding for global variable ‘read_counts’
plot_coverage: no visible binding for global variable ‘total_counts’
plot_coverage: no visible binding for global variable ‘cumpct’
plot_coverage: no visible binding for global variable ‘length_bin’
plot_coverage: no visible binding for global variable ‘min_length’
plot_coverage: no visible binding for global variable ‘max_length’
plot_coverage: no visible global function definition for ‘head’
plot_coverage: no visible binding for global variable ‘transcript’
plot_demultiplex_raw: no visible binding for global variable ‘Sample’
plot_demultiplex_raw: no visible binding for global variable
  ‘CellBarcode’
plot_demultiplex_raw: no visible binding for global variable ‘UMI’
plot_demultiplex_raw: no visible binding for global variable
  ‘UMI_count’
plot_demultiplex_raw: no visible binding for global variable
  ‘barcode_rank’
plot_demultiplex_raw: no visible binding for global variable
  ‘FlankEditDist’
plot_demultiplex_raw: no visible binding for global variable ‘n_reads’
plot_demultiplex_raw: no visible binding for global variable
  ‘BarcodeEditDist’
plot_demultiplex_raw: no visible binding for global variable ‘total
  reads’
plot_demultiplex_raw: no visible binding for global variable
  ‘demultiplexed reads’
plot_demultiplex_raw: no visible binding for global variable ‘single
  match reads’
plot_demultiplex_raw: no visible binding for global variable
  ‘undemultiplexted reads’
plot_demultiplex_raw: no visible binding for global variable
  ‘multi-matching reads’
plot_demultiplex_raw: no visible binding for global variable ‘Type’
plot_demultiplex_raw: no visible binding for global variable ‘Reads’
plot_demultiplex_raw: no visible binding for global variable ‘input’
plot_demultiplex_raw: no visible binding for global variable ‘output’
plot_demultiplex_raw: no visible binding for global variable
  ‘read1_with_adapter’
plot_demultiplex_raw: no visible binding for global variable ‘Count’
plot_flagstat: no visible global function definition for ‘everything’
plot_flagstat: no visible binding for global variable ‘name’
plot_flagstat: no visible binding for global variable ‘value’
plot_isoform_reduced_dim: no visible binding for global variable ‘x’
plot_isoform_reduced_dim: no visible binding for global variable ‘y’
plot_isoform_reduced_dim: no visible binding for global variable ‘expr’
plot_spatial: no visible binding for global variable ‘imageX’
plot_spatial: no visible binding for global variable ‘imageY’
plot_spatial_feature: no visible binding for global variable ‘imageX’
plot_spatial_feature: no visible binding for global variable ‘imageY’
plot_spatial_feature: no visible binding for global variable ‘x’
plot_spatial_feature: no visible binding for global variable ‘y’
plot_spatial_feature: no visible global function definition for
  ‘scale_alpha_continuous’
plot_spatial_feature: no visible global function definition for
  ‘scale_colour_gradient’
plot_spatial_isoform: no visible global function definition for ‘head’
plot_spatial_pie: no visible global function definition for ‘setNames’
plot_spatial_pie: no visible global function definition for ‘head’
plot_spatial_pie: no visible binding for global variable ‘imageX’
plot_spatial_pie: no visible binding for global variable ‘imageY’
sc_gene_entropy: no visible global function definition for ‘as’
sc_genotype: no visible binding for global variable ‘allele’
sc_genotype: no visible binding for global variable ‘allele_count’
sc_genotype: no visible binding for global variable ‘barcode’
sc_genotype: no visible binding for global variable ‘pct’
sc_mutations: no visible binding for global variable ‘mutation_index’
sc_mutations: no visible binding for global variable ‘bam_index’
sc_plot_genotype: no visible global function definition for ‘setNames’
sc_plot_genotype: no visible binding for global variable ‘barcode’
sc_plot_genotype: no visible binding for global variable ‘genotype’
sc_plot_genotype: no visible binding for global variable ‘x’
sc_plot_genotype: no visible binding for global variable ‘y’
sc_transcript_usage_chisq: no visible global function definition for
  ‘as’
sc_transcript_usage_chisq: no visible binding for global variable
  ‘p.value’
sc_transcript_usage_chisq: no visible binding for global variable
  ‘adj.p.value’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘total’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘test’
sc_transcript_usage_permutation: no visible global function definition
  for ‘as’
sc_transcript_usage_permutation : <anonymous>: no visible global
  function definition for ‘as’
sc_transcript_usage_permutation : <anonymous> : <anonymous>: no visible
  global function definition for ‘na.omit’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘transcript’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘p.value’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘adj.p.value’
variant_count_tb: no visible binding for global variable ‘barcode’
variant_count_tb: no visible binding for global variable ‘allele_count’
variant_count_tb: no visible binding for global variable
  ‘cell_total_reads’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘expect_cell_number’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘fastq’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘demultiplexed_fastq’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘barcodes_file’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘outdir’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible global
  function definition for ‘setNames’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline : <anonymous>: no
  visible global function definition for ‘setNames’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘expect_cell_number’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘fastq’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘demultiplexed_fastq’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘barcodes_file’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘outdir’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible global
  function definition for ‘setNames’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘j’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘genome_bam’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘minimap2’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘samtools’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘threads’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘outdir’
plot_durations,FLAMES.Pipeline: no visible binding for global variable
  ‘step’
plot_durations,FLAMES.Pipeline: no visible binding for global variable
  ‘duration’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘j’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘transcriptome_assembly’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘transcriptome_bam’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘minimap2’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘samtools’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘outdir’
resume_FLAMES,FLAMES.Pipeline : <anonymous>: no visible global function
  definition for ‘capture.output’
run_FLAMES,FLAMES.Pipeline : <anonymous>: no visible global function
  definition for ‘capture.output’
Undefined global functions or variables:
  BarcodeEditDist CellBarcode Count FSM_match FlankEditDist Freq Reads
  Sample Type UMI UMI_count adj.p.value allele allele_count as
  bam_index barcode barcode_rank barcodes_file capture.output
  cell_total_reads chisq.test count counts_no_ins cumpct demultiplexed
  reads demultiplexed_fastq download.file duration everything
  expect_cell_number expr fastq filter_res genome_bam genotype head
  imageX imageY in_tissue input j length_bin max_length min_length
  minimap2 multi-matching reads mutation_index n_reads na.omit name new
  nucleotide outdir output p.value packageVersion pct pos
  read1_with_adapter read_counts ref samtools scale_alpha_continuous
  scale_colour_gradient setNames single match reads starts_with step
  test threads total total reads total_counts tr_length transcript
  transcriptome_assembly transcriptome_bam undemultiplexted reads unzip
  value which_label x y
Consider adding
  importFrom("base", "match", "single")
  importFrom("methods", "as", "new")
  importFrom("stats", "chisq.test", "na.omit", "setNames", "step")
  importFrom("utils", "capture.output", "download.file", "head",
             "packageVersion", "unzip")
to your NAMESPACE file (and ensure that your DESCRIPTION Imports field
contains 'methods').
* checking Rd files ... OK
* checking Rd metadata ... OK
* checking Rd cross-references ... OK
* checking for missing documentation entries ... OK
* checking for code/documentation mismatches ... OK
* checking Rd \usage sections ... OK
* checking Rd contents ... OK
* checking for unstated dependencies in examples ... OK
* checking contents of ‘data’ directory ... OK
* checking data for non-ASCII characters ... OK
* checking data for ASCII and uncompressed saves ... OK
* checking line endings in shell scripts ... OK
* checking line endings in C/C++/Fortran sources/headers ... OK
* checking line endings in Makefiles ... OK
* checking compilation flags in Makevars ... OK
* checking for GNU extensions in Makefiles ... INFO
GNU make is a SystemRequirements.
* checking for portable use of $(BLAS_LIBS) and $(LAPACK_LIBS) ... OK
* checking use of PKG_*FLAGS in Makefiles ... OK
* checking include directives in Makefiles ... OK
* checking compiled code ... NOTE
Note: information on .o files is not available
File ‘/Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/FLAMES/libs/FLAMES.so’:
  Found ‘___assert_rtn’, possibly from ‘assert’ (C)
  Found ‘___stderrp’, possibly from ‘stderr’ (C)
  Found ‘___stdoutp’, possibly from ‘stdout’ (C)
  Found ‘_abort’, possibly from ‘abort’ (C)
  Found ‘_exit’, possibly from ‘exit’ (C)

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

See ‘Writing portable packages’ in the ‘Writing R Extensions’ manual.
* checking files in ‘vignettes’ ... OK
* checking examples ... OK
Examples with CPU (user + system) or elapsed time > 5s
                               user system elapsed
plot_isoform_reduced_dim     28.789  0.526  29.681
find_variants                23.637  0.624  24.671
sc_long_multisample_pipeline 16.879  3.375  17.438
MultiSampleSCPipeline        12.874  2.671  24.533
sc_plot_genotype             11.319  0.463  11.865
sc_DTU_analysis              10.076  1.350  10.516
plot_isoform_heatmap         10.456  0.676  11.430
create_sce_from_dir           7.904  2.082   9.655
blaze                         7.576  1.399  15.288
sc_long_pipeline              5.313  1.013   6.289
bulk_long_pipeline            4.413  1.086   4.295
BulkPipeline                  4.271  0.815   7.618
sc_genotype                   3.940  1.079   3.870
create_se_from_dir            3.765  1.093   5.719
plot_durations                3.391  0.613   5.833
plot_demultiplex              3.398  0.586   5.028
sc_mutations                  3.241  0.514   5.647
experiment                    2.772  0.606   5.835
get_coverage                  1.475  0.740   6.588
* 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/RtmpeKbCGS/file167d3759625b0/config_file_92115.json 
Writing configuration parameters to:  /tmp/RtmpeKbCGS/file167d3759625b0/config_file_92115.json 
Writing configuration parameters to:  /tmp/RtmpeKbCGS/file167d3759625b0/config_file_92115.json 
Writing configuration parameters to:  /tmp/RtmpeKbCGS/file167d31ec6a333/config_file_92115.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/RtmpeKbCGS/file167d326440176/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/RtmpeKbCGS/file167d338f53cb2/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/RtmpeKbCGS/file167d338f53cb2/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpeKbCGS/file167d312e87364/musc_rps24_1.fastq
Searching for barcodes...
Processing file: /tmp/RtmpeKbCGS/file167d312e87364/musc_rps24_2.fastq
Searching for barcodes...
Processing file: /tmp/RtmpeKbCGS/file167d312e87364/musc_rps24_3.fastq
Searching for barcodes...
Processing file: /tmp/RtmpeKbCGS/file167d312e87364/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/RtmpeKbCGS/file167d34ac3c27f/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/RtmpeKbCGS/file167d322fec596/config_file_92115.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 @ Sun Oct 12 22:07:57 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpeKbCGS/file167d322fec596/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpeKbCGS/file167d322fec596/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpeKbCGS/file167d322fec596/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Sun Oct 12 22:07:58 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

  |                                                                            
  |                                                                      |   0%[22:08:06] 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.

[22:08:06] 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.

[22:08:06] 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.

[22:08:06] 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.

[22:08:10] WARNING: src/learner.cc:553: 
  If you are loading a serialized model (like pickle in Python, RDS in R) generated by
  older XGBoost, please export the model by calling `Booster.save_model` from that version
  first, then load it back in current version. See:

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

  for more details about differences between saving model and serializing.

[22:08:10] WARNING: src/learner.cc:553: 
  If you are loading a serialized model (like pickle in Python, RDS in R) generated by
  older XGBoost, please export the model by calling `Booster.save_model` from that version
  first, then load it back in current version. See:

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

  for more details about differences between saving model and serializing.


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

Detected 4 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Sun Oct 12 22:08:27 2025 -------------------
Realigning sample sample1 -> /tmp/RtmpeKbCGS/file167d322fec596/sample1_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample2 -> /tmp/RtmpeKbCGS/file167d322fec596/sample2_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample3 -> /tmp/RtmpeKbCGS/file167d322fec596/sample3_realign2transcript.bam
Skipped sorting BAM files.

-- Running step: transcript_quantification @ Sun Oct 12 22:08:29 2025 ----------
2025-10-13T02:08:30.490928Z  INFO oarfish: setting user-provided filter parameters.
2025-10-13T02:08:31.031081Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpeKbCGS/file167d322fec596/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-10-13T02:08:31.046407Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-13T02:08:31.048004Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-13T02:08:31.224215Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-13T02:08:31.229558Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-13T02:08:31.759907Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-13T02:08:31.975636Z  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    │
╰─────────────────────────────────┴───────╯

2025-10-13T02:08:32.024369Z  INFO oarfish::bulk: Total number of alignment records : 116
2025-10-13T02:08:32.024635Z  INFO oarfish::bulk: number of aligned reads : 96
2025-10-13T02:08:32.024651Z  INFO oarfish::bulk: number of unique alignments : 86
2025-10-13T02:08:32.063649Z  INFO oarfish: oarfish completed successfully.
2025-10-13T02:08:32.977215Z  INFO oarfish: setting user-provided filter parameters.
2025-10-13T02:08:32.996982Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpeKbCGS/file167d322fec596/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-10-13T02:08:32.998280Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-13T02:08:32.999812Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-13T02:08:33.000028Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-13T02:08:33.001428Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-13T02:08:33.014800Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-13T02:08:33.016246Z  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    │
╰─────────────────────────────────┴───────╯

2025-10-13T02:08:33.016354Z  INFO oarfish::bulk: Total number of alignment records : 114
2025-10-13T02:08:33.016375Z  INFO oarfish::bulk: number of aligned reads : 95
2025-10-13T02:08:33.016386Z  INFO oarfish::bulk: number of unique alignments : 82
2025-10-13T02:08:33.028780Z  INFO oarfish: oarfish completed successfully.
2025-10-13T02:08:33.815210Z  INFO oarfish: setting user-provided filter parameters.
2025-10-13T02:08:33.872948Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpeKbCGS/file167d322fec596/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-10-13T02:08:33.873404Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-13T02:08:33.873496Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-13T02:08:33.879438Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-13T02:08:33.879480Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-13T02:08:33.997075Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2025-10-13T02:08:34.000267Z  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   │
╰─────────────────────────────────┴───────╯

2025-10-13T02:08:34.000399Z  INFO oarfish::bulk: Total number of alignment records : 239
2025-10-13T02:08:34.000413Z  INFO oarfish::bulk: number of aligned reads : 179
2025-10-13T02:08:34.000424Z  INFO oarfish::bulk: number of unique alignments : 143
2025-10-13T02:08:34.016230Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpeKbCGS/file167d345110c29/config_file_92115.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 @ Sun Oct 12 22:08:37 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpeKbCGS/file167d345110c29/sample1_align2genome.bam
sample2 ->/tmp/RtmpeKbCGS/file167d345110c29/sample2_align2genome.bam
sample3 ->/tmp/RtmpeKbCGS/file167d345110c29/sample3_align2genome.bam
-- Running step: isoform_identification @ Sun Oct 12 22:09:25 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 @ Sun Oct 12 22:10:02 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpeKbCGS/file167d345110c29/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpeKbCGS/file167d345110c29/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpeKbCGS/file167d345110c29/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Sun Oct 12 22:10:50 2025 ----------
2025-10-13T02:10:51.599569Z  INFO oarfish: setting user-provided filter parameters.
2025-10-13T02:10:51.825935Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpeKbCGS/file167d345110c29/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-10-13T02:10:51.847320Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-13T02:10:51.847448Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-13T02:10:51.962309Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-13T02:10:51.962390Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-13T02:10:52.012370Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-13T02:10:52.035014Z  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    │
╰─────────────────────────────────┴───────╯

2025-10-13T02:10:52.050969Z  INFO oarfish::bulk: Total number of alignment records : 116
2025-10-13T02:10:52.051041Z  INFO oarfish::bulk: number of aligned reads : 96
2025-10-13T02:10:52.051054Z  INFO oarfish::bulk: number of unique alignments : 86
2025-10-13T02:10:52.072033Z  INFO oarfish: oarfish completed successfully.
2025-10-13T02:10:52.644314Z  INFO oarfish: setting user-provided filter parameters.
2025-10-13T02:10:52.645885Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpeKbCGS/file167d345110c29/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-10-13T02:10:52.645921Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-13T02:10:52.645998Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-13T02:10:52.646309Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-13T02:10:52.646424Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-13T02:10:52.650713Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-13T02:10:52.651637Z  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    │
╰─────────────────────────────────┴───────╯

2025-10-13T02:10:52.651715Z  INFO oarfish::bulk: Total number of alignment records : 114
2025-10-13T02:10:52.651726Z  INFO oarfish::bulk: number of aligned reads : 95
2025-10-13T02:10:52.651732Z  INFO oarfish::bulk: number of unique alignments : 82
2025-10-13T02:10:52.657925Z  INFO oarfish: oarfish completed successfully.
2025-10-13T02:10:52.830072Z  INFO oarfish: setting user-provided filter parameters.
2025-10-13T02:10:52.832544Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpeKbCGS/file167d345110c29/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-10-13T02:10:52.832590Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-13T02:10:52.832607Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-13T02:10:52.832749Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-13T02:10:52.832769Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-13T02:10:52.848653Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2025-10-13T02:10:52.849232Z  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   │
╰─────────────────────────────────┴───────╯

2025-10-13T02:10:52.849315Z  INFO oarfish::bulk: Total number of alignment records : 239
2025-10-13T02:10:52.849330Z  INFO oarfish::bulk: number of aligned reads : 179
2025-10-13T02:10:52.849343Z  INFO oarfish::bulk: number of unique alignments : 143
2025-10-13T02:10:52.855393Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpeKbCGS/file167d31676dee3/config_file_92115.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 @ Sun Oct 12 22:10:54 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpeKbCGS/file167d31676dee3/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpeKbCGS/file167d31676dee3/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpeKbCGS/file167d31676dee3/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Sun Oct 12 22:10:56 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 @ Sun Oct 12 22:11:22 2025 -------------------
Realigning sample sample1 -> /tmp/RtmpeKbCGS/file167d31676dee3/sample1_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample2 -> /tmp/RtmpeKbCGS/file167d31676dee3/sample2_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample3 -> /tmp/RtmpeKbCGS/file167d31676dee3/sample3_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Sun Oct 12 22:11:23 2025 ----------
22:11:23 Sun Oct 12 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Inputs:  ['/tmp/RtmpeKbCGS/file167d31676dee3/sample3_realign2transcript.bam', '/tmp/RtmpeKbCGS/file167d31676dee3/sample2_realign2transcript.bam', '/tmp/RtmpeKbCGS/file167d31676dee3/sample1_realign2transcript.bam'] /tmp/RtmpeKbCGS/file167d31676dee3/transcript_assembly.fa.fai 5 0.4 0.4
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpeKbCGS/file167d3682b5225/config_file_92115.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 @ Sun Oct 12 22:11:43 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpeKbCGS/file167d3682b5225/sample1_align2genome.bam
sample2 ->/tmp/RtmpeKbCGS/file167d3682b5225/sample2_align2genome.bam
sample3 ->/tmp/RtmpeKbCGS/file167d3682b5225/sample3_align2genome.bam
-- Running step: isoform_identification @ Sun Oct 12 22:12:09 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 @ Sun Oct 12 22:12:32 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpeKbCGS/file167d3682b5225/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpeKbCGS/file167d3682b5225/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpeKbCGS/file167d3682b5225/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Sun Oct 12 22:12:56 2025 ----------
22:12:56 Sun Oct 12 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})
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpeKbCGS/file167d337824d5/config_file_92115.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 @ Sun Oct 12 22:12:58 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpeKbCGS/file167d337824d5/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpeKbCGS/file167d337824d5/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpeKbCGS/file167d337824d5/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Sun Oct 12 22:12:59 2025 -------------
Inputs:  ['/tmp/RtmpeKbCGS/file167d3682b5225/sample3_realign2transcript.bam', '/tmp/RtmpeKbCGS/file167d3682b5225/sample2_realign2transcript.bam', '/tmp/RtmpeKbCGS/file167d3682b5225/sample1_realign2transcript.bam'] /tmp/RtmpeKbCGS/file167d3682b5225/transcript_assembly.fa.fai 5 0.4 0.4
	Counter({'counted_reads': 375, 'not_enough_coverage': 16, 'unmapped': 2})
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sun Oct 12 22:12:59 2025 -------------------
Realigning sample sample1 -> /tmp/RtmpeKbCGS/file167d337824d5/sample1_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample2 -> /tmp/RtmpeKbCGS/file167d337824d5/sample2_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample3 -> /tmp/RtmpeKbCGS/file167d337824d5/sample3_realign2transcript.bam
Skipped sorting BAM files.

-- Running step: transcript_quantification @ Sun Oct 12 22:13:01 2025 ----------
2025-10-13T02:13:01.181382Z  INFO oarfish: setting user-provided filter parameters.
2025-10-13T02:13:01.184535Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpeKbCGS/file167d337824d5/sample1_realign2transcript.bam, contains 10 reference sequences.
2025-10-13T02:13:01.184597Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-13T02:13:01.184618Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-13T02:13:01.185735Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-13T02:13:01.185790Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-13T02:13:01.191865Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-13T02:13:01.193640Z  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    │
╰─────────────────────────────────┴───────╯

2025-10-13T02:13:01.193726Z  INFO oarfish::bulk: Total number of alignment records : 125
2025-10-13T02:13:01.193743Z  INFO oarfish::bulk: number of aligned reads : 98
2025-10-13T02:13:01.193752Z  INFO oarfish::bulk: number of unique alignments : 86
2025-10-13T02:13:01.198389Z  INFO oarfish: oarfish completed successfully.
2025-10-13T02:13:01.267510Z  INFO oarfish: setting user-provided filter parameters.
2025-10-13T02:13:01.268200Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpeKbCGS/file167d337824d5/sample2_realign2transcript.bam, contains 10 reference sequences.
2025-10-13T02:13:01.268247Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-13T02:13:01.268264Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-13T02:13:01.268550Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-13T02:13:01.268595Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-13T02:13:01.273389Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-13T02:13:01.273822Z  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    │
╰─────────────────────────────────┴───────╯

2025-10-13T02:13:01.273893Z  INFO oarfish::bulk: Total number of alignment records : 136
2025-10-13T02:13:01.273906Z  INFO oarfish::bulk: number of aligned reads : 97
2025-10-13T02:13:01.273914Z  INFO oarfish::bulk: number of unique alignments : 79
2025-10-13T02:13:01.278442Z  INFO oarfish: oarfish completed successfully.
2025-10-13T02:13:01.339668Z  INFO oarfish: setting user-provided filter parameters.
2025-10-13T02:13:01.340436Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpeKbCGS/file167d337824d5/sample3_realign2transcript.bam, contains 10 reference sequences.
2025-10-13T02:13:01.340477Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-13T02:13:01.340488Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-13T02:13:01.340612Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-13T02:13:01.340627Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-13T02:13:01.348978Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-13T02:13:01.349542Z  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   │
╰─────────────────────────────────┴───────╯

2025-10-13T02:13:01.349647Z  INFO oarfish::bulk: Total number of alignment records : 272
2025-10-13T02:13:01.349662Z  INFO oarfish::bulk: number of aligned reads : 187
2025-10-13T02:13:01.349672Z  INFO oarfish::bulk: number of unique alignments : 140
2025-10-13T02:13:01.355565Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpeKbCGS/file167d3760e64e7/config_file_92115.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 @ Sun Oct 12 22:13:01 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpeKbCGS/file167d3760e64e7/sample1_align2genome.bam
sample2 ->/tmp/RtmpeKbCGS/file167d3760e64e7/sample2_align2genome.bam
sample3 ->/tmp/RtmpeKbCGS/file167d3760e64e7/sample3_align2genome.bam
-- Running step: isoform_identification @ Sun Oct 12 22:13:29 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sun Oct 12 22:13:30 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpeKbCGS/file167d3760e64e7/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpeKbCGS/file167d3760e64e7/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpeKbCGS/file167d3760e64e7/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Sun Oct 12 22:14:00 2025 ----------
2025-10-13T02:14:00.491594Z  INFO oarfish: setting user-provided filter parameters.
2025-10-13T02:14:00.495323Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpeKbCGS/file167d3760e64e7/sample1_realign2transcript.bam, contains 10 reference sequences.
2025-10-13T02:14:00.495390Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-13T02:14:00.495436Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-13T02:14:00.496537Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-13T02:14:00.496582Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-13T02:14:00.502625Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-13T02:14:00.507115Z  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    │
╰─────────────────────────────────┴───────╯

2025-10-13T02:14:00.507324Z  INFO oarfish::bulk: Total number of alignment records : 125
2025-10-13T02:14:00.507343Z  INFO oarfish::bulk: number of aligned reads : 98
2025-10-13T02:14:00.507356Z  INFO oarfish::bulk: number of unique alignments : 86
2025-10-13T02:14:00.513708Z  INFO oarfish: oarfish completed successfully.
2025-10-13T02:14:00.583832Z  INFO oarfish: setting user-provided filter parameters.
2025-10-13T02:14:00.584554Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpeKbCGS/file167d3760e64e7/sample2_realign2transcript.bam, contains 10 reference sequences.
2025-10-13T02:14:00.584616Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-13T02:14:00.584659Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-13T02:14:00.584914Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-13T02:14:00.584946Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-13T02:14:00.588752Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-13T02:14:00.589307Z  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    │
╰─────────────────────────────────┴───────╯

2025-10-13T02:14:00.589389Z  INFO oarfish::bulk: Total number of alignment records : 136
2025-10-13T02:14:00.589405Z  INFO oarfish::bulk: number of aligned reads : 97
2025-10-13T02:14:00.589453Z  INFO oarfish::bulk: number of unique alignments : 79
2025-10-13T02:14:00.594186Z  INFO oarfish: oarfish completed successfully.
2025-10-13T02:14:00.649085Z  INFO oarfish: setting user-provided filter parameters.
2025-10-13T02:14:00.649846Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpeKbCGS/file167d3760e64e7/sample3_realign2transcript.bam, contains 10 reference sequences.
2025-10-13T02:14:00.649882Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-13T02:14:00.649919Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-13T02:14:00.650122Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-13T02:14:00.650140Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-13T02:14:00.657912Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-13T02:14:00.658543Z  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   │
╰─────────────────────────────────┴───────╯

2025-10-13T02:14:00.658773Z  INFO oarfish::bulk: Total number of alignment records : 272
2025-10-13T02:14:00.658788Z  INFO oarfish::bulk: number of aligned reads : 187
2025-10-13T02:14:00.658797Z  INFO oarfish::bulk: number of unique alignments : 140
2025-10-13T02:14:00.664138Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpeKbCGS/file167d32f3c9db3/config_file_92115.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 @ Sun Oct 12 22:14:01 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpeKbCGS/file167d32f3c9db3/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpeKbCGS/file167d32f3c9db3/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpeKbCGS/file167d32f3c9db3/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Sun Oct 12 22:14:02 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sun Oct 12 22:14:03 2025 -------------------
Realigning sample sample1 -> /tmp/RtmpeKbCGS/file167d32f3c9db3/sample1_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample2 -> /tmp/RtmpeKbCGS/file167d32f3c9db3/sample2_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample3 -> /tmp/RtmpeKbCGS/file167d32f3c9db3/sample3_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Sun Oct 12 22:14:04 2025 ----------
22:14:04 Sun Oct 12 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpeKbCGS/file167d319be516b/config_file_92115.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 @ Sun Oct 12 22:14:06 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpeKbCGS/file167d319be516b/sample1_align2genome.bam
sample2 ->/tmp/RtmpeKbCGS/file167d319be516b/sample2_align2genome.bam
sample3 ->/tmp/RtmpeKbCGS/file167d319be516b/sample3_align2genome.bam
-- Running step: isoform_identification @ Sun Oct 12 22:14:34 2025 -------------
Inputs:  ['/tmp/RtmpeKbCGS/file167d32f3c9db3/sample3_realign2transcript.bam', '/tmp/RtmpeKbCGS/file167d32f3c9db3/sample2_realign2transcript.bam', '/tmp/RtmpeKbCGS/file167d32f3c9db3/sample1_realign2transcript.bam'] /tmp/RtmpeKbCGS/file167d32f3c9db3/transcript_assembly.fa.fai 5 0.4 0.4
	Counter({'counted_reads': 391, 'not_enough_coverage': 2})
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sun Oct 12 22:14:35 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpeKbCGS/file167d319be516b/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpeKbCGS/file167d319be516b/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpeKbCGS/file167d319be516b/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Sun Oct 12 22:15:03 2025 ----------
22:15:03 Sun Oct 12 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Inputs:  ['/tmp/RtmpeKbCGS/file167d319be516b/sample3_realign2transcript.bam', '/tmp/RtmpeKbCGS/file167d319be516b/sample2_realign2transcript.bam', '/tmp/RtmpeKbCGS/file167d319be516b/sample1_realign2transcript.bam'] /tmp/RtmpeKbCGS/file167d319be516b/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/RtmpeKbCGS/file167d335201596/config_file_92115.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 @ Sun Oct 12 22:15:07 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/RtmpeKbCGS/file167d335201596/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 @ Sun Oct 12 22:15:08 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpeKbCGS/file167d335201596/matched_reads.fastq.gz -> /tmp/RtmpeKbCGS/file167d335201596/align2genome.bam
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: isoform_identification @ Sun Oct 12 22:15:08 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 @ Sun Oct 12 22:15:22 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/RtmpeKbCGS/file167d335201596/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpeKbCGS/file167d335201596/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/RtmpeKbCGS/file167d335201596/matched_reads.fastq.gz -> /tmp/RtmpeKbCGS/file167d335201596/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 @ Sun Oct 12 22:15:23 2025 ----------
2025-10-13T02:15:23.217265Z  INFO oarfish: setting user-provided filter parameters.
2025-10-13T02:15:23.220644Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpeKbCGS/file167d335201596/realign2transcript.bam, contains 5 reference sequences.
2025-10-13T02:15:23.220704Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-13T02:15:23.220722Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-13T02:15:23.225116Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-13T02:15:23.225163Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-13T02:15:23.242940Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpeKbCGS/file167d3192dcba9/config_file_92115.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 @ Sun Oct 12 22:15:24 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/RtmpeKbCGS/file167d3192dcba9/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 @ Sun Oct 12 22:15:25 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpeKbCGS/file167d3192dcba9/matched_reads.fastq.gz ->/tmp/RtmpeKbCGS/file167d3192dcba9/align2genome.bam
-- Running step: isoform_identification @ Sun Oct 12 22:15:49 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 @ Sun Oct 12 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/RtmpeKbCGS/file167d3192dcba9/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpeKbCGS/file167d3192dcba9/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/RtmpeKbCGS/file167d3192dcba9/matched_reads.fastq.gz ->/tmp/RtmpeKbCGS/file167d3192dcba9/realign2transcript.bam
-- Running step: transcript_quantification @ Sun Oct 12 22:16:29 2025 ----------
2025-10-13T02:16:30.113833Z  INFO oarfish: setting user-provided filter parameters.
2025-10-13T02:16:30.127792Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpeKbCGS/file167d3192dcba9/realign2transcript.bam, contains 5 reference sequences.
2025-10-13T02:16:30.127895Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-13T02:16:30.127944Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-13T02:16:30.130360Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-13T02:16:30.130490Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-13T02:16:30.162435Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpeKbCGS/file167d3474e0e2e/config_file_92115.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 @ Sun Oct 12 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/RtmpeKbCGS/file167d3474e0e2e/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 @ Sun Oct 12 22:16:32 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpeKbCGS/file167d3474e0e2e/matched_reads.fastq.gz -> /tmp/RtmpeKbCGS/file167d3474e0e2e/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 @ Sun Oct 12 22:16:32 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 @ Sun Oct 12 22:16:46 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/RtmpeKbCGS/file167d3474e0e2e/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpeKbCGS/file167d3474e0e2e/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/RtmpeKbCGS/file167d3474e0e2e/matched_reads.fastq.gz -> /tmp/RtmpeKbCGS/file167d3474e0e2e/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 @ Sun Oct 12 22:16:47 2025 ----------
22:16:47 Sun Oct 12 2025 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpeKbCGS/file167d35390e29/config_file_92115.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 @ Sun Oct 12 22:16:48 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/RtmpeKbCGS/file167d35390e29/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 @ Sun Oct 12 22:16:49 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpeKbCGS/file167d35390e29/matched_reads.fastq.gz ->/tmp/RtmpeKbCGS/file167d35390e29/align2genome.bam
-- Running step: isoform_identification @ Sun Oct 12 22:17:15 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 @ Sun Oct 12 22:17:25 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/RtmpeKbCGS/file167d35390e29/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpeKbCGS/file167d35390e29/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/RtmpeKbCGS/file167d35390e29/matched_reads.fastq.gz ->/tmp/RtmpeKbCGS/file167d35390e29/realign2transcript.bam
-- Running step: transcript_quantification @ Sun Oct 12 22:17:50 2025 ----------
22:17:50 Sun Oct 12 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/RtmpeKbCGS/file167d345c8a4b4/config_file_92115.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 @ Sun Oct 12 22:17:51 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/RtmpeKbCGS/file167d345c8a4b4/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 @ Sun Oct 12 22:17:52 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpeKbCGS/file167d345c8a4b4/matched_reads.fastq.gz -> /tmp/RtmpeKbCGS/file167d345c8a4b4/align2genome.bam
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: isoform_identification @ Sun Oct 12 22:17:52 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 @ Sun Oct 12 22:17:52 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/RtmpeKbCGS/file167d345c8a4b4/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpeKbCGS/file167d345c8a4b4/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/RtmpeKbCGS/file167d345c8a4b4/matched_reads.fastq.gz -> /tmp/RtmpeKbCGS/file167d345c8a4b4/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 @ Sun Oct 12 22:17:53 2025 ----------
2025-10-13T02:17:53.509838Z  INFO oarfish: setting user-provided filter parameters.
2025-10-13T02:17:53.513653Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpeKbCGS/file167d345c8a4b4/realign2transcript.bam, contains 10 reference sequences.
2025-10-13T02:17:53.513711Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-13T02:17:53.513734Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-13T02:17:53.514885Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-13T02:17:53.514977Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-13T02:17:53.536008Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpeKbCGS/file167d3cb47c58/config_file_92115.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 @ Sun Oct 12 22:17:54 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpeKbCGS/file167d3cb47c58/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 @ Sun Oct 12 22:17:55 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpeKbCGS/file167d3cb47c58/matched_reads.fastq.gz ->/tmp/RtmpeKbCGS/file167d3cb47c58/align2genome.bam
-- Running step: isoform_identification @ Sun Oct 12 22:18:22 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sun Oct 12 22:18:22 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/RtmpeKbCGS/file167d3cb47c58/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpeKbCGS/file167d3cb47c58/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/RtmpeKbCGS/file167d3cb47c58/matched_reads.fastq.gz ->/tmp/RtmpeKbCGS/file167d3cb47c58/realign2transcript.bam
-- Running step: transcript_quantification @ Sun Oct 12 22:18:50 2025 ----------
2025-10-13T02:18:50.259514Z  INFO oarfish: setting user-provided filter parameters.
2025-10-13T02:18:50.279213Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpeKbCGS/file167d3cb47c58/realign2transcript.bam, contains 10 reference sequences.
2025-10-13T02:18:50.279697Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-13T02:18:50.279970Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-13T02:18:50.288776Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-13T02:18:50.289672Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-13T02:18:50.330670Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpeKbCGS/file167d357c1b3a1/config_file_92115.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 @ Sun Oct 12 22:18:52 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpeKbCGS/file167d357c1b3a1/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 @ Sun Oct 12 22:18:54 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpeKbCGS/file167d357c1b3a1/matched_reads.fastq.gz -> /tmp/RtmpeKbCGS/file167d357c1b3a1/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 @ Sun Oct 12 22:18:56 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sun Oct 12 22:18:57 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/RtmpeKbCGS/file167d357c1b3a1/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpeKbCGS/file167d357c1b3a1/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/RtmpeKbCGS/file167d357c1b3a1/matched_reads.fastq.gz -> /tmp/RtmpeKbCGS/file167d357c1b3a1/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 @ Sun Oct 12 22:18:57 2025 ----------
22:18:57 Sun Oct 12 2025 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpeKbCGS/file167d36e6efe35/config_file_92115.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 @ Sun Oct 12 22:18:59 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpeKbCGS/file167d36e6efe35/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 @ Sun Oct 12 22:19:00 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpeKbCGS/file167d36e6efe35/matched_reads.fastq.gz ->/tmp/RtmpeKbCGS/file167d36e6efe35/align2genome.bam
-- Running step: isoform_identification @ Sun Oct 12 22:19:27 2025 -------------
	Counter({'counted_reads': 368, 'unmapped': 4})
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sun Oct 12 22:19:28 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/RtmpeKbCGS/file167d36e6efe35/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpeKbCGS/file167d36e6efe35/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/RtmpeKbCGS/file167d36e6efe35/matched_reads.fastq.gz ->/tmp/RtmpeKbCGS/file167d36e6efe35/realign2transcript.bam
-- Running step: transcript_quantification @ Sun Oct 12 22:19:53 2025 ----------
22:19:53 Sun Oct 12 2025 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpeKbCGS/file167d3648a0cea/config_file_92115.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 @ Sun Oct 12 22:19:56 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/RtmpeKbCGS/file167d3648a0cea/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpeKbCGS/file167d3648a0cea/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpeKbCGS/file167d3648a0cea/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpeKbCGS/file167d3648a0cea/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/RtmpeKbCGS/file167d3648a0cea/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpeKbCGS/file167d3648a0cea/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/RtmpeKbCGS/file167d3648a0cea/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpeKbCGS/file167d3648a0cea/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/RtmpeKbCGS/file167d3648a0cea/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpeKbCGS/file167d3648a0cea/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 @ Sun Oct 12 22:19:58 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpeKbCGS/file167d3648a0cea/sampleA_matched_reads.fastq.gz -> /tmp/RtmpeKbCGS/file167d3648a0cea/sampleA_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpeKbCGS/file167d3648a0cea/sample1_matched_reads.fastq.gz -> /tmp/RtmpeKbCGS/file167d3648a0cea/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpeKbCGS/file167d3648a0cea/sample2_matched_reads.fastq.gz -> /tmp/RtmpeKbCGS/file167d3648a0cea/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpeKbCGS/file167d3648a0cea/sample3_matched_reads.fastq.gz -> /tmp/RtmpeKbCGS/file167d3648a0cea/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Sun Oct 12 22:20:00 2025 ----------------
22:20:00 Sun Oct 12 2025 quantify genes 
Using BAM(s): '/tmp/RtmpeKbCGS/file167d3648a0cea/sampleA_align2genome.bam',
'/tmp/RtmpeKbCGS/file167d3648a0cea/sample1_align2genome.bam',
'/tmp/RtmpeKbCGS/file167d3648a0cea/sample2_align2genome.bam', and
'/tmp/RtmpeKbCGS/file167d3648a0cea/sample3_align2genome.bam'
	Counter({'counted_reads': 368, 'unmapped': 4})
parsing /tmp/RtmpeKbCGS/file167d3648a0cea/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 12.46gene_group/s]
2025-10-12 22:20:04.606 R[92115:522868117] XType: com.apple.fonts is not accessible.
2025-10-12 22:20:04.606 R[92115:522868117] XType: XTFontStaticRegistry is enabled.
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 244662.84Read/s]
parsing /tmp/RtmpeKbCGS/file167d3648a0cea/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 37.33gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1024100.01Read/s]
parsing /tmp/RtmpeKbCGS/file167d3648a0cea/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 35.26gene_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, 1009022.32Read/s]
parsing /tmp/RtmpeKbCGS/file167d3648a0cea/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 24.59gene_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, 751882.98Read/s]
-- Running step: isoform_identification @ Sun Oct 12 22:20:05 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 @ Sun Oct 12 22:20:34 2025 -------------------
Checking for fastq file(s) /tmp/RtmpeKbCGS/file167d3648a0cea/fastq, /tmp/RtmpeKbCGS/file167d3648a0cea/fastq/sample1.fq.gz, /tmp/RtmpeKbCGS/file167d3648a0cea/fastq/sample2.fq.gz, /tmp/RtmpeKbCGS/file167d3648a0cea/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpeKbCGS/file167d3648a0cea/sampleA_matched_reads.fastq.gz, /tmp/RtmpeKbCGS/file167d3648a0cea/sample1_matched_reads.fastq.gz, /tmp/RtmpeKbCGS/file167d3648a0cea/sample2_matched_reads.fastq.gz, /tmp/RtmpeKbCGS/file167d3648a0cea/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpeKbCGS/file167d3648a0cea/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpeKbCGS/file167d3648a0cea/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpeKbCGS/file167d3648a0cea/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpeKbCGS/file167d3648a0cea/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpeKbCGS/file167d3648a0cea/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpeKbCGS/file167d3648a0cea/sampleA_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpeKbCGS/file167d3648a0cea/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpeKbCGS/file167d3648a0cea/sample1_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpeKbCGS/file167d3648a0cea/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpeKbCGS/file167d3648a0cea/sample2_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpeKbCGS/file167d3648a0cea/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpeKbCGS/file167d3648a0cea/sample3_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

-- Running step: transcript_quantification @ Sun Oct 12 22:20:36 2025 ----------
2025-10-13T02:20:36.090324Z  INFO oarfish: setting user-provided filter parameters.
2025-10-13T02:20:36.093340Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpeKbCGS/file167d3648a0cea/sampleA_realign2transcript.bam, contains 5 reference sequences.
2025-10-13T02:20:36.093393Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-13T02:20:36.093408Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-13T02:20:36.094533Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-13T02:20:36.094574Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-13T02:20:36.116112Z  INFO oarfish::single_cell: Processed 100 cells.
2025-10-13T02:20:36.553062Z  INFO oarfish: setting user-provided filter parameters.
2025-10-13T02:20:36.553738Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpeKbCGS/file167d3648a0cea/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-10-13T02:20:36.553805Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-13T02:20:36.553826Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-13T02:20:36.554029Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-13T02:20:36.554138Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-13T02:20:36.963121Z  INFO oarfish: setting user-provided filter parameters.
2025-10-13T02:20:36.963950Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpeKbCGS/file167d3648a0cea/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-10-13T02:20:36.964005Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-13T02:20:36.964026Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-13T02:20:36.964170Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-13T02:20:36.964195Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-13T02:20:37.387001Z  INFO oarfish: setting user-provided filter parameters.
2025-10-13T02:20:37.387826Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpeKbCGS/file167d3648a0cea/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-10-13T02:20:37.387875Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-13T02:20:37.387893Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-13T02:20:37.388040Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-13T02:20:37.388063Z  INFO oarfish: parsed reference information for 5 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpeKbCGS/file167d32a5d8449/config_file_92115.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 @ Sun Oct 12 22:20:38 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpeKbCGS/file167d32a5d8449/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpeKbCGS/file167d32a5d8449/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpeKbCGS/file167d32a5d8449/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpeKbCGS/file167d32a5d8449/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/RtmpeKbCGS/file167d32a5d8449/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpeKbCGS/file167d32a5d8449/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/RtmpeKbCGS/file167d32a5d8449/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpeKbCGS/file167d32a5d8449/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/RtmpeKbCGS/file167d32a5d8449/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpeKbCGS/file167d32a5d8449/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 @ Sun Oct 12 22:20:40 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpeKbCGS/file167d32a5d8449/sampleA_matched_reads.fastq.gz ->/tmp/RtmpeKbCGS/file167d32a5d8449/sampleA_align2genome.bam
/tmp/RtmpeKbCGS/file167d32a5d8449/sample1_matched_reads.fastq.gz ->/tmp/RtmpeKbCGS/file167d32a5d8449/sample1_align2genome.bam
/tmp/RtmpeKbCGS/file167d32a5d8449/sample2_matched_reads.fastq.gz ->/tmp/RtmpeKbCGS/file167d32a5d8449/sample2_align2genome.bam
/tmp/RtmpeKbCGS/file167d32a5d8449/sample3_matched_reads.fastq.gz ->/tmp/RtmpeKbCGS/file167d32a5d8449/sample3_align2genome.bam
-- Running step: gene_quantification @ Sun Oct 12 22:21:04 2025 ----------------
22:21:04 Sun Oct 12 2025 quantify genes 
Using BAM(s): '/tmp/RtmpeKbCGS/file167d32a5d8449/sampleA_align2genome.bam',
'/tmp/RtmpeKbCGS/file167d32a5d8449/sample1_align2genome.bam',
'/tmp/RtmpeKbCGS/file167d32a5d8449/sample2_align2genome.bam', and
'/tmp/RtmpeKbCGS/file167d32a5d8449/sample3_align2genome.bam'
parsing /tmp/RtmpeKbCGS/file167d32a5d8449/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.99gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.99gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 196941.57Read/s]
parsing /tmp/RtmpeKbCGS/file167d32a5d8449/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 28.49gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 642982.59Read/s]
parsing /tmp/RtmpeKbCGS/file167d32a5d8449/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 36.42gene_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, 1042529.33Read/s]
parsing /tmp/RtmpeKbCGS/file167d32a5d8449/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 26.07gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 94424.62Read/s]
-- Running step: isoform_identification @ Sun Oct 12 22:21:05 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 @ Sun Oct 12 22:21:33 2025 -------------------
Checking for fastq file(s) /tmp/RtmpeKbCGS/file167d32a5d8449/fastq, /tmp/RtmpeKbCGS/file167d32a5d8449/fastq/sample1.fq.gz, /tmp/RtmpeKbCGS/file167d32a5d8449/fastq/sample2.fq.gz, /tmp/RtmpeKbCGS/file167d32a5d8449/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpeKbCGS/file167d32a5d8449/sampleA_matched_reads.fastq.gz, /tmp/RtmpeKbCGS/file167d32a5d8449/sample1_matched_reads.fastq.gz, /tmp/RtmpeKbCGS/file167d32a5d8449/sample2_matched_reads.fastq.gz, /tmp/RtmpeKbCGS/file167d32a5d8449/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpeKbCGS/file167d32a5d8449/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpeKbCGS/file167d32a5d8449/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpeKbCGS/file167d32a5d8449/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpeKbCGS/file167d32a5d8449/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpeKbCGS/file167d32a5d8449/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpeKbCGS/file167d32a5d8449/sampleA_realign2transcript.bam
/tmp/RtmpeKbCGS/file167d32a5d8449/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpeKbCGS/file167d32a5d8449/sample1_realign2transcript.bam
/tmp/RtmpeKbCGS/file167d32a5d8449/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpeKbCGS/file167d32a5d8449/sample2_realign2transcript.bam
/tmp/RtmpeKbCGS/file167d32a5d8449/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpeKbCGS/file167d32a5d8449/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Sun Oct 12 22:21:57 2025 ----------
2025-10-13T02:21:57.406630Z  INFO oarfish: setting user-provided filter parameters.
2025-10-13T02:21:57.410598Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpeKbCGS/file167d32a5d8449/sampleA_realign2transcript.bam, contains 5 reference sequences.
2025-10-13T02:21:57.410914Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-13T02:21:57.410956Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-13T02:21:57.412994Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-13T02:21:57.413065Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-13T02:21:57.430878Z  INFO oarfish::single_cell: Processed 100 cells.
2025-10-13T02:21:57.960765Z  INFO oarfish: setting user-provided filter parameters.
2025-10-13T02:21:57.961814Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpeKbCGS/file167d32a5d8449/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-10-13T02:21:57.961858Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-13T02:21:57.961873Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-13T02:21:57.962006Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-13T02:21:57.962027Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-13T02:21:58.441871Z  INFO oarfish: setting user-provided filter parameters.
2025-10-13T02:21:58.442596Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpeKbCGS/file167d32a5d8449/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-10-13T02:21:58.442654Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-13T02:21:58.442670Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-13T02:21:58.442775Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-13T02:21:58.442792Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-13T02:21:58.982105Z  INFO oarfish: setting user-provided filter parameters.
2025-10-13T02:21:58.982945Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpeKbCGS/file167d32a5d8449/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-10-13T02:21:58.982995Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-13T02:21:58.983013Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-13T02:21:58.983146Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-13T02:21:58.983160Z  INFO oarfish: parsed reference information for 5 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpeKbCGS/file167d317ea48f5/config_file_92115.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 @ Sun Oct 12 22:22:00 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpeKbCGS/file167d317ea48f5/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpeKbCGS/file167d317ea48f5/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpeKbCGS/file167d317ea48f5/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpeKbCGS/file167d317ea48f5/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/RtmpeKbCGS/file167d317ea48f5/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpeKbCGS/file167d317ea48f5/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/RtmpeKbCGS/file167d317ea48f5/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpeKbCGS/file167d317ea48f5/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/RtmpeKbCGS/file167d317ea48f5/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpeKbCGS/file167d317ea48f5/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 @ Sun Oct 12 22:22:02 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpeKbCGS/file167d317ea48f5/sampleA_matched_reads.fastq.gz -> /tmp/RtmpeKbCGS/file167d317ea48f5/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/RtmpeKbCGS/file167d317ea48f5/sample1_matched_reads.fastq.gz -> /tmp/RtmpeKbCGS/file167d317ea48f5/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/RtmpeKbCGS/file167d317ea48f5/sample2_matched_reads.fastq.gz -> /tmp/RtmpeKbCGS/file167d317ea48f5/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/RtmpeKbCGS/file167d317ea48f5/sample3_matched_reads.fastq.gz -> /tmp/RtmpeKbCGS/file167d317ea48f5/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 @ Sun Oct 12 22:22:04 2025 ----------------
22:22:04 Sun Oct 12 2025 quantify genes 
Using BAM(s): '/tmp/RtmpeKbCGS/file167d317ea48f5/sampleA_align2genome.bam',
'/tmp/RtmpeKbCGS/file167d317ea48f5/sample1_align2genome.bam',
'/tmp/RtmpeKbCGS/file167d317ea48f5/sample2_align2genome.bam', and
'/tmp/RtmpeKbCGS/file167d317ea48f5/sample3_align2genome.bam'
parsing /tmp/RtmpeKbCGS/file167d317ea48f5/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 15.93gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 374304.28Read/s]
parsing /tmp/RtmpeKbCGS/file167d317ea48f5/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 36.77gene_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, 1360373.64Read/s]
parsing /tmp/RtmpeKbCGS/file167d317ea48f5/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 39.14gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1087283.28Read/s]
parsing /tmp/RtmpeKbCGS/file167d317ea48f5/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 23.74gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 651572.73Read/s]
-- Running step: isoform_identification @ Sun Oct 12 22:22:05 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 @ Sun Oct 12 22:22:38 2025 -------------------
Checking for fastq file(s) /tmp/RtmpeKbCGS/file167d317ea48f5/fastq, /tmp/RtmpeKbCGS/file167d317ea48f5/fastq/sample1.fq.gz, /tmp/RtmpeKbCGS/file167d317ea48f5/fastq/sample2.fq.gz, /tmp/RtmpeKbCGS/file167d317ea48f5/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpeKbCGS/file167d317ea48f5/sampleA_matched_reads.fastq.gz, /tmp/RtmpeKbCGS/file167d317ea48f5/sample1_matched_reads.fastq.gz, /tmp/RtmpeKbCGS/file167d317ea48f5/sample2_matched_reads.fastq.gz, /tmp/RtmpeKbCGS/file167d317ea48f5/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpeKbCGS/file167d317ea48f5/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpeKbCGS/file167d317ea48f5/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpeKbCGS/file167d317ea48f5/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpeKbCGS/file167d317ea48f5/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpeKbCGS/file167d317ea48f5/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpeKbCGS/file167d317ea48f5/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/RtmpeKbCGS/file167d317ea48f5/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpeKbCGS/file167d317ea48f5/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/RtmpeKbCGS/file167d317ea48f5/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpeKbCGS/file167d317ea48f5/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/RtmpeKbCGS/file167d317ea48f5/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpeKbCGS/file167d317ea48f5/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 @ Sun Oct 12 22:22:39 2025 ----------
22:22:39 Sun Oct 12 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpeKbCGS/file167d317ea48f5/sample3_realign2transcript.bam...
parsing /tmp/RtmpeKbCGS/file167d317ea48f5/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpeKbCGS/file167d317ea48f5/sample3_realign2transcript.bamdone
parsing /tmp/RtmpeKbCGS/file167d317ea48f5/sampleA_realign2transcript.bam...
parsing /tmp/RtmpeKbCGS/file167d317ea48f5/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpeKbCGS/file167d317ea48f5/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpeKbCGS/file167d317ea48f5/sample2_realign2transcript.bam...
parsing /tmp/RtmpeKbCGS/file167d317ea48f5/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpeKbCGS/file167d317ea48f5/sample2_realign2transcript.bamdone
parsing /tmp/RtmpeKbCGS/file167d317ea48f5/sample1_realign2transcript.bam...
parsing /tmp/RtmpeKbCGS/file167d317ea48f5/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpeKbCGS/file167d317ea48f5/sample1_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpeKbCGS/file167d34a622d2/config_file_92115.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 @ Sun Oct 12 22:22:42 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/RtmpeKbCGS/file167d34a622d2/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpeKbCGS/file167d34a622d2/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpeKbCGS/file167d34a622d2/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpeKbCGS/file167d34a622d2/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/RtmpeKbCGS/file167d34a622d2/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpeKbCGS/file167d34a622d2/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/RtmpeKbCGS/file167d34a622d2/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpeKbCGS/file167d34a622d2/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/RtmpeKbCGS/file167d34a622d2/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpeKbCGS/file167d34a622d2/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 @ Sun Oct 12 22:22:44 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpeKbCGS/file167d34a622d2/sampleA_matched_reads.fastq.gz ->/tmp/RtmpeKbCGS/file167d34a622d2/sampleA_align2genome.bam
/tmp/RtmpeKbCGS/file167d34a622d2/sample1_matched_reads.fastq.gz ->/tmp/RtmpeKbCGS/file167d34a622d2/sample1_align2genome.bam
/tmp/RtmpeKbCGS/file167d34a622d2/sample2_matched_reads.fastq.gz ->/tmp/RtmpeKbCGS/file167d34a622d2/sample2_align2genome.bam
/tmp/RtmpeKbCGS/file167d34a622d2/sample3_matched_reads.fastq.gz ->/tmp/RtmpeKbCGS/file167d34a622d2/sample3_align2genome.bam
-- Running step: gene_quantification @ Sun Oct 12 22:23:13 2025 ----------------
22:23:13 Sun Oct 12 2025 quantify genes 
Using BAM(s): '/tmp/RtmpeKbCGS/file167d34a622d2/sampleA_align2genome.bam',
'/tmp/RtmpeKbCGS/file167d34a622d2/sample1_align2genome.bam',
'/tmp/RtmpeKbCGS/file167d34a622d2/sample2_align2genome.bam', and
'/tmp/RtmpeKbCGS/file167d34a622d2/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/RtmpeKbCGS/file167d34a622d2/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 14.59gene_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, 274187.69Read/s]
parsing /tmp/RtmpeKbCGS/file167d34a622d2/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 39.14gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 969019.50Read/s]
parsing /tmp/RtmpeKbCGS/file167d34a622d2/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 45.71gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 718596.49Read/s]
parsing /tmp/RtmpeKbCGS/file167d34a622d2/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 32.19gene_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, 459418.16Read/s]
-- Running step: isoform_identification @ Sun Oct 12 22:23:14 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 @ Sun Oct 12 22:23:41 2025 -------------------
Checking for fastq file(s) /tmp/RtmpeKbCGS/file167d34a622d2/fastq, /tmp/RtmpeKbCGS/file167d34a622d2/fastq/sample1.fq.gz, /tmp/RtmpeKbCGS/file167d34a622d2/fastq/sample2.fq.gz, /tmp/RtmpeKbCGS/file167d34a622d2/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpeKbCGS/file167d34a622d2/sampleA_matched_reads.fastq.gz, /tmp/RtmpeKbCGS/file167d34a622d2/sample1_matched_reads.fastq.gz, /tmp/RtmpeKbCGS/file167d34a622d2/sample2_matched_reads.fastq.gz, /tmp/RtmpeKbCGS/file167d34a622d2/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpeKbCGS/file167d34a622d2/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpeKbCGS/file167d34a622d2/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpeKbCGS/file167d34a622d2/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpeKbCGS/file167d34a622d2/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpeKbCGS/file167d34a622d2/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpeKbCGS/file167d34a622d2/sampleA_realign2transcript.bam
/tmp/RtmpeKbCGS/file167d34a622d2/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpeKbCGS/file167d34a622d2/sample1_realign2transcript.bam
/tmp/RtmpeKbCGS/file167d34a622d2/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpeKbCGS/file167d34a622d2/sample2_realign2transcript.bam
/tmp/RtmpeKbCGS/file167d34a622d2/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpeKbCGS/file167d34a622d2/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Sun Oct 12 22:24:05 2025 ----------
22:24:05 Sun Oct 12 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpeKbCGS/file167d34a622d2/sample3_realign2transcript.bam...
parsing /tmp/RtmpeKbCGS/file167d34a622d2/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpeKbCGS/file167d34a622d2/sample3_realign2transcript.bamdone
parsing /tmp/RtmpeKbCGS/file167d34a622d2/sampleA_realign2transcript.bam...
parsing /tmp/RtmpeKbCGS/file167d34a622d2/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpeKbCGS/file167d34a622d2/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpeKbCGS/file167d34a622d2/sample2_realign2transcript.bam...
parsing /tmp/RtmpeKbCGS/file167d34a622d2/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpeKbCGS/file167d34a622d2/sample2_realign2transcript.bamdone
parsing /tmp/RtmpeKbCGS/file167d34a622d2/sample1_realign2transcript.bam...
parsing /tmp/RtmpeKbCGS/file167d34a622d2/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpeKbCGS/file167d34a622d2/sample1_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpeKbCGS/file167d3322a0cac/config_file_92115.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 @ Sun Oct 12 22:24:08 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpeKbCGS/file167d3322a0cac/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpeKbCGS/file167d3322a0cac/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpeKbCGS/file167d3322a0cac/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpeKbCGS/file167d3322a0cac/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/RtmpeKbCGS/file167d3322a0cac/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpeKbCGS/file167d3322a0cac/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/RtmpeKbCGS/file167d3322a0cac/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpeKbCGS/file167d3322a0cac/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/RtmpeKbCGS/file167d3322a0cac/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpeKbCGS/file167d3322a0cac/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 @ Sun Oct 12 22:24:10 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpeKbCGS/file167d3322a0cac/sampleA_matched_reads.fastq.gz -> /tmp/RtmpeKbCGS/file167d3322a0cac/sampleA_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpeKbCGS/file167d3322a0cac/sample1_matched_reads.fastq.gz -> /tmp/RtmpeKbCGS/file167d3322a0cac/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpeKbCGS/file167d3322a0cac/sample2_matched_reads.fastq.gz -> /tmp/RtmpeKbCGS/file167d3322a0cac/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpeKbCGS/file167d3322a0cac/sample3_matched_reads.fastq.gz -> /tmp/RtmpeKbCGS/file167d3322a0cac/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Sun Oct 12 22:24:12 2025 ----------------
22:24:12 Sun Oct 12 2025 quantify genes 
Using BAM(s): '/tmp/RtmpeKbCGS/file167d3322a0cac/sampleA_align2genome.bam',
'/tmp/RtmpeKbCGS/file167d3322a0cac/sample1_align2genome.bam',
'/tmp/RtmpeKbCGS/file167d3322a0cac/sample2_align2genome.bam', and
'/tmp/RtmpeKbCGS/file167d3322a0cac/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/RtmpeKbCGS/file167d3322a0cac/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 16.82gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 303618.25Read/s]
parsing /tmp/RtmpeKbCGS/file167d3322a0cac/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 38.02gene_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, 1286596.32Read/s]
parsing /tmp/RtmpeKbCGS/file167d3322a0cac/sample2_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, 1017146.18Read/s]
parsing /tmp/RtmpeKbCGS/file167d3322a0cac/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 25.13gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 534197.36Read/s]
-- Running step: isoform_identification @ Sun Oct 12 22:24:13 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sun Oct 12 22:24:13 2025 -------------------
Checking for fastq file(s) /tmp/RtmpeKbCGS/file167d3322a0cac/fastq, /tmp/RtmpeKbCGS/file167d3322a0cac/fastq/sample1.fq.gz, /tmp/RtmpeKbCGS/file167d3322a0cac/fastq/sample2.fq.gz, /tmp/RtmpeKbCGS/file167d3322a0cac/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpeKbCGS/file167d3322a0cac/sampleA_matched_reads.fastq.gz, /tmp/RtmpeKbCGS/file167d3322a0cac/sample1_matched_reads.fastq.gz, /tmp/RtmpeKbCGS/file167d3322a0cac/sample2_matched_reads.fastq.gz, /tmp/RtmpeKbCGS/file167d3322a0cac/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpeKbCGS/file167d3322a0cac/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpeKbCGS/file167d3322a0cac/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpeKbCGS/file167d3322a0cac/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpeKbCGS/file167d3322a0cac/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpeKbCGS/file167d3322a0cac/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpeKbCGS/file167d3322a0cac/sampleA_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpeKbCGS/file167d3322a0cac/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpeKbCGS/file167d3322a0cac/sample1_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpeKbCGS/file167d3322a0cac/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpeKbCGS/file167d3322a0cac/sample2_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpeKbCGS/file167d3322a0cac/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpeKbCGS/file167d3322a0cac/sample3_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

-- Running step: transcript_quantification @ Sun Oct 12 22:24:16 2025 ----------
2025-10-13T02:24:16.289499Z  INFO oarfish: setting user-provided filter parameters.
2025-10-13T02:24:16.293027Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpeKbCGS/file167d3322a0cac/sampleA_realign2transcript.bam, contains 14 reference sequences.
2025-10-13T02:24:16.293087Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-13T02:24:16.293101Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-13T02:24:16.294562Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-13T02:24:16.294730Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-10-13T02:24:16.319014Z  INFO oarfish::single_cell: Processed 100 cells.
2025-10-13T02:24:17.092406Z  INFO oarfish: setting user-provided filter parameters.
2025-10-13T02:24:17.093235Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpeKbCGS/file167d3322a0cac/sample1_realign2transcript.bam, contains 14 reference sequences.
2025-10-13T02:24:17.093292Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-13T02:24:17.093305Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-13T02:24:17.093478Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-13T02:24:17.093501Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-10-13T02:24:17.841674Z  INFO oarfish: setting user-provided filter parameters.
2025-10-13T02:24:17.842406Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpeKbCGS/file167d3322a0cac/sample2_realign2transcript.bam, contains 14 reference sequences.
2025-10-13T02:24:17.842449Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-13T02:24:17.842462Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-13T02:24:17.842657Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-13T02:24:17.842681Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-10-13T02:24:18.537829Z  INFO oarfish: setting user-provided filter parameters.
2025-10-13T02:24:18.538550Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpeKbCGS/file167d3322a0cac/sample3_realign2transcript.bam, contains 14 reference sequences.
2025-10-13T02:24:18.538591Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-13T02:24:18.538605Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-13T02:24:18.538778Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-13T02:24:18.538798Z  INFO oarfish: parsed reference information for 14 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpeKbCGS/file167d31e9ccff/config_file_92115.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 @ Sun Oct 12 22:24:19 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/RtmpeKbCGS/file167d31e9ccff/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpeKbCGS/file167d31e9ccff/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpeKbCGS/file167d31e9ccff/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpeKbCGS/file167d31e9ccff/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/RtmpeKbCGS/file167d31e9ccff/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpeKbCGS/file167d31e9ccff/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/RtmpeKbCGS/file167d31e9ccff/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpeKbCGS/file167d31e9ccff/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/RtmpeKbCGS/file167d31e9ccff/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpeKbCGS/file167d31e9ccff/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 @ Sun Oct 12 22:24:21 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpeKbCGS/file167d31e9ccff/sampleA_matched_reads.fastq.gz ->/tmp/RtmpeKbCGS/file167d31e9ccff/sampleA_align2genome.bam
/tmp/RtmpeKbCGS/file167d31e9ccff/sample1_matched_reads.fastq.gz ->/tmp/RtmpeKbCGS/file167d31e9ccff/sample1_align2genome.bam
/tmp/RtmpeKbCGS/file167d31e9ccff/sample2_matched_reads.fastq.gz ->/tmp/RtmpeKbCGS/file167d31e9ccff/sample2_align2genome.bam
/tmp/RtmpeKbCGS/file167d31e9ccff/sample3_matched_reads.fastq.gz ->/tmp/RtmpeKbCGS/file167d31e9ccff/sample3_align2genome.bam
-- Running step: gene_quantification @ Sun Oct 12 22:24:44 2025 ----------------
22:24:44 Sun Oct 12 2025 quantify genes 
Using BAM(s): '/tmp/RtmpeKbCGS/file167d31e9ccff/sampleA_align2genome.bam',
'/tmp/RtmpeKbCGS/file167d31e9ccff/sample1_align2genome.bam',
'/tmp/RtmpeKbCGS/file167d31e9ccff/sample2_align2genome.bam', and
'/tmp/RtmpeKbCGS/file167d31e9ccff/sample3_align2genome.bam'
parsing /tmp/RtmpeKbCGS/file167d31e9ccff/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 13.67gene_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, 299114.56Read/s]
parsing /tmp/RtmpeKbCGS/file167d31e9ccff/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 37.82gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 958654.23Read/s]
parsing /tmp/RtmpeKbCGS/file167d31e9ccff/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 33.93gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 901535.55Read/s]
parsing /tmp/RtmpeKbCGS/file167d31e9ccff/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 22.99gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 435093.78Read/s]
-- Running step: isoform_identification @ Sun Oct 12 22:24:46 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sun Oct 12 22:24:46 2025 -------------------
Checking for fastq file(s) /tmp/RtmpeKbCGS/file167d31e9ccff/fastq, /tmp/RtmpeKbCGS/file167d31e9ccff/fastq/sample1.fq.gz, /tmp/RtmpeKbCGS/file167d31e9ccff/fastq/sample2.fq.gz, /tmp/RtmpeKbCGS/file167d31e9ccff/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpeKbCGS/file167d31e9ccff/sampleA_matched_reads.fastq.gz, /tmp/RtmpeKbCGS/file167d31e9ccff/sample1_matched_reads.fastq.gz, /tmp/RtmpeKbCGS/file167d31e9ccff/sample2_matched_reads.fastq.gz, /tmp/RtmpeKbCGS/file167d31e9ccff/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpeKbCGS/file167d31e9ccff/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpeKbCGS/file167d31e9ccff/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpeKbCGS/file167d31e9ccff/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpeKbCGS/file167d31e9ccff/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpeKbCGS/file167d31e9ccff/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpeKbCGS/file167d31e9ccff/sampleA_realign2transcript.bam
/tmp/RtmpeKbCGS/file167d31e9ccff/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpeKbCGS/file167d31e9ccff/sample1_realign2transcript.bam
/tmp/RtmpeKbCGS/file167d31e9ccff/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpeKbCGS/file167d31e9ccff/sample2_realign2transcript.bam
/tmp/RtmpeKbCGS/file167d31e9ccff/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpeKbCGS/file167d31e9ccff/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Sun Oct 12 22:25:10 2025 ----------
2025-10-13T02:25:10.419453Z  INFO oarfish: setting user-provided filter parameters.
2025-10-13T02:25:10.420258Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpeKbCGS/file167d31e9ccff/sampleA_realign2transcript.bam, contains 14 reference sequences.
2025-10-13T02:25:10.420301Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-13T02:25:10.420314Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-13T02:25:10.420453Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-13T02:25:10.420471Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-10-13T02:25:10.438103Z  INFO oarfish::single_cell: Processed 100 cells.
2025-10-13T02:25:11.398123Z  INFO oarfish: setting user-provided filter parameters.
2025-10-13T02:25:11.399055Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpeKbCGS/file167d31e9ccff/sample1_realign2transcript.bam, contains 14 reference sequences.
2025-10-13T02:25:11.399116Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-13T02:25:11.399134Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-13T02:25:11.399313Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-13T02:25:11.399340Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-10-13T02:25:12.227971Z  INFO oarfish: setting user-provided filter parameters.
2025-10-13T02:25:12.228633Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpeKbCGS/file167d31e9ccff/sample2_realign2transcript.bam, contains 14 reference sequences.
2025-10-13T02:25:12.228688Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-13T02:25:12.228709Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-13T02:25:12.228905Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-13T02:25:12.228936Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-10-13T02:25:12.942325Z  INFO oarfish: setting user-provided filter parameters.
2025-10-13T02:25:12.943400Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpeKbCGS/file167d31e9ccff/sample3_realign2transcript.bam, contains 14 reference sequences.
2025-10-13T02:25:12.943439Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-13T02:25:12.943452Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-13T02:25:12.943623Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-13T02:25:12.943641Z  INFO oarfish: parsed reference information for 14 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpeKbCGS/file167d3581ef0f6/config_file_92115.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 @ Sun Oct 12 22:25:14 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpeKbCGS/file167d3581ef0f6/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpeKbCGS/file167d3581ef0f6/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpeKbCGS/file167d3581ef0f6/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpeKbCGS/file167d3581ef0f6/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/RtmpeKbCGS/file167d3581ef0f6/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpeKbCGS/file167d3581ef0f6/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/RtmpeKbCGS/file167d3581ef0f6/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpeKbCGS/file167d3581ef0f6/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/RtmpeKbCGS/file167d3581ef0f6/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpeKbCGS/file167d3581ef0f6/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 @ Sun Oct 12 22:25:16 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpeKbCGS/file167d3581ef0f6/sampleA_matched_reads.fastq.gz -> /tmp/RtmpeKbCGS/file167d3581ef0f6/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/RtmpeKbCGS/file167d3581ef0f6/sample1_matched_reads.fastq.gz -> /tmp/RtmpeKbCGS/file167d3581ef0f6/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/RtmpeKbCGS/file167d3581ef0f6/sample2_matched_reads.fastq.gz -> /tmp/RtmpeKbCGS/file167d3581ef0f6/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/RtmpeKbCGS/file167d3581ef0f6/sample3_matched_reads.fastq.gz -> /tmp/RtmpeKbCGS/file167d3581ef0f6/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 @ Sun Oct 12 22:25:18 2025 ----------------
22:25:18 Sun Oct 12 2025 quantify genes 
Using BAM(s): '/tmp/RtmpeKbCGS/file167d3581ef0f6/sampleA_align2genome.bam',
'/tmp/RtmpeKbCGS/file167d3581ef0f6/sample1_align2genome.bam',
'/tmp/RtmpeKbCGS/file167d3581ef0f6/sample2_align2genome.bam', and
'/tmp/RtmpeKbCGS/file167d3581ef0f6/sample3_align2genome.bam'
parsing /tmp/RtmpeKbCGS/file167d3581ef0f6/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 17.34gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 208888.00Read/s]
parsing /tmp/RtmpeKbCGS/file167d3581ef0f6/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 41.88gene_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, 1308920.23Read/s]
parsing /tmp/RtmpeKbCGS/file167d3581ef0f6/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 38.92gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1103299.66Read/s]
parsing /tmp/RtmpeKbCGS/file167d3581ef0f6/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  6.71gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  6.70gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 558436.38Read/s]
-- Running step: isoform_identification @ Sun Oct 12 22:25:19 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sun Oct 12 22:25:19 2025 -------------------
Checking for fastq file(s) /tmp/RtmpeKbCGS/file167d3581ef0f6/fastq, /tmp/RtmpeKbCGS/file167d3581ef0f6/fastq/sample1.fq.gz, /tmp/RtmpeKbCGS/file167d3581ef0f6/fastq/sample2.fq.gz, /tmp/RtmpeKbCGS/file167d3581ef0f6/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpeKbCGS/file167d3581ef0f6/sampleA_matched_reads.fastq.gz, /tmp/RtmpeKbCGS/file167d3581ef0f6/sample1_matched_reads.fastq.gz, /tmp/RtmpeKbCGS/file167d3581ef0f6/sample2_matched_reads.fastq.gz, /tmp/RtmpeKbCGS/file167d3581ef0f6/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpeKbCGS/file167d3581ef0f6/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpeKbCGS/file167d3581ef0f6/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpeKbCGS/file167d3581ef0f6/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpeKbCGS/file167d3581ef0f6/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpeKbCGS/file167d3581ef0f6/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpeKbCGS/file167d3581ef0f6/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/RtmpeKbCGS/file167d3581ef0f6/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpeKbCGS/file167d3581ef0f6/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/RtmpeKbCGS/file167d3581ef0f6/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpeKbCGS/file167d3581ef0f6/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/RtmpeKbCGS/file167d3581ef0f6/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpeKbCGS/file167d3581ef0f6/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 @ Sun Oct 12 22:25:21 2025 ----------
22:25:21 Sun Oct 12 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpeKbCGS/file167d3581ef0f6/sample3_realign2transcript.bam...
parsing /tmp/RtmpeKbCGS/file167d3581ef0f6/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpeKbCGS/file167d3581ef0f6/sample3_realign2transcript.bamdone
parsing /tmp/RtmpeKbCGS/file167d3581ef0f6/sampleA_realign2transcript.bam...
parsing /tmp/RtmpeKbCGS/file167d3581ef0f6/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpeKbCGS/file167d3581ef0f6/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpeKbCGS/file167d3581ef0f6/sample2_realign2transcript.bam...
parsing /tmp/RtmpeKbCGS/file167d3581ef0f6/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpeKbCGS/file167d3581ef0f6/sample2_realign2transcript.bamdone
parsing /tmp/RtmpeKbCGS/file167d3581ef0f6/sample1_realign2transcript.bam...
parsing /tmp/RtmpeKbCGS/file167d3581ef0f6/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpeKbCGS/file167d3581ef0f6/sample1_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpeKbCGS/file167d347cd543d/config_file_92115.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 @ Sun Oct 12 22:25:25 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/RtmpeKbCGS/file167d347cd543d/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpeKbCGS/file167d347cd543d/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpeKbCGS/file167d347cd543d/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpeKbCGS/file167d347cd543d/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/RtmpeKbCGS/file167d347cd543d/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpeKbCGS/file167d347cd543d/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/RtmpeKbCGS/file167d347cd543d/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpeKbCGS/file167d347cd543d/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/RtmpeKbCGS/file167d347cd543d/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpeKbCGS/file167d347cd543d/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 @ Sun Oct 12 22:25:27 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpeKbCGS/file167d347cd543d/sampleA_matched_reads.fastq.gz ->/tmp/RtmpeKbCGS/file167d347cd543d/sampleA_align2genome.bam
/tmp/RtmpeKbCGS/file167d347cd543d/sample1_matched_reads.fastq.gz ->/tmp/RtmpeKbCGS/file167d347cd543d/sample1_align2genome.bam
/tmp/RtmpeKbCGS/file167d347cd543d/sample2_matched_reads.fastq.gz ->/tmp/RtmpeKbCGS/file167d347cd543d/sample2_align2genome.bam
/tmp/RtmpeKbCGS/file167d347cd543d/sample3_matched_reads.fastq.gz ->/tmp/RtmpeKbCGS/file167d347cd543d/sample3_align2genome.bam
-- Running step: gene_quantification @ Sun Oct 12 22:25:50 2025 ----------------
22:25:50 Sun Oct 12 2025 quantify genes 
Using BAM(s): '/tmp/RtmpeKbCGS/file167d347cd543d/sampleA_align2genome.bam',
'/tmp/RtmpeKbCGS/file167d347cd543d/sample1_align2genome.bam',
'/tmp/RtmpeKbCGS/file167d347cd543d/sample2_align2genome.bam', and
'/tmp/RtmpeKbCGS/file167d347cd543d/sample3_align2genome.bam'
	Counter({'counted_reads': 176})
	Counter({'counted_reads': 358})
	Counter({'counted_reads': 95})
	Counter({'counted_reads': 91})
parsing /tmp/RtmpeKbCGS/file167d347cd543d/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 16.78gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 310587.96Read/s]
parsing /tmp/RtmpeKbCGS/file167d347cd543d/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 38.06gene_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, 1157368.65Read/s]
parsing /tmp/RtmpeKbCGS/file167d347cd543d/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 36.12gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1182160.09Read/s]
parsing /tmp/RtmpeKbCGS/file167d347cd543d/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 26.43gene_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, 604018.43Read/s]
-- Running step: isoform_identification @ Sun Oct 12 22:25:51 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sun Oct 12 22:25:52 2025 -------------------
Checking for fastq file(s) /tmp/RtmpeKbCGS/file167d347cd543d/fastq, /tmp/RtmpeKbCGS/file167d347cd543d/fastq/sample1.fq.gz, /tmp/RtmpeKbCGS/file167d347cd543d/fastq/sample2.fq.gz, /tmp/RtmpeKbCGS/file167d347cd543d/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpeKbCGS/file167d347cd543d/sampleA_matched_reads.fastq.gz, /tmp/RtmpeKbCGS/file167d347cd543d/sample1_matched_reads.fastq.gz, /tmp/RtmpeKbCGS/file167d347cd543d/sample2_matched_reads.fastq.gz, /tmp/RtmpeKbCGS/file167d347cd543d/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpeKbCGS/file167d347cd543d/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpeKbCGS/file167d347cd543d/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpeKbCGS/file167d347cd543d/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpeKbCGS/file167d347cd543d/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpeKbCGS/file167d347cd543d/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpeKbCGS/file167d347cd543d/sampleA_realign2transcript.bam
/tmp/RtmpeKbCGS/file167d347cd543d/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpeKbCGS/file167d347cd543d/sample1_realign2transcript.bam
/tmp/RtmpeKbCGS/file167d347cd543d/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpeKbCGS/file167d347cd543d/sample2_realign2transcript.bam
/tmp/RtmpeKbCGS/file167d347cd543d/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpeKbCGS/file167d347cd543d/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Sun Oct 12 22:26:14 2025 ----------
22:26:14 Sun Oct 12 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpeKbCGS/file167d347cd543d/sample3_realign2transcript.bam...
parsing /tmp/RtmpeKbCGS/file167d347cd543d/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpeKbCGS/file167d347cd543d/sample3_realign2transcript.bamdone
parsing /tmp/RtmpeKbCGS/file167d347cd543d/sampleA_realign2transcript.bam...
parsing /tmp/RtmpeKbCGS/file167d347cd543d/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpeKbCGS/file167d347cd543d/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpeKbCGS/file167d347cd543d/sample2_realign2transcript.bam...
parsing /tmp/RtmpeKbCGS/file167d347cd543d/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpeKbCGS/file167d347cd543d/sample2_realign2transcript.bamdone
parsing /tmp/RtmpeKbCGS/file167d347cd543d/sample1_realign2transcript.bam...
parsing /tmp/RtmpeKbCGS/file167d347cd543d/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpeKbCGS/file167d347cd543d/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 
 845.800   89.326 1128.666 
/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
BulkPipeline4.2710.8157.618
MultiSampleSCPipeline12.874 2.67124.533
SingleCellPipeline3.5270.3132.926
add_gene_counts0.3290.0070.338
annotation_to_fasta0.2250.0060.232
blaze 7.576 1.39915.288
bulk_long_pipeline4.4131.0864.295
combine_sce1.0570.1001.169
config-set0.3120.2300.609
config0.4060.6781.989
controllers-set0.5400.1390.855
controllers0.3950.2650.777
convolution_filter0.0010.0010.001
create_config0.0140.0030.017
create_sce_from_dir7.9042.0829.655
create_se_from_dir3.7651.0935.719
cutadapt0.2000.1060.984
example_pipeline0.3810.0460.470
experiment2.7720.6065.835
filter_annotation0.0430.0050.053
filter_coverage1.1840.3672.659
find_barcode1.9810.3472.837
find_bin0.0080.0110.035
find_variants23.637 0.62424.671
get_coverage1.4750.7406.588
index_genome0.2910.2561.040
mutation_positions1.5430.0541.687
plot_coverage3.2620.4074.106
plot_demultiplex3.3980.5865.028
plot_demultiplex_raw2.0700.1744.905
plot_durations3.3910.6135.833
plot_isoform_heatmap10.456 0.67611.430
plot_isoform_reduced_dim28.789 0.52629.681
plot_isoforms4.3930.0394.468
resume_FLAMES2.8390.5953.830
run_FLAMES2.7050.5533.627
run_step1.3440.3021.786
sc_DTU_analysis10.076 1.35010.516
sc_gene_entropy1.7060.0612.106
sc_genotype3.9401.0793.870
sc_impute_transcript0.8350.0190.870
sc_long_multisample_pipeline16.879 3.37517.438
sc_long_pipeline5.3131.0136.289
sc_mutations3.2410.5145.647
sc_plot_genotype11.319 0.46311.865
show-FLAMESPipeline0.3880.0410.455
steps-set0.5750.0430.650
steps0.1940.0430.286
weight_transcripts0.0280.0390.069