Back to Multiple platform build/check report for BioC 3.22:   simplified   long
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This page was generated on 2025-10-09 12:04 -0400 (Thu, 09 Oct 2025).

HostnameOSArch (*)R versionInstalled pkgs
nebbiolo2Linux (Ubuntu 24.04.3 LTS)x86_644.5.1 Patched (2025-08-23 r88802) -- "Great Square Root" 4854
lconwaymacOS 12.7.1 Montereyx86_644.5.1 Patched (2025-09-10 r88807) -- "Great Square Root" 4642
kjohnson3macOS 13.7.7 Venturaarm644.5.1 Patched (2025-09-10 r88807) -- "Great Square Root" 4587
taishanLinux (openEuler 24.03 LTS)aarch644.5.0 (2025-04-11) -- "How About a Twenty-Six" 4584
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 737/2341HostnameOS / ArchINSTALLBUILDCHECKBUILD BIN
FLAMES 2.3.5  (landing page)
Changqing Wang
Snapshot Date: 2025-10-08 14:17 -0400 (Wed, 08 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: 2025-10-08 21:57:41 -0400 (Wed, 08 Oct 2025)
EndedAt: 2025-10-08 22:26:07 -0400 (Wed, 08 Oct 2025)
EllapsedTime: 1706.1 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     29.259  0.501  30.115
find_variants                25.303  0.386  25.161
sc_long_multisample_pipeline 15.717  3.103  16.009
MultiSampleSCPipeline        14.044  2.140  18.137
sc_plot_genotype             12.182  0.359  11.612
sc_DTU_analysis              10.558  1.369  10.627
plot_isoform_heatmap         10.063  0.571  10.774
blaze                         8.424  1.257  10.733
create_sce_from_dir           7.463  1.564   7.837
sc_long_pipeline              5.414  0.855   5.005
bulk_long_pipeline            4.705  1.147   4.588
BulkPipeline                  4.536  0.652   5.632
sc_genotype                   4.030  1.093   4.027
plot_demultiplex              3.764  0.579   5.183
* 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/RtmpauyNMT/fileb64745586d8c/config_file_46663.json 
Writing configuration parameters to:  /tmp/RtmpauyNMT/fileb64745586d8c/config_file_46663.json 
Writing configuration parameters to:  /tmp/RtmpauyNMT/fileb64745586d8c/config_file_46663.json 
Writing configuration parameters to:  /tmp/RtmpauyNMT/fileb64726058aaf/config_file_46663.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/RtmpauyNMT/fileb6477fda6289/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/RtmpauyNMT/fileb647365e55b8/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/RtmpauyNMT/fileb647365e55b8/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpauyNMT/fileb6473d312baf/musc_rps24_1.fastq
Searching for barcodes...
Processing file: /tmp/RtmpauyNMT/fileb6473d312baf/musc_rps24_2.fastq
Searching for barcodes...
Processing file: /tmp/RtmpauyNMT/fileb6473d312baf/musc_rps24_3.fastq
Searching for barcodes...
Processing file: /tmp/RtmpauyNMT/fileb6473d312baf/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/RtmpauyNMT/fileb64741b8d976/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/RtmpauyNMT/fileb6472dc1672/config_file_46663.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 @ Wed Oct  8 22:09:10 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpauyNMT/fileb6472dc1672/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpauyNMT/fileb6472dc1672/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpauyNMT/fileb6472dc1672/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Wed Oct  8 22:09:11 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

  |                                                                            
  |                                                                      |   0%[22:09:18] 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.

[22:09:18] 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.

[22:09:18] 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.

[22:09:18] 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.

[22:09:20] 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.

[22:09:20] 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 @ Wed Oct  8 22:09:40 2025 -------------------
Realigning sample sample1 -> /tmp/RtmpauyNMT/fileb6472dc1672/sample1_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample2 -> /tmp/RtmpauyNMT/fileb6472dc1672/sample2_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample3 -> /tmp/RtmpauyNMT/fileb6472dc1672/sample3_realign2transcript.bam
Skipped sorting BAM files.

-- Running step: transcript_quantification @ Wed Oct  8 22:09:42 2025 ----------
2025-10-09T02:09:42.776117Z  INFO oarfish: setting user-provided filter parameters.
2025-10-09T02:09:42.804472Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpauyNMT/fileb6472dc1672/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-10-09T02:09:42.807407Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-09T02:09:42.807914Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-09T02:09:42.875471Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-09T02:09:42.875530Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-09T02:09:42.886327Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-09T02:09:42.889542Z  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    │
╰─────────────────────────────────┴───────╯

2025-10-09T02:09:42.892924Z  INFO oarfish::bulk: Total number of alignment records : 116
2025-10-09T02:09:42.892989Z  INFO oarfish::bulk: number of aligned reads : 96
2025-10-09T02:09:42.893012Z  INFO oarfish::bulk: number of unique alignments : 86
2025-10-09T02:09:42.907907Z  INFO oarfish: oarfish completed successfully.
2025-10-09T02:09:43.013584Z  INFO oarfish: setting user-provided filter parameters.
2025-10-09T02:09:43.014680Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpauyNMT/fileb6472dc1672/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-10-09T02:09:43.014783Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-09T02:09:43.014801Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-09T02:09:43.015861Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-09T02:09:43.015907Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-09T02:09:43.020459Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-09T02:09:43.021880Z  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    │
╰─────────────────────────────────┴───────╯

2025-10-09T02:09:43.021983Z  INFO oarfish::bulk: Total number of alignment records : 114
2025-10-09T02:09:43.022004Z  INFO oarfish::bulk: number of aligned reads : 95
2025-10-09T02:09:43.022015Z  INFO oarfish::bulk: number of unique alignments : 82
2025-10-09T02:09:43.026097Z  INFO oarfish: oarfish completed successfully.
2025-10-09T02:09:43.095415Z  INFO oarfish: setting user-provided filter parameters.
2025-10-09T02:09:43.096501Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpauyNMT/fileb6472dc1672/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-10-09T02:09:43.096576Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-09T02:09:43.096630Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-09T02:09:43.097035Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-09T02:09:43.097062Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-09T02:09:43.102945Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2025-10-09T02:09:43.103793Z  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   │
╰─────────────────────────────────┴───────╯

2025-10-09T02:09:43.103948Z  INFO oarfish::bulk: Total number of alignment records : 239
2025-10-09T02:09:43.103974Z  INFO oarfish::bulk: number of aligned reads : 179
2025-10-09T02:09:43.103986Z  INFO oarfish::bulk: number of unique alignments : 143
2025-10-09T02:09:43.108650Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpauyNMT/fileb647b8b4a3f/config_file_46663.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 @ Wed Oct  8 22:09:44 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpauyNMT/fileb647b8b4a3f/sample1_align2genome.bam
sample2 ->/tmp/RtmpauyNMT/fileb647b8b4a3f/sample2_align2genome.bam
sample3 ->/tmp/RtmpauyNMT/fileb647b8b4a3f/sample3_align2genome.bam
-- Running step: isoform_identification @ Wed Oct  8 22:10:11 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

  |                                                                            
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  |                                                                            
  |=======================                                               |  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 @ Wed Oct  8 22:10:35 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpauyNMT/fileb647b8b4a3f/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpauyNMT/fileb647b8b4a3f/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpauyNMT/fileb647b8b4a3f/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Wed Oct  8 22:11:20 2025 ----------
2025-10-09T02:11:22.024402Z  INFO oarfish: setting user-provided filter parameters.
2025-10-09T02:11:22.195416Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpauyNMT/fileb647b8b4a3f/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-10-09T02:11:22.218318Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-09T02:11:22.218358Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-09T02:11:22.290547Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-09T02:11:22.290599Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-09T02:11:22.384651Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-09T02:11:22.462548Z  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    │
╰─────────────────────────────────┴───────╯

2025-10-09T02:11:22.475490Z  INFO oarfish::bulk: Total number of alignment records : 116
2025-10-09T02:11:22.475513Z  INFO oarfish::bulk: number of aligned reads : 96
2025-10-09T02:11:22.475523Z  INFO oarfish::bulk: number of unique alignments : 86
2025-10-09T02:11:22.503595Z  INFO oarfish: oarfish completed successfully.
2025-10-09T02:11:22.584640Z  INFO oarfish: setting user-provided filter parameters.
2025-10-09T02:11:22.587245Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpauyNMT/fileb647b8b4a3f/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-10-09T02:11:22.587285Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-09T02:11:22.587301Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-09T02:11:22.587434Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-09T02:11:22.587455Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-09T02:11:22.596301Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-09T02:11:22.597006Z  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    │
╰─────────────────────────────────┴───────╯

2025-10-09T02:11:22.597186Z  INFO oarfish::bulk: Total number of alignment records : 114
2025-10-09T02:11:22.597233Z  INFO oarfish::bulk: number of aligned reads : 95
2025-10-09T02:11:22.597243Z  INFO oarfish::bulk: number of unique alignments : 82
2025-10-09T02:11:22.602943Z  INFO oarfish: oarfish completed successfully.
2025-10-09T02:11:22.675460Z  INFO oarfish: setting user-provided filter parameters.
2025-10-09T02:11:22.676254Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpauyNMT/fileb647b8b4a3f/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-10-09T02:11:22.676296Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-09T02:11:22.676308Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-09T02:11:22.676428Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-09T02:11:22.676442Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-09T02:11:22.694715Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2025-10-09T02:11:22.695390Z  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   │
╰─────────────────────────────────┴───────╯

2025-10-09T02:11:22.696015Z  INFO oarfish::bulk: Total number of alignment records : 239
2025-10-09T02:11:22.696045Z  INFO oarfish::bulk: number of aligned reads : 179
2025-10-09T02:11:22.696056Z  INFO oarfish::bulk: number of unique alignments : 143
2025-10-09T02:11:22.702827Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpauyNMT/fileb64733dfc6d0/config_file_46663.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 @ Wed Oct  8 22:11:24 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpauyNMT/fileb64733dfc6d0/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpauyNMT/fileb64733dfc6d0/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpauyNMT/fileb64733dfc6d0/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Wed Oct  8 22:11:26 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 4 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Wed Oct  8 22:11:55 2025 -------------------
Realigning sample sample1 -> /tmp/RtmpauyNMT/fileb64733dfc6d0/sample1_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample2 -> /tmp/RtmpauyNMT/fileb64733dfc6d0/sample2_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample3 -> /tmp/RtmpauyNMT/fileb64733dfc6d0/sample3_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Wed Oct  8 22:11:56 2025 ----------
22:11:56 Wed Oct 08 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Inputs:  ['/tmp/RtmpauyNMT/fileb64733dfc6d0/sample3_realign2transcript.bam', '/tmp/RtmpauyNMT/fileb64733dfc6d0/sample2_realign2transcript.bam', '/tmp/RtmpauyNMT/fileb64733dfc6d0/sample1_realign2transcript.bam'] /tmp/RtmpauyNMT/fileb64733dfc6d0/transcript_assembly.fa.fai 5 0.4 0.4
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpauyNMT/fileb6471cb36baf/config_file_46663.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 @ Wed Oct  8 22:12:22 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpauyNMT/fileb6471cb36baf/sample1_align2genome.bam
sample2 ->/tmp/RtmpauyNMT/fileb6471cb36baf/sample2_align2genome.bam
sample3 ->/tmp/RtmpauyNMT/fileb6471cb36baf/sample3_align2genome.bam
-- Running step: isoform_identification @ Wed Oct  8 22:12:58 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

  |                                                                            
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  |                                                                            
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  |===============================================                       |  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 @ Wed Oct  8 22:13:22 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpauyNMT/fileb6471cb36baf/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpauyNMT/fileb6471cb36baf/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpauyNMT/fileb6471cb36baf/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Wed Oct  8 22:13:48 2025 ----------
22:13:48 Wed Oct 08 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	Counter({'counted_reads': 375, 'not_enough_coverage': 16, 'unmapped': 2})
Inputs:  ['/tmp/RtmpauyNMT/fileb6471cb36baf/sample3_realign2transcript.bam', '/tmp/RtmpauyNMT/fileb6471cb36baf/sample2_realign2transcript.bam', '/tmp/RtmpauyNMT/fileb6471cb36baf/sample1_realign2transcript.bam'] /tmp/RtmpauyNMT/fileb6471cb36baf/transcript_assembly.fa.fai 5 0.4 0.4
	Counter({'counted_reads': 375, 'not_enough_coverage': 16, 'unmapped': 2})
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpauyNMT/fileb6477c1da565/config_file_46663.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 @ Wed Oct  8 22:13:50 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpauyNMT/fileb6477c1da565/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpauyNMT/fileb6477c1da565/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpauyNMT/fileb6477c1da565/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Wed Oct  8 22:13:51 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Wed Oct  8 22:13:51 2025 -------------------
Realigning sample sample1 -> /tmp/RtmpauyNMT/fileb6477c1da565/sample1_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample2 -> /tmp/RtmpauyNMT/fileb6477c1da565/sample2_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample3 -> /tmp/RtmpauyNMT/fileb6477c1da565/sample3_realign2transcript.bam
Skipped sorting BAM files.

-- Running step: transcript_quantification @ Wed Oct  8 22:13:53 2025 ----------
2025-10-09T02:13:53.221223Z  INFO oarfish: setting user-provided filter parameters.
2025-10-09T02:13:53.240165Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpauyNMT/fileb6477c1da565/sample1_realign2transcript.bam, contains 10 reference sequences.
2025-10-09T02:13:53.240241Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-09T02:13:53.240265Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-09T02:13:53.244366Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-09T02:13:53.244432Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-09T02:13:53.253249Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-09T02:13:53.258616Z  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    │
╰─────────────────────────────────┴───────╯

2025-10-09T02:13:53.260296Z  INFO oarfish::bulk: Total number of alignment records : 125
2025-10-09T02:13:53.260338Z  INFO oarfish::bulk: number of aligned reads : 98
2025-10-09T02:13:53.260348Z  INFO oarfish::bulk: number of unique alignments : 86
2025-10-09T02:13:53.265020Z  INFO oarfish: oarfish completed successfully.
2025-10-09T02:13:53.332593Z  INFO oarfish: setting user-provided filter parameters.
2025-10-09T02:13:53.333551Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpauyNMT/fileb6477c1da565/sample2_realign2transcript.bam, contains 10 reference sequences.
2025-10-09T02:13:53.333598Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-09T02:13:53.333614Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-09T02:13:53.333781Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-09T02:13:53.333806Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-09T02:13:53.338309Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-09T02:13:53.338732Z  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    │
╰─────────────────────────────────┴───────╯

2025-10-09T02:13:53.338802Z  INFO oarfish::bulk: Total number of alignment records : 136
2025-10-09T02:13:53.338812Z  INFO oarfish::bulk: number of aligned reads : 97
2025-10-09T02:13:53.338819Z  INFO oarfish::bulk: number of unique alignments : 79
2025-10-09T02:13:53.343497Z  INFO oarfish: oarfish completed successfully.
2025-10-09T02:13:53.437207Z  INFO oarfish: setting user-provided filter parameters.
2025-10-09T02:13:53.438112Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpauyNMT/fileb6477c1da565/sample3_realign2transcript.bam, contains 10 reference sequences.
2025-10-09T02:13:53.438169Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-09T02:13:53.438180Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-09T02:13:53.438306Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-09T02:13:53.438320Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-09T02:13:53.446343Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-09T02:13:53.447053Z  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   │
╰─────────────────────────────────┴───────╯

2025-10-09T02:13:53.447124Z  INFO oarfish::bulk: Total number of alignment records : 272
2025-10-09T02:13:53.447136Z  INFO oarfish::bulk: number of aligned reads : 187
2025-10-09T02:13:53.447145Z  INFO oarfish::bulk: number of unique alignments : 140
2025-10-09T02:13:53.452849Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpauyNMT/fileb6471305d87f/config_file_46663.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 @ Wed Oct  8 22:13:54 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpauyNMT/fileb6471305d87f/sample1_align2genome.bam
sample2 ->/tmp/RtmpauyNMT/fileb6471305d87f/sample2_align2genome.bam
sample3 ->/tmp/RtmpauyNMT/fileb6471305d87f/sample3_align2genome.bam
-- Running step: isoform_identification @ Wed Oct  8 22:14:18 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Wed Oct  8 22:14:18 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpauyNMT/fileb6471305d87f/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpauyNMT/fileb6471305d87f/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpauyNMT/fileb6471305d87f/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Wed Oct  8 22:14:42 2025 ----------
2025-10-09T02:14:42.534689Z  INFO oarfish: setting user-provided filter parameters.
2025-10-09T02:14:42.548757Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpauyNMT/fileb6471305d87f/sample1_realign2transcript.bam, contains 10 reference sequences.
2025-10-09T02:14:42.549392Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-09T02:14:42.549472Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-09T02:14:42.554259Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-09T02:14:42.554410Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-09T02:14:42.565962Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-09T02:14:42.571436Z  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    │
╰─────────────────────────────────┴───────╯

2025-10-09T02:14:42.572066Z  INFO oarfish::bulk: Total number of alignment records : 125
2025-10-09T02:14:42.572157Z  INFO oarfish::bulk: number of aligned reads : 98
2025-10-09T02:14:42.572177Z  INFO oarfish::bulk: number of unique alignments : 86
2025-10-09T02:14:42.581125Z  INFO oarfish: oarfish completed successfully.
2025-10-09T02:14:42.710285Z  INFO oarfish: setting user-provided filter parameters.
2025-10-09T02:14:42.713213Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpauyNMT/fileb6471305d87f/sample2_realign2transcript.bam, contains 10 reference sequences.
2025-10-09T02:14:42.713296Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-09T02:14:42.713351Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-09T02:14:42.713668Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-09T02:14:42.713711Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-09T02:14:42.720665Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-09T02:14:42.722264Z  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    │
╰─────────────────────────────────┴───────╯

2025-10-09T02:14:42.722337Z  INFO oarfish::bulk: Total number of alignment records : 136
2025-10-09T02:14:42.722352Z  INFO oarfish::bulk: number of aligned reads : 97
2025-10-09T02:14:42.722362Z  INFO oarfish::bulk: number of unique alignments : 79
2025-10-09T02:14:42.727530Z  INFO oarfish: oarfish completed successfully.
2025-10-09T02:14:42.869522Z  INFO oarfish: setting user-provided filter parameters.
2025-10-09T02:14:42.872663Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpauyNMT/fileb6471305d87f/sample3_realign2transcript.bam, contains 10 reference sequences.
2025-10-09T02:14:42.872707Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-09T02:14:42.872722Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-09T02:14:42.872891Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-09T02:14:42.872907Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-09T02:14:42.880861Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-09T02:14:42.881360Z  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   │
╰─────────────────────────────────┴───────╯

2025-10-09T02:14:42.881424Z  INFO oarfish::bulk: Total number of alignment records : 272
2025-10-09T02:14:42.881433Z  INFO oarfish::bulk: number of aligned reads : 187
2025-10-09T02:14:42.881439Z  INFO oarfish::bulk: number of unique alignments : 140
2025-10-09T02:14:42.885774Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpauyNMT/fileb6471c47e99a/config_file_46663.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 @ Wed Oct  8 22:14:44 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpauyNMT/fileb6471c47e99a/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpauyNMT/fileb6471c47e99a/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpauyNMT/fileb6471c47e99a/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Wed Oct  8 22:14:45 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Wed Oct  8 22:14:46 2025 -------------------
Realigning sample sample1 -> /tmp/RtmpauyNMT/fileb6471c47e99a/sample1_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample2 -> /tmp/RtmpauyNMT/fileb6471c47e99a/sample2_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample3 -> /tmp/RtmpauyNMT/fileb6471c47e99a/sample3_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Wed Oct  8 22:14:46 2025 ----------
22:14:46 Wed Oct 08 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/RtmpauyNMT/fileb647318f6433/config_file_46663.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 @ Wed Oct  8 22:14:48 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpauyNMT/fileb647318f6433/sample1_align2genome.bam
sample2 ->/tmp/RtmpauyNMT/fileb647318f6433/sample2_align2genome.bam
sample3 ->/tmp/RtmpauyNMT/fileb647318f6433/sample3_align2genome.bam
-- Running step: isoform_identification @ Wed Oct  8 22:15:13 2025 -------------
Inputs:  ['/tmp/RtmpauyNMT/fileb6471c47e99a/sample3_realign2transcript.bam', '/tmp/RtmpauyNMT/fileb6471c47e99a/sample2_realign2transcript.bam', '/tmp/RtmpauyNMT/fileb6471c47e99a/sample1_realign2transcript.bam'] /tmp/RtmpauyNMT/fileb6471c47e99a/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 @ Wed Oct  8 22:15:13 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpauyNMT/fileb647318f6433/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpauyNMT/fileb647318f6433/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpauyNMT/fileb647318f6433/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Wed Oct  8 22:15:35 2025 ----------
22:15:35 Wed Oct 08 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpauyNMT/fileb64739b0bfe1/config_file_46663.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 @ Wed Oct  8 22:15:37 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpauyNMT/fileb64739b0bfe1/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 @ Wed Oct  8 22:15:37 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpauyNMT/fileb64739b0bfe1/matched_reads.fastq.gz -> /tmp/RtmpauyNMT/fileb64739b0bfe1/align2genome.bam
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: isoform_identification @ Wed Oct  8 22:15:38 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 @ Wed Oct  8 22:15:49 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/RtmpauyNMT/fileb64739b0bfe1/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpauyNMT/fileb64739b0bfe1/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/RtmpauyNMT/fileb64739b0bfe1/matched_reads.fastq.gz -> /tmp/RtmpauyNMT/fileb64739b0bfe1/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 @ Wed Oct  8 22:15:50 2025 ----------
2025-10-09T02:15:50.077703Z  INFO oarfish: setting user-provided filter parameters.
2025-10-09T02:15:50.081263Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpauyNMT/fileb64739b0bfe1/realign2transcript.bam, contains 5 reference sequences.
2025-10-09T02:15:50.081333Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-09T02:15:50.081355Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-09T02:15:50.082519Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-09T02:15:50.082571Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-09T02:15:50.097352Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpauyNMT/fileb64777e4f21/config_file_46663.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 @ Wed Oct  8 22:15:50 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpauyNMT/fileb64777e4f21/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 @ Wed Oct  8 22:15:51 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpauyNMT/fileb64777e4f21/matched_reads.fastq.gz ->/tmp/RtmpauyNMT/fileb64777e4f21/align2genome.bam
-- Running step: isoform_identification @ Wed Oct  8 22:16:16 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---
Detected 2 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Wed Oct  8 22:16:28 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/RtmpauyNMT/fileb64777e4f21/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpauyNMT/fileb64777e4f21/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/RtmpauyNMT/fileb64777e4f21/matched_reads.fastq.gz ->/tmp/RtmpauyNMT/fileb64777e4f21/realign2transcript.bam
-- Running step: transcript_quantification @ Wed Oct  8 22:16:52 2025 ----------
2025-10-09T02:16:53.018415Z  INFO oarfish: setting user-provided filter parameters.
2025-10-09T02:16:53.023894Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpauyNMT/fileb64777e4f21/realign2transcript.bam, contains 5 reference sequences.
2025-10-09T02:16:53.024117Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-09T02:16:53.024153Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-09T02:16:53.026680Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-09T02:16:53.026786Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-09T02:16:53.048059Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpauyNMT/fileb647760566ab/config_file_46663.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 @ Wed Oct  8 22:16:53 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/RtmpauyNMT/fileb647760566ab/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 @ Wed Oct  8 22:16:54 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpauyNMT/fileb647760566ab/matched_reads.fastq.gz -> /tmp/RtmpauyNMT/fileb647760566ab/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 @ Wed Oct  8 22:16:54 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 @ Wed Oct  8 22:17:06 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/RtmpauyNMT/fileb647760566ab/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpauyNMT/fileb647760566ab/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/RtmpauyNMT/fileb647760566ab/matched_reads.fastq.gz -> /tmp/RtmpauyNMT/fileb647760566ab/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 @ Wed Oct  8 22:17:07 2025 ----------
22:17:07 Wed Oct 08 2025 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Inputs:  ['/tmp/RtmpauyNMT/fileb647318f6433/sample3_realign2transcript.bam', '/tmp/RtmpauyNMT/fileb647318f6433/sample2_realign2transcript.bam', '/tmp/RtmpauyNMT/fileb647318f6433/sample1_realign2transcript.bam'] /tmp/RtmpauyNMT/fileb647318f6433/transcript_assembly.fa.fai 5 0.4 0.4
	Counter({'counted_reads': 391, 'not_enough_coverage': 2})
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpauyNMT/fileb64737a047a3/config_file_46663.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 @ Wed Oct  8 22:17:08 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpauyNMT/fileb64737a047a3/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 @ Wed Oct  8 22:17:08 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpauyNMT/fileb64737a047a3/matched_reads.fastq.gz ->/tmp/RtmpauyNMT/fileb64737a047a3/align2genome.bam
-- Running step: isoform_identification @ Wed Oct  8 22:17:30 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 @ Wed Oct  8 22:17:42 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/RtmpauyNMT/fileb64737a047a3/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpauyNMT/fileb64737a047a3/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/RtmpauyNMT/fileb64737a047a3/matched_reads.fastq.gz ->/tmp/RtmpauyNMT/fileb64737a047a3/realign2transcript.bam
-- Running step: transcript_quantification @ Wed Oct  8 22:18:03 2025 ----------
22:18:03 Wed Oct 08 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/RtmpauyNMT/fileb64768b6c4a6/config_file_46663.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 @ Wed Oct  8 22:18:04 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpauyNMT/fileb64768b6c4a6/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 @ Wed Oct  8 22:18:05 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpauyNMT/fileb64768b6c4a6/matched_reads.fastq.gz -> /tmp/RtmpauyNMT/fileb64768b6c4a6/align2genome.bam
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: isoform_identification @ Wed Oct  8 22:18:05 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 @ Wed Oct  8 22:18:05 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/RtmpauyNMT/fileb64768b6c4a6/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpauyNMT/fileb64768b6c4a6/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/RtmpauyNMT/fileb64768b6c4a6/matched_reads.fastq.gz -> /tmp/RtmpauyNMT/fileb64768b6c4a6/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 @ Wed Oct  8 22:18:06 2025 ----------
2025-10-09T02:18:06.428094Z  INFO oarfish: setting user-provided filter parameters.
2025-10-09T02:18:06.431810Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpauyNMT/fileb64768b6c4a6/realign2transcript.bam, contains 10 reference sequences.
2025-10-09T02:18:06.431925Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-09T02:18:06.431989Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-09T02:18:06.433235Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-09T02:18:06.433290Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-09T02:18:06.453615Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpauyNMT/fileb6476ac756fb/config_file_46663.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 @ Wed Oct  8 22:18:07 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/RtmpauyNMT/fileb6476ac756fb/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 @ Wed Oct  8 22:18:07 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpauyNMT/fileb6476ac756fb/matched_reads.fastq.gz ->/tmp/RtmpauyNMT/fileb6476ac756fb/align2genome.bam
-- Running step: isoform_identification @ Wed Oct  8 22:18:33 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Wed Oct  8 22:18:33 2025 -------------------
Checking for fastq file(s) /Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpauyNMT/fileb6476ac756fb/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpauyNMT/fileb6476ac756fb/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/RtmpauyNMT/fileb6476ac756fb/matched_reads.fastq.gz ->/tmp/RtmpauyNMT/fileb6476ac756fb/realign2transcript.bam
-- Running step: transcript_quantification @ Wed Oct  8 22:18:55 2025 ----------
2025-10-09T02:18:55.381966Z  INFO oarfish: setting user-provided filter parameters.
2025-10-09T02:18:55.386781Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpauyNMT/fileb6476ac756fb/realign2transcript.bam, contains 10 reference sequences.
2025-10-09T02:18:55.386900Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-09T02:18:55.386981Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-09T02:18:55.389961Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-09T02:18:55.390093Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-09T02:18:55.418779Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpauyNMT/fileb64731ec11d8/config_file_46663.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 @ Wed Oct  8 22:18:56 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 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpauyNMT/fileb64731ec11d8/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 @ Wed Oct  8 22:18:57 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpauyNMT/fileb64731ec11d8/matched_reads.fastq.gz -> /tmp/RtmpauyNMT/fileb64731ec11d8/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 @ Wed Oct  8 22:18:57 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Wed Oct  8 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/RtmpauyNMT/fileb64731ec11d8/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpauyNMT/fileb64731ec11d8/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/RtmpauyNMT/fileb64731ec11d8/matched_reads.fastq.gz -> /tmp/RtmpauyNMT/fileb64731ec11d8/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 @ Wed Oct  8 22:18:58 2025 ----------
22:18:58 Wed Oct 08 2025 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpauyNMT/fileb64733ee3468/config_file_46663.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 @ Wed Oct  8 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/RtmpauyNMT/fileb64733ee3468/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 @ Wed Oct  8 22:19:00 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpauyNMT/fileb64733ee3468/matched_reads.fastq.gz ->/tmp/RtmpauyNMT/fileb64733ee3468/align2genome.bam
-- Running step: isoform_identification @ Wed Oct  8 22:19:21 2025 -------------
	Counter({'counted_reads': 368, 'unmapped': 4})
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Wed Oct  8 22:19:21 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/RtmpauyNMT/fileb64733ee3468/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpauyNMT/fileb64733ee3468/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/RtmpauyNMT/fileb64733ee3468/matched_reads.fastq.gz ->/tmp/RtmpauyNMT/fileb64733ee3468/realign2transcript.bam
-- Running step: transcript_quantification @ Wed Oct  8 22:19:43 2025 ----------
22:19:43 Wed Oct 08 2025 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpauyNMT/fileb6479137f94/config_file_46663.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 @ Wed Oct  8 22:19:45 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpauyNMT/fileb6479137f94/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpauyNMT/fileb6479137f94/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpauyNMT/fileb6479137f94/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpauyNMT/fileb6479137f94/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/RtmpauyNMT/fileb6479137f94/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpauyNMT/fileb6479137f94/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/RtmpauyNMT/fileb6479137f94/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpauyNMT/fileb6479137f94/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/RtmpauyNMT/fileb6479137f94/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpauyNMT/fileb6479137f94/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 @ Wed Oct  8 22:19:47 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpauyNMT/fileb6479137f94/sampleA_matched_reads.fastq.gz -> /tmp/RtmpauyNMT/fileb6479137f94/sampleA_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpauyNMT/fileb6479137f94/sample1_matched_reads.fastq.gz -> /tmp/RtmpauyNMT/fileb6479137f94/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpauyNMT/fileb6479137f94/sample2_matched_reads.fastq.gz -> /tmp/RtmpauyNMT/fileb6479137f94/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpauyNMT/fileb6479137f94/sample3_matched_reads.fastq.gz -> /tmp/RtmpauyNMT/fileb6479137f94/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Wed Oct  8 22:19:49 2025 ----------------
22:19:49 Wed Oct 08 2025 quantify genes 
Using BAM(s): '/tmp/RtmpauyNMT/fileb6479137f94/sampleA_align2genome.bam',
'/tmp/RtmpauyNMT/fileb6479137f94/sample1_align2genome.bam',
'/tmp/RtmpauyNMT/fileb6479137f94/sample2_align2genome.bam', and
'/tmp/RtmpauyNMT/fileb6479137f94/sample3_align2genome.bam'
	Counter({'counted_reads': 368, 'unmapped': 4})
parsing /tmp/RtmpauyNMT/fileb6479137f94/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 13.87gene_group/s]
2025-10-08 22:19:52.057 R[46663:492459099] XType: com.apple.fonts is not accessible.
2025-10-08 22:19:52.057 R[46663:492459099] 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, 231739.75Read/s]
parsing /tmp/RtmpauyNMT/fileb6479137f94/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 42.05gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1005153.37Read/s]
parsing /tmp/RtmpauyNMT/fileb6479137f94/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 35.54gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1250687.02Read/s]
parsing /tmp/RtmpauyNMT/fileb6479137f94/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 28.22gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 626389.49Read/s]
-- Running step: isoform_identification @ Wed Oct  8 22:19:53 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 @ Wed Oct  8 22:20:21 2025 -------------------
Checking for fastq file(s) /tmp/RtmpauyNMT/fileb6479137f94/fastq, /tmp/RtmpauyNMT/fileb6479137f94/fastq/sample1.fq.gz, /tmp/RtmpauyNMT/fileb6479137f94/fastq/sample2.fq.gz, /tmp/RtmpauyNMT/fileb6479137f94/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpauyNMT/fileb6479137f94/sampleA_matched_reads.fastq.gz, /tmp/RtmpauyNMT/fileb6479137f94/sample1_matched_reads.fastq.gz, /tmp/RtmpauyNMT/fileb6479137f94/sample2_matched_reads.fastq.gz, /tmp/RtmpauyNMT/fileb6479137f94/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpauyNMT/fileb6479137f94/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpauyNMT/fileb6479137f94/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpauyNMT/fileb6479137f94/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpauyNMT/fileb6479137f94/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpauyNMT/fileb6479137f94/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpauyNMT/fileb6479137f94/sampleA_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpauyNMT/fileb6479137f94/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpauyNMT/fileb6479137f94/sample1_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpauyNMT/fileb6479137f94/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpauyNMT/fileb6479137f94/sample2_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpauyNMT/fileb6479137f94/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpauyNMT/fileb6479137f94/sample3_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

-- Running step: transcript_quantification @ Wed Oct  8 22:20:22 2025 ----------
2025-10-09T02:20:22.773827Z  INFO oarfish: setting user-provided filter parameters.
2025-10-09T02:20:22.776969Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpauyNMT/fileb6479137f94/sampleA_realign2transcript.bam, contains 5 reference sequences.
2025-10-09T02:20:22.777025Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-09T02:20:22.777045Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-09T02:20:22.778419Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-09T02:20:22.778471Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-09T02:20:22.793500Z  INFO oarfish::single_cell: Processed 100 cells.
2025-10-09T02:20:23.210342Z  INFO oarfish: setting user-provided filter parameters.
2025-10-09T02:20:23.211409Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpauyNMT/fileb6479137f94/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-10-09T02:20:23.211462Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-09T02:20:23.211474Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-09T02:20:23.211617Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-09T02:20:23.211639Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-09T02:20:23.630787Z  INFO oarfish: setting user-provided filter parameters.
2025-10-09T02:20:23.631695Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpauyNMT/fileb6479137f94/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-10-09T02:20:23.631754Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-09T02:20:23.631773Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-09T02:20:23.632020Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-09T02:20:23.632067Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-09T02:20:24.079208Z  INFO oarfish: setting user-provided filter parameters.
2025-10-09T02:20:24.079946Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpauyNMT/fileb6479137f94/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-10-09T02:20:24.080000Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-09T02:20:24.080017Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-09T02:20:24.080151Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-09T02:20:24.080175Z  INFO oarfish: parsed reference information for 5 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpauyNMT/fileb64762ff8bdf/config_file_46663.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 @ Wed Oct  8 22:20:24 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 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpauyNMT/fileb64762ff8bdf/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpauyNMT/fileb64762ff8bdf/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpauyNMT/fileb64762ff8bdf/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpauyNMT/fileb64762ff8bdf/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/RtmpauyNMT/fileb64762ff8bdf/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpauyNMT/fileb64762ff8bdf/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/RtmpauyNMT/fileb64762ff8bdf/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpauyNMT/fileb64762ff8bdf/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/RtmpauyNMT/fileb64762ff8bdf/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpauyNMT/fileb64762ff8bdf/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 @ Wed Oct  8 22:20:26 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpauyNMT/fileb64762ff8bdf/sampleA_matched_reads.fastq.gz ->/tmp/RtmpauyNMT/fileb64762ff8bdf/sampleA_align2genome.bam
/tmp/RtmpauyNMT/fileb64762ff8bdf/sample1_matched_reads.fastq.gz ->/tmp/RtmpauyNMT/fileb64762ff8bdf/sample1_align2genome.bam
/tmp/RtmpauyNMT/fileb64762ff8bdf/sample2_matched_reads.fastq.gz ->/tmp/RtmpauyNMT/fileb64762ff8bdf/sample2_align2genome.bam
/tmp/RtmpauyNMT/fileb64762ff8bdf/sample3_matched_reads.fastq.gz ->/tmp/RtmpauyNMT/fileb64762ff8bdf/sample3_align2genome.bam
-- Running step: gene_quantification @ Wed Oct  8 22:20:49 2025 ----------------
22:20:49 Wed Oct 08 2025 quantify genes 
Using BAM(s): '/tmp/RtmpauyNMT/fileb64762ff8bdf/sampleA_align2genome.bam',
'/tmp/RtmpauyNMT/fileb64762ff8bdf/sample1_align2genome.bam',
'/tmp/RtmpauyNMT/fileb64762ff8bdf/sample2_align2genome.bam', and
'/tmp/RtmpauyNMT/fileb64762ff8bdf/sample3_align2genome.bam'
parsing /tmp/RtmpauyNMT/fileb64762ff8bdf/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 16.41gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 295681.70Read/s]
parsing /tmp/RtmpauyNMT/fileb64762ff8bdf/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 42.08gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1224399.81Read/s]
parsing /tmp/RtmpauyNMT/fileb64762ff8bdf/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 40.06gene_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, 1108548.47Read/s]
parsing /tmp/RtmpauyNMT/fileb64762ff8bdf/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 26.70gene_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, 571026.52Read/s]
-- Running step: isoform_identification @ Wed Oct  8 22:20:51 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 @ Wed Oct  8 22:21:19 2025 -------------------
Checking for fastq file(s) /tmp/RtmpauyNMT/fileb64762ff8bdf/fastq, /tmp/RtmpauyNMT/fileb64762ff8bdf/fastq/sample1.fq.gz, /tmp/RtmpauyNMT/fileb64762ff8bdf/fastq/sample2.fq.gz, /tmp/RtmpauyNMT/fileb64762ff8bdf/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpauyNMT/fileb64762ff8bdf/sampleA_matched_reads.fastq.gz, /tmp/RtmpauyNMT/fileb64762ff8bdf/sample1_matched_reads.fastq.gz, /tmp/RtmpauyNMT/fileb64762ff8bdf/sample2_matched_reads.fastq.gz, /tmp/RtmpauyNMT/fileb64762ff8bdf/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpauyNMT/fileb64762ff8bdf/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpauyNMT/fileb64762ff8bdf/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpauyNMT/fileb64762ff8bdf/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpauyNMT/fileb64762ff8bdf/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpauyNMT/fileb64762ff8bdf/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpauyNMT/fileb64762ff8bdf/sampleA_realign2transcript.bam
/tmp/RtmpauyNMT/fileb64762ff8bdf/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpauyNMT/fileb64762ff8bdf/sample1_realign2transcript.bam
/tmp/RtmpauyNMT/fileb64762ff8bdf/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpauyNMT/fileb64762ff8bdf/sample2_realign2transcript.bam
/tmp/RtmpauyNMT/fileb64762ff8bdf/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpauyNMT/fileb64762ff8bdf/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Wed Oct  8 22:21:41 2025 ----------
2025-10-09T02:21:41.820908Z  INFO oarfish: setting user-provided filter parameters.
2025-10-09T02:21:41.821806Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpauyNMT/fileb64762ff8bdf/sampleA_realign2transcript.bam, contains 5 reference sequences.
2025-10-09T02:21:41.821870Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-09T02:21:41.821887Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-09T02:21:41.822097Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-09T02:21:41.822164Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-09T02:21:41.830957Z  INFO oarfish::single_cell: Processed 100 cells.
2025-10-09T02:21:42.347713Z  INFO oarfish: setting user-provided filter parameters.
2025-10-09T02:21:42.348452Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpauyNMT/fileb64762ff8bdf/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-10-09T02:21:42.348507Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-09T02:21:42.348525Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-09T02:21:42.348673Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-09T02:21:42.348695Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-09T02:21:42.824142Z  INFO oarfish: setting user-provided filter parameters.
2025-10-09T02:21:42.824703Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpauyNMT/fileb64762ff8bdf/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-10-09T02:21:42.824740Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-09T02:21:42.824752Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-09T02:21:42.824853Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-09T02:21:42.824867Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-09T02:21:43.316052Z  INFO oarfish: setting user-provided filter parameters.
2025-10-09T02:21:43.316701Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpauyNMT/fileb64762ff8bdf/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-10-09T02:21:43.316758Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-09T02:21:43.316777Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-09T02:21:43.316929Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-09T02:21:43.316957Z  INFO oarfish: parsed reference information for 5 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpauyNMT/fileb64751f45fd4/config_file_46663.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 @ Wed Oct  8 22:21:44 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/RtmpauyNMT/fileb64751f45fd4/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpauyNMT/fileb64751f45fd4/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpauyNMT/fileb64751f45fd4/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpauyNMT/fileb64751f45fd4/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/RtmpauyNMT/fileb64751f45fd4/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpauyNMT/fileb64751f45fd4/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/RtmpauyNMT/fileb64751f45fd4/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpauyNMT/fileb64751f45fd4/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/RtmpauyNMT/fileb64751f45fd4/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpauyNMT/fileb64751f45fd4/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 @ Wed Oct  8 22:21:46 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpauyNMT/fileb64751f45fd4/sampleA_matched_reads.fastq.gz -> /tmp/RtmpauyNMT/fileb64751f45fd4/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/RtmpauyNMT/fileb64751f45fd4/sample1_matched_reads.fastq.gz -> /tmp/RtmpauyNMT/fileb64751f45fd4/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/RtmpauyNMT/fileb64751f45fd4/sample2_matched_reads.fastq.gz -> /tmp/RtmpauyNMT/fileb64751f45fd4/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/RtmpauyNMT/fileb64751f45fd4/sample3_matched_reads.fastq.gz -> /tmp/RtmpauyNMT/fileb64751f45fd4/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 @ Wed Oct  8 22:21:48 2025 ----------------
22:21:48 Wed Oct 08 2025 quantify genes 
Using BAM(s): '/tmp/RtmpauyNMT/fileb64751f45fd4/sampleA_align2genome.bam',
'/tmp/RtmpauyNMT/fileb64751f45fd4/sample1_align2genome.bam',
'/tmp/RtmpauyNMT/fileb64751f45fd4/sample2_align2genome.bam', and
'/tmp/RtmpauyNMT/fileb64751f45fd4/sample3_align2genome.bam'
parsing /tmp/RtmpauyNMT/fileb64751f45fd4/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 16.47gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 393890.54Read/s]
parsing /tmp/RtmpauyNMT/fileb64751f45fd4/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 36.75gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1232169.21Read/s]
parsing /tmp/RtmpauyNMT/fileb64751f45fd4/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 43.59gene_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, 1125564.62Read/s]
parsing /tmp/RtmpauyNMT/fileb64751f45fd4/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 29.79gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 652221.19Read/s]
-- Running step: isoform_identification @ Wed Oct  8 22:21:49 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 6 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Wed Oct  8 22:22:19 2025 -------------------
Checking for fastq file(s) /tmp/RtmpauyNMT/fileb64751f45fd4/fastq, /tmp/RtmpauyNMT/fileb64751f45fd4/fastq/sample1.fq.gz, /tmp/RtmpauyNMT/fileb64751f45fd4/fastq/sample2.fq.gz, /tmp/RtmpauyNMT/fileb64751f45fd4/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpauyNMT/fileb64751f45fd4/sampleA_matched_reads.fastq.gz, /tmp/RtmpauyNMT/fileb64751f45fd4/sample1_matched_reads.fastq.gz, /tmp/RtmpauyNMT/fileb64751f45fd4/sample2_matched_reads.fastq.gz, /tmp/RtmpauyNMT/fileb64751f45fd4/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpauyNMT/fileb64751f45fd4/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpauyNMT/fileb64751f45fd4/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpauyNMT/fileb64751f45fd4/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpauyNMT/fileb64751f45fd4/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpauyNMT/fileb64751f45fd4/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpauyNMT/fileb64751f45fd4/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/RtmpauyNMT/fileb64751f45fd4/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpauyNMT/fileb64751f45fd4/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/RtmpauyNMT/fileb64751f45fd4/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpauyNMT/fileb64751f45fd4/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/RtmpauyNMT/fileb64751f45fd4/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpauyNMT/fileb64751f45fd4/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 @ Wed Oct  8 22:22:20 2025 ----------
22:22:20 Wed Oct 08 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpauyNMT/fileb64751f45fd4/sample3_realign2transcript.bam...
parsing /tmp/RtmpauyNMT/fileb64751f45fd4/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpauyNMT/fileb64751f45fd4/sample3_realign2transcript.bamdone
parsing /tmp/RtmpauyNMT/fileb64751f45fd4/sampleA_realign2transcript.bam...
parsing /tmp/RtmpauyNMT/fileb64751f45fd4/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpauyNMT/fileb64751f45fd4/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpauyNMT/fileb64751f45fd4/sample2_realign2transcript.bam...
parsing /tmp/RtmpauyNMT/fileb64751f45fd4/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpauyNMT/fileb64751f45fd4/sample2_realign2transcript.bamdone
parsing /tmp/RtmpauyNMT/fileb64751f45fd4/sample1_realign2transcript.bam...
parsing /tmp/RtmpauyNMT/fileb64751f45fd4/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpauyNMT/fileb64751f45fd4/sample1_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpauyNMT/fileb647eb58797/config_file_46663.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 @ Wed Oct  8 22:22:24 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 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpauyNMT/fileb647eb58797/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpauyNMT/fileb647eb58797/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpauyNMT/fileb647eb58797/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpauyNMT/fileb647eb58797/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/RtmpauyNMT/fileb647eb58797/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpauyNMT/fileb647eb58797/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/RtmpauyNMT/fileb647eb58797/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpauyNMT/fileb647eb58797/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/RtmpauyNMT/fileb647eb58797/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpauyNMT/fileb647eb58797/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 @ Wed Oct  8 22:22:26 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpauyNMT/fileb647eb58797/sampleA_matched_reads.fastq.gz ->/tmp/RtmpauyNMT/fileb647eb58797/sampleA_align2genome.bam
/tmp/RtmpauyNMT/fileb647eb58797/sample1_matched_reads.fastq.gz ->/tmp/RtmpauyNMT/fileb647eb58797/sample1_align2genome.bam
/tmp/RtmpauyNMT/fileb647eb58797/sample2_matched_reads.fastq.gz ->/tmp/RtmpauyNMT/fileb647eb58797/sample2_align2genome.bam
/tmp/RtmpauyNMT/fileb647eb58797/sample3_matched_reads.fastq.gz ->/tmp/RtmpauyNMT/fileb647eb58797/sample3_align2genome.bam
-- Running step: gene_quantification @ Wed Oct  8 22:22:49 2025 ----------------
22:22:49 Wed Oct 08 2025 quantify genes 
Using BAM(s): '/tmp/RtmpauyNMT/fileb647eb58797/sampleA_align2genome.bam',
'/tmp/RtmpauyNMT/fileb647eb58797/sample1_align2genome.bam',
'/tmp/RtmpauyNMT/fileb647eb58797/sample2_align2genome.bam', and
'/tmp/RtmpauyNMT/fileb647eb58797/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/RtmpauyNMT/fileb647eb58797/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  7.71gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  7.70gene_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, 394572.34Read/s]
parsing /tmp/RtmpauyNMT/fileb647eb58797/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 39.06gene_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, 1396053.79Read/s]
parsing /tmp/RtmpauyNMT/fileb647eb58797/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 39.67gene_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, 1284075.43Read/s]
parsing /tmp/RtmpauyNMT/fileb647eb58797/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 26.32gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 616954.58Read/s]
-- Running step: isoform_identification @ Wed Oct  8 22:22:50 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 @ Wed Oct  8 22:23:19 2025 -------------------
Checking for fastq file(s) /tmp/RtmpauyNMT/fileb647eb58797/fastq, /tmp/RtmpauyNMT/fileb647eb58797/fastq/sample1.fq.gz, /tmp/RtmpauyNMT/fileb647eb58797/fastq/sample2.fq.gz, /tmp/RtmpauyNMT/fileb647eb58797/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpauyNMT/fileb647eb58797/sampleA_matched_reads.fastq.gz, /tmp/RtmpauyNMT/fileb647eb58797/sample1_matched_reads.fastq.gz, /tmp/RtmpauyNMT/fileb647eb58797/sample2_matched_reads.fastq.gz, /tmp/RtmpauyNMT/fileb647eb58797/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpauyNMT/fileb647eb58797/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpauyNMT/fileb647eb58797/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpauyNMT/fileb647eb58797/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpauyNMT/fileb647eb58797/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpauyNMT/fileb647eb58797/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpauyNMT/fileb647eb58797/sampleA_realign2transcript.bam
/tmp/RtmpauyNMT/fileb647eb58797/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpauyNMT/fileb647eb58797/sample1_realign2transcript.bam
/tmp/RtmpauyNMT/fileb647eb58797/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpauyNMT/fileb647eb58797/sample2_realign2transcript.bam
/tmp/RtmpauyNMT/fileb647eb58797/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpauyNMT/fileb647eb58797/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Wed Oct  8 22:23:42 2025 ----------
22:23:42 Wed Oct 08 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpauyNMT/fileb647eb58797/sample3_realign2transcript.bam...
parsing /tmp/RtmpauyNMT/fileb647eb58797/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpauyNMT/fileb647eb58797/sample3_realign2transcript.bamdone
parsing /tmp/RtmpauyNMT/fileb647eb58797/sampleA_realign2transcript.bam...
parsing /tmp/RtmpauyNMT/fileb647eb58797/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpauyNMT/fileb647eb58797/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpauyNMT/fileb647eb58797/sample2_realign2transcript.bam...
parsing /tmp/RtmpauyNMT/fileb647eb58797/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpauyNMT/fileb647eb58797/sample2_realign2transcript.bamdone
parsing /tmp/RtmpauyNMT/fileb647eb58797/sample1_realign2transcript.bam...
parsing /tmp/RtmpauyNMT/fileb647eb58797/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpauyNMT/fileb647eb58797/sample1_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpauyNMT/fileb6474f023904/config_file_46663.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 @ Wed Oct  8 22:23:45 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpauyNMT/fileb6474f023904/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpauyNMT/fileb6474f023904/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpauyNMT/fileb6474f023904/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpauyNMT/fileb6474f023904/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/RtmpauyNMT/fileb6474f023904/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpauyNMT/fileb6474f023904/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/RtmpauyNMT/fileb6474f023904/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpauyNMT/fileb6474f023904/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/RtmpauyNMT/fileb6474f023904/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpauyNMT/fileb6474f023904/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 @ Wed Oct  8 22:23:47 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpauyNMT/fileb6474f023904/sampleA_matched_reads.fastq.gz -> /tmp/RtmpauyNMT/fileb6474f023904/sampleA_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpauyNMT/fileb6474f023904/sample1_matched_reads.fastq.gz -> /tmp/RtmpauyNMT/fileb6474f023904/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpauyNMT/fileb6474f023904/sample2_matched_reads.fastq.gz -> /tmp/RtmpauyNMT/fileb6474f023904/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpauyNMT/fileb6474f023904/sample3_matched_reads.fastq.gz -> /tmp/RtmpauyNMT/fileb6474f023904/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Wed Oct  8 22:23:49 2025 ----------------
22:23:49 Wed Oct 08 2025 quantify genes 
Using BAM(s): '/tmp/RtmpauyNMT/fileb6474f023904/sampleA_align2genome.bam',
'/tmp/RtmpauyNMT/fileb6474f023904/sample1_align2genome.bam',
'/tmp/RtmpauyNMT/fileb6474f023904/sample2_align2genome.bam', and
'/tmp/RtmpauyNMT/fileb6474f023904/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/RtmpauyNMT/fileb6474f023904/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 15.17gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 369112.93Read/s]
parsing /tmp/RtmpauyNMT/fileb6474f023904/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 41.61gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 840070.50Read/s]
parsing /tmp/RtmpauyNMT/fileb6474f023904/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 38.87gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1063464.50Read/s]
parsing /tmp/RtmpauyNMT/fileb6474f023904/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 26.82gene_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, 674585.69Read/s]
-- Running step: isoform_identification @ Wed Oct  8 22:23:50 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Wed Oct  8 22:23:50 2025 -------------------
Checking for fastq file(s) /tmp/RtmpauyNMT/fileb6474f023904/fastq, /tmp/RtmpauyNMT/fileb6474f023904/fastq/sample1.fq.gz, /tmp/RtmpauyNMT/fileb6474f023904/fastq/sample2.fq.gz, /tmp/RtmpauyNMT/fileb6474f023904/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpauyNMT/fileb6474f023904/sampleA_matched_reads.fastq.gz, /tmp/RtmpauyNMT/fileb6474f023904/sample1_matched_reads.fastq.gz, /tmp/RtmpauyNMT/fileb6474f023904/sample2_matched_reads.fastq.gz, /tmp/RtmpauyNMT/fileb6474f023904/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpauyNMT/fileb6474f023904/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpauyNMT/fileb6474f023904/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpauyNMT/fileb6474f023904/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpauyNMT/fileb6474f023904/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpauyNMT/fileb6474f023904/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpauyNMT/fileb6474f023904/sampleA_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpauyNMT/fileb6474f023904/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpauyNMT/fileb6474f023904/sample1_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpauyNMT/fileb6474f023904/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpauyNMT/fileb6474f023904/sample2_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpauyNMT/fileb6474f023904/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpauyNMT/fileb6474f023904/sample3_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

-- Running step: transcript_quantification @ Wed Oct  8 22:23:53 2025 ----------
2025-10-09T02:23:53.495640Z  INFO oarfish: setting user-provided filter parameters.
2025-10-09T02:23:53.501610Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpauyNMT/fileb6474f023904/sampleA_realign2transcript.bam, contains 14 reference sequences.
2025-10-09T02:23:53.501718Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-09T02:23:53.501749Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-09T02:23:53.503655Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-09T02:23:53.503699Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-10-09T02:23:53.534258Z  INFO oarfish::single_cell: Processed 100 cells.
2025-10-09T02:23:54.312285Z  INFO oarfish: setting user-provided filter parameters.
2025-10-09T02:23:54.313027Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpauyNMT/fileb6474f023904/sample1_realign2transcript.bam, contains 14 reference sequences.
2025-10-09T02:23:54.313080Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-09T02:23:54.313101Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-09T02:23:54.313301Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-09T02:23:54.313335Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-10-09T02:23:55.058569Z  INFO oarfish: setting user-provided filter parameters.
2025-10-09T02:23:55.059592Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpauyNMT/fileb6474f023904/sample2_realign2transcript.bam, contains 14 reference sequences.
2025-10-09T02:23:55.059651Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-09T02:23:55.059671Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-09T02:23:55.059871Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-09T02:23:55.059900Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-10-09T02:23:55.842399Z  INFO oarfish: setting user-provided filter parameters.
2025-10-09T02:23:55.842971Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpauyNMT/fileb6474f023904/sample3_realign2transcript.bam, contains 14 reference sequences.
2025-10-09T02:23:55.843007Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-09T02:23:55.843020Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-09T02:23:55.843145Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-09T02:23:55.843162Z  INFO oarfish: parsed reference information for 14 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpauyNMT/fileb6474251fa84/config_file_46663.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 @ Wed Oct  8 22:23:57 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpauyNMT/fileb6474251fa84/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpauyNMT/fileb6474251fa84/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpauyNMT/fileb6474251fa84/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpauyNMT/fileb6474251fa84/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/RtmpauyNMT/fileb6474251fa84/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpauyNMT/fileb6474251fa84/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/RtmpauyNMT/fileb6474251fa84/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpauyNMT/fileb6474251fa84/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/RtmpauyNMT/fileb6474251fa84/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpauyNMT/fileb6474251fa84/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 @ Wed Oct  8 22:23:58 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpauyNMT/fileb6474251fa84/sampleA_matched_reads.fastq.gz ->/tmp/RtmpauyNMT/fileb6474251fa84/sampleA_align2genome.bam
/tmp/RtmpauyNMT/fileb6474251fa84/sample1_matched_reads.fastq.gz ->/tmp/RtmpauyNMT/fileb6474251fa84/sample1_align2genome.bam
/tmp/RtmpauyNMT/fileb6474251fa84/sample2_matched_reads.fastq.gz ->/tmp/RtmpauyNMT/fileb6474251fa84/sample2_align2genome.bam
/tmp/RtmpauyNMT/fileb6474251fa84/sample3_matched_reads.fastq.gz ->/tmp/RtmpauyNMT/fileb6474251fa84/sample3_align2genome.bam
-- Running step: gene_quantification @ Wed Oct  8 22:24:22 2025 ----------------
22:24:22 Wed Oct 08 2025 quantify genes 
Using BAM(s): '/tmp/RtmpauyNMT/fileb6474251fa84/sampleA_align2genome.bam',
'/tmp/RtmpauyNMT/fileb6474251fa84/sample1_align2genome.bam',
'/tmp/RtmpauyNMT/fileb6474251fa84/sample2_align2genome.bam', and
'/tmp/RtmpauyNMT/fileb6474251fa84/sample3_align2genome.bam'
parsing /tmp/RtmpauyNMT/fileb6474251fa84/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 14.17gene_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, 383798.54Read/s]
parsing /tmp/RtmpauyNMT/fileb6474251fa84/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 36.07gene_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, 914748.32Read/s]
parsing /tmp/RtmpauyNMT/fileb6474251fa84/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 42.38gene_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, 1154692.21Read/s]
parsing /tmp/RtmpauyNMT/fileb6474251fa84/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 25.74gene_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, 554098.50Read/s]
-- Running step: isoform_identification @ Wed Oct  8 22:24:23 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Wed Oct  8 22:24:23 2025 -------------------
Checking for fastq file(s) /tmp/RtmpauyNMT/fileb6474251fa84/fastq, /tmp/RtmpauyNMT/fileb6474251fa84/fastq/sample1.fq.gz, /tmp/RtmpauyNMT/fileb6474251fa84/fastq/sample2.fq.gz, /tmp/RtmpauyNMT/fileb6474251fa84/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpauyNMT/fileb6474251fa84/sampleA_matched_reads.fastq.gz, /tmp/RtmpauyNMT/fileb6474251fa84/sample1_matched_reads.fastq.gz, /tmp/RtmpauyNMT/fileb6474251fa84/sample2_matched_reads.fastq.gz, /tmp/RtmpauyNMT/fileb6474251fa84/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpauyNMT/fileb6474251fa84/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpauyNMT/fileb6474251fa84/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpauyNMT/fileb6474251fa84/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpauyNMT/fileb6474251fa84/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpauyNMT/fileb6474251fa84/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpauyNMT/fileb6474251fa84/sampleA_realign2transcript.bam
/tmp/RtmpauyNMT/fileb6474251fa84/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpauyNMT/fileb6474251fa84/sample1_realign2transcript.bam
/tmp/RtmpauyNMT/fileb6474251fa84/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpauyNMT/fileb6474251fa84/sample2_realign2transcript.bam
/tmp/RtmpauyNMT/fileb6474251fa84/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpauyNMT/fileb6474251fa84/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Wed Oct  8 22:24:47 2025 ----------
2025-10-09T02:24:47.189363Z  INFO oarfish: setting user-provided filter parameters.
2025-10-09T02:24:47.191139Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpauyNMT/fileb6474251fa84/sampleA_realign2transcript.bam, contains 14 reference sequences.
2025-10-09T02:24:47.191195Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-09T02:24:47.191219Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-09T02:24:47.191486Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-09T02:24:47.191525Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-10-09T02:24:47.207120Z  INFO oarfish::single_cell: Processed 100 cells.
2025-10-09T02:24:48.132742Z  INFO oarfish: setting user-provided filter parameters.
2025-10-09T02:24:48.133628Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpauyNMT/fileb6474251fa84/sample1_realign2transcript.bam, contains 14 reference sequences.
2025-10-09T02:24:48.133680Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-09T02:24:48.133733Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-09T02:24:48.134107Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-09T02:24:48.134151Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-10-09T02:24:48.961873Z  INFO oarfish: setting user-provided filter parameters.
2025-10-09T02:24:48.962652Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpauyNMT/fileb6474251fa84/sample2_realign2transcript.bam, contains 14 reference sequences.
2025-10-09T02:24:48.962929Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-09T02:24:48.962949Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-09T02:24:48.963105Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-09T02:24:48.963127Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-10-09T02:24:49.702561Z  INFO oarfish: setting user-provided filter parameters.
2025-10-09T02:24:49.703294Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpauyNMT/fileb6474251fa84/sample3_realign2transcript.bam, contains 14 reference sequences.
2025-10-09T02:24:49.703340Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-09T02:24:49.703360Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-09T02:24:49.703539Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-09T02:24:49.703565Z  INFO oarfish: parsed reference information for 14 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpauyNMT/fileb6477180c79f/config_file_46663.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 @ Wed Oct  8 22:24:51 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 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpauyNMT/fileb6477180c79f/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpauyNMT/fileb6477180c79f/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpauyNMT/fileb6477180c79f/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpauyNMT/fileb6477180c79f/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/RtmpauyNMT/fileb6477180c79f/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpauyNMT/fileb6477180c79f/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/RtmpauyNMT/fileb6477180c79f/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpauyNMT/fileb6477180c79f/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/RtmpauyNMT/fileb6477180c79f/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpauyNMT/fileb6477180c79f/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 @ Wed Oct  8 22:24:52 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpauyNMT/fileb6477180c79f/sampleA_matched_reads.fastq.gz -> /tmp/RtmpauyNMT/fileb6477180c79f/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/RtmpauyNMT/fileb6477180c79f/sample1_matched_reads.fastq.gz -> /tmp/RtmpauyNMT/fileb6477180c79f/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/RtmpauyNMT/fileb6477180c79f/sample2_matched_reads.fastq.gz -> /tmp/RtmpauyNMT/fileb6477180c79f/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/RtmpauyNMT/fileb6477180c79f/sample3_matched_reads.fastq.gz -> /tmp/RtmpauyNMT/fileb6477180c79f/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 @ Wed Oct  8 22:24:55 2025 ----------------
22:24:55 Wed Oct 08 2025 quantify genes 
Using BAM(s): '/tmp/RtmpauyNMT/fileb6477180c79f/sampleA_align2genome.bam',
'/tmp/RtmpauyNMT/fileb6477180c79f/sample1_align2genome.bam',
'/tmp/RtmpauyNMT/fileb6477180c79f/sample2_align2genome.bam', and
'/tmp/RtmpauyNMT/fileb6477180c79f/sample3_align2genome.bam'
parsing /tmp/RtmpauyNMT/fileb6477180c79f/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 16.80gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 363861.48Read/s]
parsing /tmp/RtmpauyNMT/fileb6477180c79f/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 37.39gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1074801.15Read/s]
parsing /tmp/RtmpauyNMT/fileb6477180c79f/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 37.39gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1139508.80Read/s]
parsing /tmp/RtmpauyNMT/fileb6477180c79f/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  6.42gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  6.41gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 486781.49Read/s]
-- Running step: isoform_identification @ Wed Oct  8 22:24:56 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Wed Oct  8 22:24:56 2025 -------------------
Checking for fastq file(s) /tmp/RtmpauyNMT/fileb6477180c79f/fastq, /tmp/RtmpauyNMT/fileb6477180c79f/fastq/sample1.fq.gz, /tmp/RtmpauyNMT/fileb6477180c79f/fastq/sample2.fq.gz, /tmp/RtmpauyNMT/fileb6477180c79f/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpauyNMT/fileb6477180c79f/sampleA_matched_reads.fastq.gz, /tmp/RtmpauyNMT/fileb6477180c79f/sample1_matched_reads.fastq.gz, /tmp/RtmpauyNMT/fileb6477180c79f/sample2_matched_reads.fastq.gz, /tmp/RtmpauyNMT/fileb6477180c79f/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpauyNMT/fileb6477180c79f/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpauyNMT/fileb6477180c79f/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpauyNMT/fileb6477180c79f/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpauyNMT/fileb6477180c79f/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpauyNMT/fileb6477180c79f/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpauyNMT/fileb6477180c79f/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/RtmpauyNMT/fileb6477180c79f/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpauyNMT/fileb6477180c79f/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/RtmpauyNMT/fileb6477180c79f/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpauyNMT/fileb6477180c79f/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/RtmpauyNMT/fileb6477180c79f/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpauyNMT/fileb6477180c79f/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 @ Wed Oct  8 22:24:58 2025 ----------
22:24:58 Wed Oct 08 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpauyNMT/fileb6477180c79f/sample3_realign2transcript.bam...
parsing /tmp/RtmpauyNMT/fileb6477180c79f/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpauyNMT/fileb6477180c79f/sample3_realign2transcript.bamdone
parsing /tmp/RtmpauyNMT/fileb6477180c79f/sampleA_realign2transcript.bam...
parsing /tmp/RtmpauyNMT/fileb6477180c79f/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpauyNMT/fileb6477180c79f/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpauyNMT/fileb6477180c79f/sample2_realign2transcript.bam...
parsing /tmp/RtmpauyNMT/fileb6477180c79f/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpauyNMT/fileb6477180c79f/sample2_realign2transcript.bamdone
parsing /tmp/RtmpauyNMT/fileb6477180c79f/sample1_realign2transcript.bam...
parsing /tmp/RtmpauyNMT/fileb6477180c79f/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpauyNMT/fileb6477180c79f/sample1_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpauyNMT/fileb6472806190b/config_file_46663.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 @ Wed Oct  8 22:25:02 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/RtmpauyNMT/fileb6472806190b/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpauyNMT/fileb6472806190b/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpauyNMT/fileb6472806190b/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpauyNMT/fileb6472806190b/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/RtmpauyNMT/fileb6472806190b/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpauyNMT/fileb6472806190b/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/RtmpauyNMT/fileb6472806190b/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpauyNMT/fileb6472806190b/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/RtmpauyNMT/fileb6472806190b/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpauyNMT/fileb6472806190b/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 @ Wed Oct  8 22:25:04 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpauyNMT/fileb6472806190b/sampleA_matched_reads.fastq.gz ->/tmp/RtmpauyNMT/fileb6472806190b/sampleA_align2genome.bam
/tmp/RtmpauyNMT/fileb6472806190b/sample1_matched_reads.fastq.gz ->/tmp/RtmpauyNMT/fileb6472806190b/sample1_align2genome.bam
/tmp/RtmpauyNMT/fileb6472806190b/sample2_matched_reads.fastq.gz ->/tmp/RtmpauyNMT/fileb6472806190b/sample2_align2genome.bam
/tmp/RtmpauyNMT/fileb6472806190b/sample3_matched_reads.fastq.gz ->/tmp/RtmpauyNMT/fileb6472806190b/sample3_align2genome.bam
-- Running step: gene_quantification @ Wed Oct  8 22:25:27 2025 ----------------
22:25:27 Wed Oct 08 2025 quantify genes 
Using BAM(s): '/tmp/RtmpauyNMT/fileb6472806190b/sampleA_align2genome.bam',
'/tmp/RtmpauyNMT/fileb6472806190b/sample1_align2genome.bam',
'/tmp/RtmpauyNMT/fileb6472806190b/sample2_align2genome.bam', and
'/tmp/RtmpauyNMT/fileb6472806190b/sample3_align2genome.bam'
	Counter({'counted_reads': 176})
	Counter({'counted_reads': 358})
	Counter({'counted_reads': 95})
	Counter({'counted_reads': 91})
parsing /tmp/RtmpauyNMT/fileb6472806190b/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 16.94gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 305849.96Read/s]
parsing /tmp/RtmpauyNMT/fileb6472806190b/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 37.84gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1167029.49Read/s]
parsing /tmp/RtmpauyNMT/fileb6472806190b/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 37.20gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 980344.05Read/s]
parsing /tmp/RtmpauyNMT/fileb6472806190b/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 27.98gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 666820.99Read/s]
-- Running step: isoform_identification @ Wed Oct  8 22:25:28 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Wed Oct  8 22:25:29 2025 -------------------
Checking for fastq file(s) /tmp/RtmpauyNMT/fileb6472806190b/fastq, /tmp/RtmpauyNMT/fileb6472806190b/fastq/sample1.fq.gz, /tmp/RtmpauyNMT/fileb6472806190b/fastq/sample2.fq.gz, /tmp/RtmpauyNMT/fileb6472806190b/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpauyNMT/fileb6472806190b/sampleA_matched_reads.fastq.gz, /tmp/RtmpauyNMT/fileb6472806190b/sample1_matched_reads.fastq.gz, /tmp/RtmpauyNMT/fileb6472806190b/sample2_matched_reads.fastq.gz, /tmp/RtmpauyNMT/fileb6472806190b/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpauyNMT/fileb6472806190b/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpauyNMT/fileb6472806190b/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpauyNMT/fileb6472806190b/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpauyNMT/fileb6472806190b/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpauyNMT/fileb6472806190b/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpauyNMT/fileb6472806190b/sampleA_realign2transcript.bam
/tmp/RtmpauyNMT/fileb6472806190b/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpauyNMT/fileb6472806190b/sample1_realign2transcript.bam
/tmp/RtmpauyNMT/fileb6472806190b/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpauyNMT/fileb6472806190b/sample2_realign2transcript.bam
/tmp/RtmpauyNMT/fileb6472806190b/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpauyNMT/fileb6472806190b/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Wed Oct  8 22:25:50 2025 ----------
22:25:50 Wed Oct 08 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpauyNMT/fileb6472806190b/sample3_realign2transcript.bam...
parsing /tmp/RtmpauyNMT/fileb6472806190b/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpauyNMT/fileb6472806190b/sample3_realign2transcript.bamdone
parsing /tmp/RtmpauyNMT/fileb6472806190b/sampleA_realign2transcript.bam...
parsing /tmp/RtmpauyNMT/fileb6472806190b/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpauyNMT/fileb6472806190b/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpauyNMT/fileb6472806190b/sample2_realign2transcript.bam...
parsing /tmp/RtmpauyNMT/fileb6472806190b/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpauyNMT/fileb6472806190b/sample2_realign2transcript.bamdone
parsing /tmp/RtmpauyNMT/fileb6472806190b/sample1_realign2transcript.bam...
parsing /tmp/RtmpauyNMT/fileb6472806190b/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpauyNMT/fileb6472806190b/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 
 861.176   71.337 1028.727 
/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
BulkPipeline4.5360.6525.632
MultiSampleSCPipeline14.044 2.14018.137
SingleCellPipeline3.9640.3213.223
add_gene_counts0.3980.0090.412
annotation_to_fasta0.2580.0070.267
blaze 8.424 1.25710.733
bulk_long_pipeline4.7051.1474.588
combine_sce1.0190.0891.120
config-set0.3130.1680.529
config0.2920.1720.504
controllers-set0.4510.0770.589
controllers0.3450.1630.542
convolution_filter0.0010.0000.000
create_config0.0120.0020.014
create_sce_from_dir7.4631.5647.837
create_se_from_dir3.7150.7864.890
cutadapt0.1690.0630.259
example_pipeline0.4380.0450.520
experiment3.0550.4633.821
filter_annotation0.0610.0050.067
filter_coverage1.4110.2671.796
find_barcode1.8100.2822.265
find_bin0.0060.0100.029
find_variants25.303 0.38625.161
get_coverage1.3870.2611.761
index_genome0.3150.1880.561
mutation_positions1.6210.0291.662
plot_coverage3.8430.3734.390
plot_demultiplex3.7640.5795.183
plot_demultiplex_raw2.2610.1392.602
plot_durations3.3900.5634.501
plot_isoform_heatmap10.063 0.57110.774
plot_isoform_reduced_dim29.259 0.50130.115
plot_isoforms4.5030.0534.615
resume_FLAMES3.2670.4894.308
run_FLAMES3.0430.5074.082
run_step1.4980.3021.994
sc_DTU_analysis10.558 1.36910.627
sc_gene_entropy1.7240.0602.122
sc_genotype4.0301.0934.027
sc_impute_transcript0.7850.0150.803
sc_long_multisample_pipeline15.717 3.10316.009
sc_long_pipeline5.4140.8555.005
sc_mutations3.3490.4373.280
sc_plot_genotype12.182 0.35911.612
show-FLAMESPipeline0.4090.0390.498
steps-set0.6260.0550.757
steps0.1980.0440.286
weight_transcripts0.0240.0210.045