Back to Multiple platform build/check report for BioC 3.22:   simplified   long
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This page was generated on 2025-10-04 12:03 -0400 (Sat, 04 Oct 2025).

HostnameOSArch (*)R versionInstalled pkgs
nebbiolo2Linux (Ubuntu 24.04.3 LTS)x86_644.5.1 Patched (2025-08-23 r88802) -- "Great Square Root" 4853
lconwaymacOS 12.7.1 Montereyx86_644.5.1 Patched (2025-09-10 r88807) -- "Great Square Root" 4640
kjohnson3macOS 13.7.7 Venturaarm644.5.1 Patched (2025-09-10 r88807) -- "Great Square Root" 4585
taishanLinux (openEuler 24.03 LTS)aarch644.5.0 (2025-04-11) -- "How About a Twenty-Six" 4576
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: 2025-10-03 13:45 -0400 (Fri, 03 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 nebbiolo2

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: /home/biocbuild/bbs-3.22-bioc/R/bin/R CMD check --install=check:FLAMES.install-out.txt --library=/home/biocbuild/bbs-3.22-bioc/R/site-library --timings FLAMES_2.3.5.tar.gz
StartedAt: 2025-10-03 23:48:55 -0400 (Fri, 03 Oct 2025)
EndedAt: 2025-10-04 00:10:58 -0400 (Sat, 04 Oct 2025)
EllapsedTime: 1322.5 seconds
RetCode: 0
Status:   OK  
CheckDir: FLAMES.Rcheck
Warnings: 0

Command output

##############################################################################
##############################################################################
###
### Running command:
###
###   /home/biocbuild/bbs-3.22-bioc/R/bin/R CMD check --install=check:FLAMES.install-out.txt --library=/home/biocbuild/bbs-3.22-bioc/R/site-library --timings FLAMES_2.3.5.tar.gz
###
##############################################################################
##############################################################################


* using log directory ‘/home/biocbuild/bbs-3.22-bioc/meat/FLAMES.Rcheck’
* using R version 4.5.1 Patched (2025-08-23 r88802)
* using platform: x86_64-pc-linux-gnu
* R was compiled by
    gcc (Ubuntu 13.3.0-6ubuntu2~24.04) 13.3.0
    GNU Fortran (Ubuntu 13.3.0-6ubuntu2~24.04) 13.3.0
* running under: Ubuntu 24.04.3 LTS
* using session charset: UTF-8
* 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 ‘/home/biocbuild/bbs-3.22-bioc/meat/FLAMES.Rcheck/00install.out’ for details.
* used C++ compiler: ‘g++ (Ubuntu 13.3.0-6ubuntu2~24.04) 13.3.0’
* checking C++ specification ... OK
* checking installed package size ... INFO
  installed size is  6.0Mb
  sub-directories of 1Mb or more:
    bin    1.1Mb
    data   1.8Mb
    libs   1.5Mb
* 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 loading without being on the library search path ... 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 ‘/home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/libs/FLAMES.so’:
  Found ‘abort’, possibly from ‘abort’ (C)
  Found ‘exit’, possibly from ‘exit’ (C)
  Found ‘stderr’, possibly from ‘stderr’ (C)
  Found ‘stdout’, possibly from ‘stdout’ (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     23.863  0.193  24.057
blaze                         4.415 18.654  12.735
find_variants                20.436  0.066  19.889
bulk_long_pipeline            2.331 13.356   2.484
sc_long_multisample_pipeline  8.126  7.033   8.446
sc_plot_genotype             11.609  0.704  11.142
MultiSampleSCPipeline         9.981  0.663  11.150
sc_DTU_analysis               7.110  2.108   7.027
plot_isoform_heatmap          7.113  0.108   7.220
create_sce_from_dir           3.593  2.575   3.834
sc_long_pipeline              3.223  2.220   2.938
* 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 re-building of vignette outputs ... OK
* checking PDF version of manual ... OK
* DONE

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


Installation output

FLAMES.Rcheck/00install.out

##############################################################################
##############################################################################
###
### Running command:
###
###   /home/biocbuild/bbs-3.22-bioc/R/bin/R CMD INSTALL FLAMES
###
##############################################################################
##############################################################################


* installing to library ‘/home/biocbuild/bbs-3.22-bioc/R/site-library’
* installing *source* package ‘FLAMES’ ...
** this is package ‘FLAMES’ version ‘2.3.5’
** using non-staged installation via StagedInstall field
** libs
using C++ compiler: ‘g++ (Ubuntu 13.3.0-6ubuntu2~24.04) 13.3.0’
using C++17
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c RcppExports.cpp -o RcppExports.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c RcppFunctions.cpp -o RcppFunctions.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c classes/BamRecord.cpp -o classes/BamRecord.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c classes/GFFRecord.cpp -o classes/GFFRecord.o
In file included from classes/GFFRecord.cpp:8:
classes/../utility/utility.h: In function ‘std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > parseSpace(const std::string&)’:
classes/../utility/utility.h:255:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  255 |         for (int i = 0; i < s.size(); i++) {
      |                         ~~^~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c classes/GeneAnnotationParser.cpp -o classes/GeneAnnotationParser.o
In file included from classes/GeneAnnotationParser.cpp:15:
classes/../utility/utility.h: In function ‘std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > parseSpace(const std::string&)’:
classes/../utility/utility.h:255:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  255 |         for (int i = 0; i < s.size(); i++) {
      |                         ~~^~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c classes/Isoforms.cpp -o classes/Isoforms.o
In file included from classes/Isoforms.cpp:16:
classes/../utility/utility.h: In function ‘std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > parseSpace(const std::string&)’:
classes/../utility/utility.h:255:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  255 |         for (int i = 0; i < s.size(); i++) {
      |                         ~~^~~~~~~~~~
classes/Isoforms.cpp: In member function ‘void Isoforms::update_all_splice()’:
classes/Isoforms.cpp:233:35: warning: comparison of integer expressions of different signedness: ‘std::vector<StartEndPair>::size_type’ {aka ‘long unsigned int’} and ‘int’ [-Wsign-compare]
  233 |                 if (blocks.size() >= (int)this->Min_sup_cnt) {
      |                     ~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~~~~~~
classes/Isoforms.cpp: In member function ‘void Isoforms::filter_TSS_TES(std::ofstream&, DoubleJunctions, float)’:
classes/Isoforms.cpp:368:33: warning: comparison of integer expressions of different signedness: ‘std::unordered_map<int, int>::size_type’ {aka ‘long unsigned int’} and ‘int’ [-Wsign-compare]
  368 |         if ((left_counts.size() < (int)this->Min_sup_cnt) ||
      |              ~~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~~~~~
classes/Isoforms.cpp:369:38: warning: comparison of integer expressions of different signedness: ‘std::unordered_map<int, int>::size_type’ {aka ‘long unsigned int’} and ‘int’ [-Wsign-compare]
  369 |                 (right_counts.size() < (int)this->Min_sup_cnt)) {
      |                  ~~~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~~~~~
classes/Isoforms.cpp: In member function ‘void Isoforms::match_known_annotation(const std::unordered_map<std::__cxx11::basic_string<char>, Junctions>&, const std::unordered_map<std::__cxx11::basic_string<char>, Pos>&, const std::unordered_map<std::__cxx11::basic_string<char>, std::vector<StartEndPair> >&, GeneBlocks, std::unordered_map<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> >)’:
classes/Isoforms.cpp:662:51: warning: comparison of integer expressions of different signedness: ‘int’ and ‘const long unsigned int’ [-Wsign-compare]
  662 |                                 for (int j = 0; j < std::min(raw_iso_key.size() - 1, junction.junctions.size()); j++) {
      |                                                 ~~^~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
classes/Isoforms.cpp:713:43: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<int>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  713 |                         for (int i = 0; i < new_exons.size(); ++i) {
      |                                         ~~^~~~~~~~~~~~~~~~~~
classes/Isoforms.cpp:725:46: warning: comparisons like ‘X<=Y<=Z’ do not have their mathematical meaning [-Wparentheses]
  725 |                                 } else if (0 < i < raw_iso_key.size() - 1) { // a site from the middle somewhere
      |                                            ~~^~~
classes/Isoforms.cpp: In member function ‘std::string Isoforms::isoform_to_gff3(float)’:
classes/Isoforms.cpp:1140:43: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<int>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
 1140 |                         for (int i = 0; i < exons.size(); i+=2) {
      |                                         ~~^~~~~~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c classes/junctions.cpp -o classes/junctions.o
In file included from classes/junctions.cpp:12:
classes/../utility/utility.h: In function ‘std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > parseSpace(const std::string&)’:
classes/../utility/utility.h:255:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  255 |         for (int i = 0; i < s.size(); i++) {
      |                         ~~^~~~~~~~~~
classes/junctions.cpp: In function ‘std::unordered_map<std::__cxx11::basic_string<char>, std::vector<StartEndPair> > get_gene_flat(const std::unordered_map<std::__cxx11::basic_string<char>, std::vector<std::__cxx11::basic_string<char> > >&, const std::unordered_map<std::__cxx11::basic_string<char>, std::vector<StartEndPair> >&)’:
classes/junctions.cpp:166:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<StartEndPair>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  166 |         for (int i = 1; i < exons.size(); i++) {
      |                         ~~^~~~~~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c main-functions/find_isoform.cpp -o main-functions/find_isoform.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c main-functions/flexiplex.cpp -o main-functions/flexiplex.o
main-functions/flexiplex.cpp: In function ‘unsigned int edit_distance(const std::string&, const std::string&, unsigned int&, int)’:
main-functions/flexiplex.cpp:123:21: warning: comparison of integer expressions of different signedness: ‘unsigned int’ and ‘int’ [-Wsign-compare]
  123 |       if (min_value <= max_editd)
      |           ~~~~~~~~~~^~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘std::string get_umi(const std::string&, const std::vector<std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > >&, const std::vector<int>&, int, int, bool, int, int)’:
main-functions/flexiplex.cpp:164:22: warning: comparison of integer expressions of different signedness: ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} and ‘int’ [-Wsign-compare]
  164 |     if (seq.length() < umi_start + umi_length) {
      |         ~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:186:36: warning: comparison of integer expressions of different signedness: ‘std::vector<int>::size_type’ {aka ‘long unsigned int’} and ‘int’ [-Wsign-compare]
  186 |     if (read_to_subpatterns.size() > umi_index + 1) {
      |         ~~~~~~~~~~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘Barcode get_barcode(const std::string&, const std::unordered_set<std::__cxx11::basic_string<char> >&, int, int, const std::vector<std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > >&)’:
main-functions/flexiplex.cpp:299:19: warning: comparison of integer expressions of different signedness: ‘int’ and ‘__gnu_cxx::__alloc_traits<std::allocator<long unsigned int>, long unsigned int>::value_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  299 |     if (i_pattern >= subpattern_ends[i_subpattern]) {
main-functions/flexiplex.cpp:358:22: warning: comparison of integer expressions of different signedness: ‘unsigned int’ and ‘int’ [-Wsign-compare]
  358 |     if (editDistance == barcode.editd) {
      |         ~~~~~~~~~~~~~^~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:360:29: warning: comparison of integer expressions of different signedness: ‘unsigned int’ and ‘int’ [-Wsign-compare]
  360 |     } else if (editDistance < barcode.editd &&
      |                ~~~~~~~~~~~~~^~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:361:29: warning: comparison of integer expressions of different signedness: ‘unsigned int’ and ‘int’ [-Wsign-compare]
  361 |                editDistance <= barcode_max_editd) { // if best so far, update
      |                ~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘std::vector<Barcode> big_barcode_search(const std::string&, const std::unordered_set<std::__cxx11::basic_string<char> >&, int, int, const std::vector<std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > >&)’:
main-functions/flexiplex.cpp:400:29: warning: comparison of integer expressions of different signedness: ‘const int’ and ‘const std::__cxx11::basic_string<char>::size_type’ {aka ‘const long unsigned int’} [-Wsign-compare]
  400 |       if (barcode.flank_end == std::string::npos) {
      |           ~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘void write_bgzfstring(BGZF*, const std::string&)’:
main-functions/flexiplex.cpp:423:21: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  423 |   if (bytes_written != str.size()) {
      |       ~~~~~~~~~~~~~~^~~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘void print_stats(const std::string&, const std::vector<Barcode>&, BGZF*)’:
main-functions/flexiplex.cpp:438:28: warning: comparison of integer expressions of different signedness: ‘const int’ and ‘const std::__cxx11::basic_string<char>::size_type’ {aka ‘const long unsigned int’} [-Wsign-compare]
  438 |         (barcode.flank_end == std::string::npos ? "True" : "False") + "\n";
      |          ~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘void print_read(const std::string&, const std::string&, const std::string&, const std::vector<Barcode>&, BGZF*, std::unordered_set<std::__cxx11::basic_string<char> >&, bool, bool, bool)’:
main-functions/flexiplex.cpp:475:21: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<Barcode>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  475 |   for (int b = 0; b < vec_bc.size(); b++) {
      |                   ~~^~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:490:32: warning: comparison of integer expressions of different signedness: ‘const int’ and ‘const std::__cxx11::basic_string<char>::size_type’ {aka ‘const long unsigned int’} [-Wsign-compare]
  490 |     if (vec_bc.at(b).flank_end == std::string::npos) {
      |         ~~~~~~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:495:23: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<Barcode>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  495 |     for (int f = 0; f < vec_bc.size(); f++) {
      |                     ~~^~~~~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘Rcpp::IntegerVector flexiplex_cpp(Rcpp::StringVector, Rcpp::String, bool, int, int, Rcpp::StringVector, Rcpp::String, Rcpp::String, Rcpp::String, bool, int)’:
main-functions/flexiplex.cpp:754:25: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<std::vector<SearchResult> >::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  754 |       for (int t = 0; t < sr_v.size();
      |                       ~~^~~~~~~~~~~~~
main-functions/flexiplex.cpp:759:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<SearchResult>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  759 |         for (int r = 0; r < sr_v[t].size(); r++) { // loop over the reads
      |                         ~~^~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:761:29: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<Barcode>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  761 |           for (int b = 0; b < sr_v[t][r].vec_bc_for.size(); b++)
      |                           ~~^~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:763:29: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<Barcode>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  763 |           for (int b = 0; b < sr_v[t][r].vec_bc_rev.size(); b++)
      |                           ~~^~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c main-functions/get_transcript_seq.cpp -o main-functions/get_transcript_seq.o
main-functions/get_transcript_seq.cpp: In function ‘void write_fa(const std::string&, const std::string&, const std::string&, int)’:
main-functions/get_transcript_seq.cpp:87:14: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
   87 |     while (i < seq.size()) {
      |            ~~^~~~~~~~~~~~
main-functions/get_transcript_seq.cpp:88:26: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
   88 |         if (i + wrap_len > seq.size()) {
      |             ~~~~~~~~~~~~~^~~~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c main-functions/group_bam2isoform.cpp -o main-functions/group_bam2isoform.o
In file included from main-functions/group_bam2isoform.cpp:18:
main-functions/../utility/utility.h: In function ‘std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > parseSpace(const std::string&)’:
main-functions/../utility/utility.h:255:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  255 |         for (int i = 0; i < s.size(); i++) {
      |                         ~~^~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c main-functions/pileup_readid.cpp -o main-functions/pileup_readid.o
main-functions/pileup_readid.cpp: In function ‘Rcpp::NumericMatrix variant_count_matrix_cpp(Rcpp::String, Rcpp::String, int, bool, bool)’:
main-functions/pileup_readid.cpp:268:21: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  268 |   for (int i = 0; i < BASES.size(); i++) {
      |                   ~~^~~~~~~~~~~~~~
main-functions/pileup_readid.cpp: In instantiation of ‘std::unordered_map<std::__cxx11::basic_string<char>, std::vector<std::vector<std::__cxx11::basic_string<char> > > > group_umis(const TableType&, int) [with TableType = std::unordered_map<std::__cxx11::basic_string<char>, std::unordered_map<std::__cxx11::basic_string<char>, std::unordered_map<std::__cxx11::basic_string<char>, unsigned int> > >]’:
main-functions/pileup_readid.cpp:342:30:   required from here
main-functions/pileup_readid.cpp:86:16: warning: unused variable ‘end’ [-Wunused-variable]
   86 |   unsigned int end;
      |                ^~~
main-functions/pileup_readid.cpp: In instantiation of ‘std::unordered_map<std::__cxx11::basic_string<char>, std::vector<std::vector<std::__cxx11::basic_string<char> > > > group_umis(const TableType&, int) [with TableType = std::unordered_map<std::__cxx11::basic_string<char>, std::unordered_map<std::__cxx11::basic_string<char>, std::array<unsigned int, 5> > >]’:
main-functions/pileup_readid.cpp:344:30:   required from here
main-functions/pileup_readid.cpp:86:16: warning: unused variable ‘end’ [-Wunused-variable]
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c tests/test-junctions.cpp -o tests/test-junctions.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c tests/test-parsing.cpp -o tests/test-parsing.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c utility/cigars.cpp -o utility/cigars.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c utility/edlib-1.2.7/edlib.cpp -o utility/edlib-1.2.7/edlib.o
gcc -std=gnu2x -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security -c utility/bam.c -o utility/bam.o
g++ -std=gnu++17 -shared -L/home/biocbuild/bbs-3.22-bioc/R/lib -L/usr/local/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 /home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/usrlib/libhts.a -lcurl -lbz2 -llzma -lz -L/home/biocbuild/bbs-3.22-bioc/R/lib -lR
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="-g -O2  -Wall -Werror=format-security -Wno-unused-result" minimap2)
make[1]: Entering directory '/home/biocbuild/bbs-3.22-bioc/meat/FLAMES/src/submodule/minimap2'
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  main.c -o main.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  kthread.c -o kthread.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  kalloc.c -o kalloc.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  misc.c -o misc.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  bseq.c -o bseq.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  sketch.c -o sketch.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  sdust.c -o sdust.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  options.c -o options.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  index.c -o index.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  lchain.c -o lchain.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  align.c -o align.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  hit.c -o hit.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  seed.c -o seed.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  jump.c -o jump.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  map.c -o map.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  format.c -o format.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  pe.c -o pe.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  esterr.c -o esterr.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  splitidx.c -o splitidx.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse2 -DHAVE_KALLOC  ksw2_ll_sse.c -o ksw2_ll_sse.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH  ksw2_extz2_sse.c -o ksw2_extz2_sse41.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH  ksw2_extd2_sse.c -o ksw2_extd2_sse41.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH  ksw2_exts2_sse.c -o ksw2_exts2_sse41.o
cc -c -g -O2  -Wall -Werror=format-security -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 -g -O2  -Wall -Werror=format-security -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 -g -O2  -Wall -Werror=format-security -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 -g -O2  -Wall -Werror=format-security -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
ar: `u' modifier ignored since `D' is the default (see `U')
cc -g -O2  -Wall -Werror=format-security -Wno-unused-result main.o -o minimap2 -L. -lminimap2 -lm -lz -lpthread
make[1]: Leaving directory '/home/biocbuild/bbs-3.22-bioc/meat/FLAMES/src/submodule/minimap2'
echo "Installing binary to /home/biocbuild/bbs-3.22-bioc/meat/FLAMES/src/../inst/bin"
Installing binary to /home/biocbuild/bbs-3.22-bioc/meat/FLAMES/src/../inst/bin
mkdir -p ../inst/bin
cp submodule/minimap2/minimap2 ../inst/bin/
Rust version too old for edition 2024, skipping oarfish installation.
installing to /home/biocbuild/bbs-3.22-bioc/R/site-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 (2025-08-23 r88802) -- "Great Square Root"
Copyright (C) 2025 The R Foundation for Statistical Computing
Platform: x86_64-pc-linux-gnu

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/Rtmp271HBk/file11b6825f419d66/config_file_1160834.json 
Writing configuration parameters to:  /tmp/Rtmp271HBk/file11b6825f419d66/config_file_1160834.json 
Writing configuration parameters to:  /tmp/Rtmp271HBk/file11b6825f419d66/config_file_1160834.json 
Writing configuration parameters to:  /tmp/Rtmp271HBk/file11b68255e488e3/config_file_1160834.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/Rtmp271HBk/file11b6823d4b9486/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-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/Rtmp271HBk/file11b682b1eaaa0/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-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/Rtmp271HBk/file11b682b1eaaa0/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp271HBk/file11b68255cd02a2/musc_rps24_1.fastq
Searching for barcodes...
Processing file: /tmp/Rtmp271HBk/file11b68255cd02a2/musc_rps24_2.fastq
Searching for barcodes...
Processing file: /tmp/Rtmp271HBk/file11b68255cd02a2/musc_rps24_3.fastq
Searching for barcodes...
Processing file: /tmp/Rtmp271HBk/file11b68255cd02a2/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/Rtmp271HBk/file11b6826ce59aae/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-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/Rtmp271HBk/file11b6822b04985a/config_file_1160834.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Fri Oct  3 23:57:59 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/Rtmp271HBk/file11b6822b04985a/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/Rtmp271HBk/file11b6822b04985a/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/Rtmp271HBk/file11b6822b04985a/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Fri Oct  3 23:58:01 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

  |                                                                            
  |                                                                      |   0%[23:58:07] 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.

[23:58:07] 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.

[23:58:08] WARNING: src/learner.cc:553: 
  If you are loading a serialized model (like pickle in Python, RDS in R) generated by
  older XGBoost, please export the model by calling `Booster.save_model` from that version
  first, then load it back in current version. See:

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

  for more details about differences between saving model and serializing.

[23:58:08] WARNING: src/learner.cc:553: 
  If you are loading a serialized model (like pickle in Python, RDS in R) generated by
  older XGBoost, please export the model by calling `Booster.save_model` from that version
  first, then load it back in current version. See:

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

  for more details about differences between saving model and serializing.

[23:58:09] 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.

[23:58:09] WARNING: src/learner.cc:553: 
  If you are loading a serialized model (like pickle in Python, RDS in R) generated by
  older XGBoost, please export the model by calling `Booster.save_model` from that version
  first, then load it back in current version. See:

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

  for more details about differences between saving model and serializing.


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

Detected 4 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Fri Oct  3 23:58:26 2025 -------------------
Realigning sample sample1 -> /tmp/Rtmp271HBk/file11b6822b04985a/sample1_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample2 -> /tmp/Rtmp271HBk/file11b6822b04985a/sample2_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample3 -> /tmp/Rtmp271HBk/file11b6822b04985a/sample3_realign2transcript.bam
Skipped sorting BAM files.

-- Running step: transcript_quantification @ Fri Oct  3 23:58:27 2025 ----------
2025-10-04T03:58:27.333137Z  INFO oarfish: setting user-provided filter parameters.
2025-10-04T03:58:27.333515Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp271HBk/file11b6822b04985a/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-10-04T03:58:27.333527Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-04T03:58:27.333531Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-04T03:58:27.333587Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-04T03:58:27.333593Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-04T03:58:27.335190Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-04T03:58:27.335320Z  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    │
╰─────────────────────────────────┴───────╯

2025-10-04T03:58:27.335342Z  INFO oarfish::bulk: Total number of alignment records : 116
2025-10-04T03:58:27.335356Z  INFO oarfish::bulk: number of aligned reads : 96
2025-10-04T03:58:27.335358Z  INFO oarfish::bulk: number of unique alignments : 86
2025-10-04T03:58:27.335987Z  INFO oarfish: oarfish completed successfully.
2025-10-04T03:58:27.343288Z  INFO oarfish: setting user-provided filter parameters.
2025-10-04T03:58:27.343740Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp271HBk/file11b6822b04985a/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-10-04T03:58:27.343748Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-04T03:58:27.343751Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-04T03:58:27.343807Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-04T03:58:27.343812Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-04T03:58:27.345433Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-04T03:58:27.345579Z  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    │
╰─────────────────────────────────┴───────╯

2025-10-04T03:58:27.345615Z  INFO oarfish::bulk: Total number of alignment records : 114
2025-10-04T03:58:27.345618Z  INFO oarfish::bulk: number of aligned reads : 95
2025-10-04T03:58:27.345620Z  INFO oarfish::bulk: number of unique alignments : 82
2025-10-04T03:58:27.346223Z  INFO oarfish: oarfish completed successfully.
2025-10-04T03:58:27.353609Z  INFO oarfish: setting user-provided filter parameters.
2025-10-04T03:58:27.354039Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp271HBk/file11b6822b04985a/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-10-04T03:58:27.354047Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-04T03:58:27.354050Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-04T03:58:27.354107Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-04T03:58:27.354113Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-04T03:58:27.356833Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2025-10-04T03:58:27.356988Z  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   │
╰─────────────────────────────────┴───────╯

2025-10-04T03:58:27.357025Z  INFO oarfish::bulk: Total number of alignment records : 239
2025-10-04T03:58:27.357027Z  INFO oarfish::bulk: number of aligned reads : 179
2025-10-04T03:58:27.357029Z  INFO oarfish::bulk: number of unique alignments : 143
2025-10-04T03:58:27.357739Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp271HBk/file11b68210755295/config_file_1160834.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Fri Oct  3 23:58:27 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/Rtmp271HBk/file11b68210755295/sample1_align2genome.bam
sample2 ->/tmp/Rtmp271HBk/file11b68210755295/sample2_align2genome.bam
sample3 ->/tmp/Rtmp271HBk/file11b68210755295/sample3_align2genome.bam
-- Running step: isoform_identification @ Fri Oct  3 23:58:46 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 4 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Fri Oct  3 23:59:07 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/Rtmp271HBk/file11b68210755295/sample1_realign2transcript.bam
sample2 ->/tmp/Rtmp271HBk/file11b68210755295/sample2_realign2transcript.bam
sample3 ->/tmp/Rtmp271HBk/file11b68210755295/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Fri Oct  3 23:59:27 2025 ----------
2025-10-04T03:59:27.675847Z  INFO oarfish: setting user-provided filter parameters.
2025-10-04T03:59:27.676231Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp271HBk/file11b68210755295/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-10-04T03:59:27.676253Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-04T03:59:27.676257Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-04T03:59:27.676306Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-04T03:59:27.676311Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-04T03:59:27.677797Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-04T03:59:27.677912Z  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    │
╰─────────────────────────────────┴───────╯

2025-10-04T03:59:27.677932Z  INFO oarfish::bulk: Total number of alignment records : 116
2025-10-04T03:59:27.677934Z  INFO oarfish::bulk: number of aligned reads : 96
2025-10-04T03:59:27.677936Z  INFO oarfish::bulk: number of unique alignments : 86
2025-10-04T03:59:27.678538Z  INFO oarfish: oarfish completed successfully.
2025-10-04T03:59:27.687458Z  INFO oarfish: setting user-provided filter parameters.
2025-10-04T03:59:27.687795Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp271HBk/file11b68210755295/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-10-04T03:59:27.687802Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-04T03:59:27.687805Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-04T03:59:27.687854Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-04T03:59:27.687859Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-04T03:59:27.689389Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-04T03:59:27.689522Z  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    │
╰─────────────────────────────────┴───────╯

2025-10-04T03:59:27.689547Z  INFO oarfish::bulk: Total number of alignment records : 114
2025-10-04T03:59:27.689559Z  INFO oarfish::bulk: number of aligned reads : 95
2025-10-04T03:59:27.689561Z  INFO oarfish::bulk: number of unique alignments : 82
2025-10-04T03:59:27.690141Z  INFO oarfish: oarfish completed successfully.
2025-10-04T03:59:27.699535Z  INFO oarfish: setting user-provided filter parameters.
2025-10-04T03:59:27.699902Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp271HBk/file11b68210755295/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-10-04T03:59:27.699912Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-04T03:59:27.699915Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-04T03:59:27.699974Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-04T03:59:27.699979Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-04T03:59:27.702650Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2025-10-04T03:59:27.702830Z  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   │
╰─────────────────────────────────┴───────╯

2025-10-04T03:59:27.702859Z  INFO oarfish::bulk: Total number of alignment records : 239
2025-10-04T03:59:27.702862Z  INFO oarfish::bulk: number of aligned reads : 179
2025-10-04T03:59:27.702864Z  INFO oarfish::bulk: number of unique alignments : 143
2025-10-04T03:59:27.703589Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp271HBk/file11b68245bb27ff/config_file_1160834.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Fri Oct  3 23:59:27 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/Rtmp271HBk/file11b68245bb27ff/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/Rtmp271HBk/file11b68245bb27ff/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/Rtmp271HBk/file11b68245bb27ff/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Fri Oct  3 23:59:28 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 4 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Fri Oct  3 23:59:48 2025 -------------------
Realigning sample sample1 -> /tmp/Rtmp271HBk/file11b68245bb27ff/sample1_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample2 -> /tmp/Rtmp271HBk/file11b68245bb27ff/sample2_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample3 -> /tmp/Rtmp271HBk/file11b68245bb27ff/sample3_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Fri Oct  3 23:59:48 2025 ----------
23:59:48 Fri Oct 03 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp271HBk/file11b68249802efd/config_file_1160834.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Fri Oct  3 23:59:49 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/Rtmp271HBk/file11b68249802efd/sample1_align2genome.bam
sample2 ->/tmp/Rtmp271HBk/file11b68249802efd/sample2_align2genome.bam
sample3 ->/tmp/Rtmp271HBk/file11b68249802efd/sample3_align2genome.bam
-- Running step: isoform_identification @ Sat Oct  4 00:00:09 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 4 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Sat Oct  4 00:00:28 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/Rtmp271HBk/file11b68249802efd/sample1_realign2transcript.bam
sample2 ->/tmp/Rtmp271HBk/file11b68249802efd/sample2_realign2transcript.bam
sample3 ->/tmp/Rtmp271HBk/file11b68249802efd/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Sat Oct  4 00:00:45 2025 ----------
00:00:45 Sat Oct 04 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Inputs:  ['/tmp/Rtmp271HBk/file11b68245bb27ff/sample1_realign2transcript.bam', '/tmp/Rtmp271HBk/file11b68245bb27ff/sample2_realign2transcript.bam', '/tmp/Rtmp271HBk/file11b68245bb27ff/sample3_realign2transcript.bam'] /tmp/Rtmp271HBk/file11b68245bb27ff/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/Rtmp271HBk/file11b68267e2ef7f/config_file_1160834.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 @ Sat Oct  4 00:00:46 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/Rtmp271HBk/file11b68267e2ef7f/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/Rtmp271HBk/file11b68267e2ef7f/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/Rtmp271HBk/file11b68267e2ef7f/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Sat Oct  4 00:00:47 2025 -------------
Inputs:  ['/tmp/Rtmp271HBk/file11b68249802efd/sample1_realign2transcript.bam', '/tmp/Rtmp271HBk/file11b68249802efd/sample2_realign2transcript.bam', '/tmp/Rtmp271HBk/file11b68249802efd/sample3_realign2transcript.bam'] /tmp/Rtmp271HBk/file11b68249802efd/transcript_assembly.fa.fai 5 0.4 0.4
	Counter({'counted_reads': 375, 'not_enough_coverage': 16, 'unmapped': 2})
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sat Oct  4 00:00:47 2025 -------------------
Realigning sample sample1 -> /tmp/Rtmp271HBk/file11b68267e2ef7f/sample1_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample2 -> /tmp/Rtmp271HBk/file11b68267e2ef7f/sample2_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample3 -> /tmp/Rtmp271HBk/file11b68267e2ef7f/sample3_realign2transcript.bam
Skipped sorting BAM files.

-- Running step: transcript_quantification @ Sat Oct  4 00:00:48 2025 ----------
2025-10-04T04:00:48.771238Z  INFO oarfish: setting user-provided filter parameters.
2025-10-04T04:00:48.771646Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp271HBk/file11b68267e2ef7f/sample1_realign2transcript.bam, contains 10 reference sequences.
2025-10-04T04:00:48.771656Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-04T04:00:48.771660Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-04T04:00:48.771732Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-04T04:00:48.771740Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-04T04:00:48.774396Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-04T04:00:48.774522Z  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    │
╰─────────────────────────────────┴───────╯

2025-10-04T04:00:48.774543Z  INFO oarfish::bulk: Total number of alignment records : 125
2025-10-04T04:00:48.774546Z  INFO oarfish::bulk: number of aligned reads : 98
2025-10-04T04:00:48.774548Z  INFO oarfish::bulk: number of unique alignments : 86
2025-10-04T04:00:48.775169Z  INFO oarfish: oarfish completed successfully.
2025-10-04T04:00:48.784491Z  INFO oarfish: setting user-provided filter parameters.
2025-10-04T04:00:48.784855Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp271HBk/file11b68267e2ef7f/sample2_realign2transcript.bam, contains 10 reference sequences.
2025-10-04T04:00:48.784864Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-04T04:00:48.784867Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-04T04:00:48.784941Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-04T04:00:48.784948Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-04T04:00:48.787651Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-04T04:00:48.787788Z  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    │
╰─────────────────────────────────┴───────╯

2025-10-04T04:00:48.787812Z  INFO oarfish::bulk: Total number of alignment records : 136
2025-10-04T04:00:48.787815Z  INFO oarfish::bulk: number of aligned reads : 97
2025-10-04T04:00:48.787817Z  INFO oarfish::bulk: number of unique alignments : 79
2025-10-04T04:00:48.788432Z  INFO oarfish: oarfish completed successfully.
2025-10-04T04:00:48.796931Z  INFO oarfish: setting user-provided filter parameters.
2025-10-04T04:00:48.797338Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp271HBk/file11b68267e2ef7f/sample3_realign2transcript.bam, contains 10 reference sequences.
2025-10-04T04:00:48.797348Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-04T04:00:48.797352Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-04T04:00:48.797414Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-04T04:00:48.797420Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-04T04:00:48.801701Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-04T04:00:48.801857Z  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   │
╰─────────────────────────────────┴───────╯

2025-10-04T04:00:48.801884Z  INFO oarfish::bulk: Total number of alignment records : 272
2025-10-04T04:00:48.801886Z  INFO oarfish::bulk: number of aligned reads : 187
2025-10-04T04:00:48.801888Z  INFO oarfish::bulk: number of unique alignments : 140
2025-10-04T04:00:48.802583Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp271HBk/file11b6821747a383/config_file_1160834.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 @ Sat Oct  4 00:00:48 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/Rtmp271HBk/file11b6821747a383/sample1_align2genome.bam
sample2 ->/tmp/Rtmp271HBk/file11b6821747a383/sample2_align2genome.bam
sample3 ->/tmp/Rtmp271HBk/file11b6821747a383/sample3_align2genome.bam
-- Running step: isoform_identification @ Sat Oct  4 00:01:07 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sat Oct  4 00:01:07 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/Rtmp271HBk/file11b6821747a383/sample1_realign2transcript.bam
sample2 ->/tmp/Rtmp271HBk/file11b6821747a383/sample2_realign2transcript.bam
sample3 ->/tmp/Rtmp271HBk/file11b6821747a383/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Sat Oct  4 00:01:26 2025 ----------
2025-10-04T04:01:26.066770Z  INFO oarfish: setting user-provided filter parameters.
2025-10-04T04:01:26.067152Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp271HBk/file11b6821747a383/sample1_realign2transcript.bam, contains 10 reference sequences.
2025-10-04T04:01:26.067164Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-04T04:01:26.067179Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-04T04:01:26.067251Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-04T04:01:26.067257Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-04T04:01:26.069876Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-04T04:01:26.069990Z  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    │
╰─────────────────────────────────┴───────╯

2025-10-04T04:01:26.070009Z  INFO oarfish::bulk: Total number of alignment records : 125
2025-10-04T04:01:26.070012Z  INFO oarfish::bulk: number of aligned reads : 98
2025-10-04T04:01:26.070014Z  INFO oarfish::bulk: number of unique alignments : 86
2025-10-04T04:01:26.070616Z  INFO oarfish: oarfish completed successfully.
2025-10-04T04:01:26.080187Z  INFO oarfish: setting user-provided filter parameters.
2025-10-04T04:01:26.080649Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp271HBk/file11b6821747a383/sample2_realign2transcript.bam, contains 10 reference sequences.
2025-10-04T04:01:26.080657Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-04T04:01:26.080661Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-04T04:01:26.080729Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-04T04:01:26.080735Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-04T04:01:26.083418Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-04T04:01:26.083551Z  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    │
╰─────────────────────────────────┴───────╯

2025-10-04T04:01:26.083575Z  INFO oarfish::bulk: Total number of alignment records : 136
2025-10-04T04:01:26.083578Z  INFO oarfish::bulk: number of aligned reads : 97
2025-10-04T04:01:26.083589Z  INFO oarfish::bulk: number of unique alignments : 79
2025-10-04T04:01:26.084196Z  INFO oarfish: oarfish completed successfully.
2025-10-04T04:01:26.093542Z  INFO oarfish: setting user-provided filter parameters.
2025-10-04T04:01:26.093890Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp271HBk/file11b6821747a383/sample3_realign2transcript.bam, contains 10 reference sequences.
2025-10-04T04:01:26.093898Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-04T04:01:26.093901Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-04T04:01:26.093968Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-04T04:01:26.093975Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-04T04:01:26.098263Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-04T04:01:26.098415Z  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   │
╰─────────────────────────────────┴───────╯

2025-10-04T04:01:26.098442Z  INFO oarfish::bulk: Total number of alignment records : 272
2025-10-04T04:01:26.098444Z  INFO oarfish::bulk: number of aligned reads : 187
2025-10-04T04:01:26.098446Z  INFO oarfish::bulk: number of unique alignments : 140
2025-10-04T04:01:26.099164Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp271HBk/file11b6822e451160/config_file_1160834.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 @ Sat Oct  4 00:01:26 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/Rtmp271HBk/file11b6822e451160/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/Rtmp271HBk/file11b6822e451160/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/Rtmp271HBk/file11b6822e451160/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Sat Oct  4 00:01:27 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sat Oct  4 00:01:27 2025 -------------------
Realigning sample sample1 -> /tmp/Rtmp271HBk/file11b6822e451160/sample1_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample2 -> /tmp/Rtmp271HBk/file11b6822e451160/sample2_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample3 -> /tmp/Rtmp271HBk/file11b6822e451160/sample3_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Sat Oct  4 00:01:28 2025 ----------
00:01:28 Sat Oct 04 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/Rtmp271HBk/file11b68295df5ae/config_file_1160834.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 @ Sat Oct  4 00:01:29 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/Rtmp271HBk/file11b68295df5ae/sample1_align2genome.bam
sample2 ->/tmp/Rtmp271HBk/file11b68295df5ae/sample2_align2genome.bam
sample3 ->/tmp/Rtmp271HBk/file11b68295df5ae/sample3_align2genome.bam
-- Running step: isoform_identification @ Sat Oct  4 00:01:48 2025 -------------
Inputs:  ['/tmp/Rtmp271HBk/file11b6822e451160/sample1_realign2transcript.bam', '/tmp/Rtmp271HBk/file11b6822e451160/sample2_realign2transcript.bam', '/tmp/Rtmp271HBk/file11b6822e451160/sample3_realign2transcript.bam'] /tmp/Rtmp271HBk/file11b6822e451160/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 @ Sat Oct  4 00:01:48 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/Rtmp271HBk/file11b68295df5ae/sample1_realign2transcript.bam
sample2 ->/tmp/Rtmp271HBk/file11b68295df5ae/sample2_realign2transcript.bam
sample3 ->/tmp/Rtmp271HBk/file11b68295df5ae/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Sat Oct  4 00:02:06 2025 ----------
00:02:06 Sat Oct 04 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/Rtmp271HBk/file11b6823efe0aa9/config_file_1160834.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 @ Sat Oct  4 00:02:07 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/Rtmp271HBk/file11b6823efe0aa9/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-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 @ Sat Oct  4 00:02:07 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmp271HBk/file11b6823efe0aa9/matched_reads.fastq.gz -> /tmp/Rtmp271HBk/file11b6823efe0aa9/align2genome.bam
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: isoform_identification @ Sat Oct  4 00:02:08 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---
Detected 2 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Sat Oct  4 00:02:18 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp271HBk/file11b6823efe0aa9/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp271HBk/file11b6823efe0aa9/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/Rtmp271HBk/file11b6823efe0aa9/matched_reads.fastq.gz -> /tmp/Rtmp271HBk/file11b6823efe0aa9/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 @ Sat Oct  4 00:02:18 2025 ----------
2025-10-04T04:02:18.485953Z  INFO oarfish: setting user-provided filter parameters.
2025-10-04T04:02:18.486428Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp271HBk/file11b6823efe0aa9/realign2transcript.bam, contains 5 reference sequences.
2025-10-04T04:02:18.486438Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-04T04:02:18.486440Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-04T04:02:18.486490Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-04T04:02:18.486495Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-04T04:02:18.492636Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp271HBk/file11b6822224d550/config_file_1160834.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 @ Sat Oct  4 00:02:18 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmp271HBk/file11b6822224d550/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-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 @ Sat Oct  4 00:02:19 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmp271HBk/file11b6822224d550/matched_reads.fastq.gz ->/tmp/Rtmp271HBk/file11b6822224d550/align2genome.bam
-- Running step: isoform_identification @ Sat Oct  4 00:02:36 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 @ Sat Oct  4 00:02:47 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp271HBk/file11b6822224d550/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp271HBk/file11b6822224d550/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/Rtmp271HBk/file11b6822224d550/matched_reads.fastq.gz ->/tmp/Rtmp271HBk/file11b6822224d550/realign2transcript.bam
-- Running step: transcript_quantification @ Sat Oct  4 00:03:04 2025 ----------
2025-10-04T04:03:04.424092Z  INFO oarfish: setting user-provided filter parameters.
2025-10-04T04:03:04.424527Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp271HBk/file11b6822224d550/realign2transcript.bam, contains 5 reference sequences.
2025-10-04T04:03:04.424538Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-04T04:03:04.424541Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-04T04:03:04.424597Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-04T04:03:04.424603Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-04T04:03:04.431387Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp271HBk/file11b682142cc7b8/config_file_1160834.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 @ Sat Oct  4 00:03:04 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/Rtmp271HBk/file11b682142cc7b8/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-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 @ Sat Oct  4 00:03:05 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmp271HBk/file11b682142cc7b8/matched_reads.fastq.gz -> /tmp/Rtmp271HBk/file11b682142cc7b8/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 @ Sat Oct  4 00:03:05 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 @ Sat Oct  4 00:03:15 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp271HBk/file11b682142cc7b8/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp271HBk/file11b682142cc7b8/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/Rtmp271HBk/file11b682142cc7b8/matched_reads.fastq.gz -> /tmp/Rtmp271HBk/file11b682142cc7b8/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 @ Sat Oct  4 00:03:15 2025 ----------
00:03:15 Sat Oct 04 2025 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Inputs:  ['/tmp/Rtmp271HBk/file11b68295df5ae/sample1_realign2transcript.bam', '/tmp/Rtmp271HBk/file11b68295df5ae/sample2_realign2transcript.bam', '/tmp/Rtmp271HBk/file11b68295df5ae/sample3_realign2transcript.bam'] /tmp/Rtmp271HBk/file11b68295df5ae/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/Rtmp271HBk/file11b6822ce4573f/config_file_1160834.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 @ Sat Oct  4 00:03:16 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmp271HBk/file11b6822ce4573f/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-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 @ Sat Oct  4 00:03:16 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmp271HBk/file11b6822ce4573f/matched_reads.fastq.gz ->/tmp/Rtmp271HBk/file11b6822ce4573f/align2genome.bam
-- Running step: isoform_identification @ Sat Oct  4 00:03:34 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---
Detected 2 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Sat Oct  4 00:03:44 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp271HBk/file11b6822ce4573f/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp271HBk/file11b6822ce4573f/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/Rtmp271HBk/file11b6822ce4573f/matched_reads.fastq.gz ->/tmp/Rtmp271HBk/file11b6822ce4573f/realign2transcript.bam
-- Running step: transcript_quantification @ Sat Oct  4 00:04:02 2025 ----------
00:04:02 Sat Oct 04 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/Rtmp271HBk/file11b6824fa5d84f/config_file_1160834.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 @ Sat Oct  4 00:04:02 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 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmp271HBk/file11b6824fa5d84f/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-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 @ Sat Oct  4 00:04:03 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmp271HBk/file11b6824fa5d84f/matched_reads.fastq.gz -> /tmp/Rtmp271HBk/file11b6824fa5d84f/align2genome.bam
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: isoform_identification @ Sat Oct  4 00:04:03 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 @ Sat Oct  4 00:04:03 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp271HBk/file11b6824fa5d84f/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp271HBk/file11b6824fa5d84f/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/Rtmp271HBk/file11b6824fa5d84f/matched_reads.fastq.gz -> /tmp/Rtmp271HBk/file11b6824fa5d84f/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 @ Sat Oct  4 00:04:04 2025 ----------
2025-10-04T04:04:04.012709Z  INFO oarfish: setting user-provided filter parameters.
2025-10-04T04:04:04.013099Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp271HBk/file11b6824fa5d84f/realign2transcript.bam, contains 10 reference sequences.
2025-10-04T04:04:04.013107Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-04T04:04:04.013110Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-04T04:04:04.013183Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-04T04:04:04.013193Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-04T04:04:04.022531Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp271HBk/file11b68267af8fa6/config_file_1160834.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 @ Sat Oct  4 00:04:04 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/Rtmp271HBk/file11b68267af8fa6/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-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 @ Sat Oct  4 00:04:05 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmp271HBk/file11b68267af8fa6/matched_reads.fastq.gz ->/tmp/Rtmp271HBk/file11b68267af8fa6/align2genome.bam
-- Running step: isoform_identification @ Sat Oct  4 00:04:22 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sat Oct  4 00:04:23 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp271HBk/file11b68267af8fa6/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp271HBk/file11b68267af8fa6/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/Rtmp271HBk/file11b68267af8fa6/matched_reads.fastq.gz ->/tmp/Rtmp271HBk/file11b68267af8fa6/realign2transcript.bam
-- Running step: transcript_quantification @ Sat Oct  4 00:04:40 2025 ----------
2025-10-04T04:04:40.910630Z  INFO oarfish: setting user-provided filter parameters.
2025-10-04T04:04:40.911026Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp271HBk/file11b68267af8fa6/realign2transcript.bam, contains 10 reference sequences.
2025-10-04T04:04:40.911038Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-04T04:04:40.911043Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-04T04:04:40.911253Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-04T04:04:40.911262Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-04T04:04:40.921253Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp271HBk/file11b682791aec01/config_file_1160834.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 @ Sat Oct  4 00:04:41 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmp271HBk/file11b682791aec01/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-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 @ Sat Oct  4 00:04:41 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmp271HBk/file11b682791aec01/matched_reads.fastq.gz -> /tmp/Rtmp271HBk/file11b682791aec01/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 @ Sat Oct  4 00:04:42 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sat Oct  4 00:04:42 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp271HBk/file11b682791aec01/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp271HBk/file11b682791aec01/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/Rtmp271HBk/file11b682791aec01/matched_reads.fastq.gz -> /tmp/Rtmp271HBk/file11b682791aec01/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 @ Sat Oct  4 00:04:42 2025 ----------
00:04:42 Sat Oct 04 2025 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp271HBk/file11b6825bd34517/config_file_1160834.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 @ Sat Oct  4 00:04:44 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 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmp271HBk/file11b6825bd34517/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-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 @ Sat Oct  4 00:04:44 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmp271HBk/file11b6825bd34517/matched_reads.fastq.gz ->/tmp/Rtmp271HBk/file11b6825bd34517/align2genome.bam
-- Running step: isoform_identification @ Sat Oct  4 00:05:01 2025 -------------
	Counter({'counted_reads': 368, 'unmapped': 4})
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sat Oct  4 00:05:01 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp271HBk/file11b6825bd34517/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp271HBk/file11b6825bd34517/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/Rtmp271HBk/file11b6825bd34517/matched_reads.fastq.gz ->/tmp/Rtmp271HBk/file11b6825bd34517/realign2transcript.bam
-- Running step: transcript_quantification @ Sat Oct  4 00:05:20 2025 ----------
00:05:20 Sat Oct 04 2025 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp271HBk/file11b682549b2b6d/config_file_1160834.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 @ Sat Oct  4 00:05:21 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/Rtmp271HBk/file11b682549b2b6d/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp271HBk/file11b682549b2b6d/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmp271HBk/file11b682549b2b6d/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmp271HBk/file11b682549b2b6d/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/Rtmp271HBk/file11b682549b2b6d/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp271HBk/file11b682549b2b6d/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/Rtmp271HBk/file11b682549b2b6d/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp271HBk/file11b682549b2b6d/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/Rtmp271HBk/file11b682549b2b6d/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp271HBk/file11b682549b2b6d/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 @ Sat Oct  4 00:05:22 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmp271HBk/file11b682549b2b6d/sampleA_matched_reads.fastq.gz -> /tmp/Rtmp271HBk/file11b682549b2b6d/sampleA_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmp271HBk/file11b682549b2b6d/sample1_matched_reads.fastq.gz -> /tmp/Rtmp271HBk/file11b682549b2b6d/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmp271HBk/file11b682549b2b6d/sample2_matched_reads.fastq.gz -> /tmp/Rtmp271HBk/file11b682549b2b6d/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmp271HBk/file11b682549b2b6d/sample3_matched_reads.fastq.gz -> /tmp/Rtmp271HBk/file11b682549b2b6d/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Sat Oct  4 00:05:24 2025 ----------------
00:05:24 Sat Oct 04 2025 quantify genes 
Using BAM(s): '/tmp/Rtmp271HBk/file11b682549b2b6d/sampleA_align2genome.bam',
'/tmp/Rtmp271HBk/file11b682549b2b6d/sample1_align2genome.bam',
'/tmp/Rtmp271HBk/file11b682549b2b6d/sample2_align2genome.bam', and
'/tmp/Rtmp271HBk/file11b682549b2b6d/sample3_align2genome.bam'
	Counter({'counted_reads': 368, 'unmapped': 4})
parsing /tmp/Rtmp271HBk/file11b682549b2b6d/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 20.79gene_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, 359372.13Read/s]
parsing /tmp/Rtmp271HBk/file11b682549b2b6d/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 50.54gene_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, 1344328.21Read/s]
parsing /tmp/Rtmp271HBk/file11b682549b2b6d/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 50.84gene_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, 1222401.49Read/s]
parsing /tmp/Rtmp271HBk/file11b682549b2b6d/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 35.41gene_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, 683735.00Read/s]
-- Running step: isoform_identification @ Sat Oct  4 00:05:25 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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  |                                                                            
  |==================                                                    |  25%
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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 @ Sat Oct  4 00:05:50 2025 -------------------
Checking for fastq file(s) /tmp/Rtmp271HBk/file11b682549b2b6d/fastq, /tmp/Rtmp271HBk/file11b682549b2b6d/fastq/sample1.fq.gz, /tmp/Rtmp271HBk/file11b682549b2b6d/fastq/sample2.fq.gz, /tmp/Rtmp271HBk/file11b682549b2b6d/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp271HBk/file11b682549b2b6d/sampleA_matched_reads.fastq.gz, /tmp/Rtmp271HBk/file11b682549b2b6d/sample1_matched_reads.fastq.gz, /tmp/Rtmp271HBk/file11b682549b2b6d/sample2_matched_reads.fastq.gz, /tmp/Rtmp271HBk/file11b682549b2b6d/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp271HBk/file11b682549b2b6d/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmp271HBk/file11b682549b2b6d/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmp271HBk/file11b682549b2b6d/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmp271HBk/file11b682549b2b6d/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/Rtmp271HBk/file11b682549b2b6d/sampleA_matched_reads_dedup.fastq.gz -> /tmp/Rtmp271HBk/file11b682549b2b6d/sampleA_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/Rtmp271HBk/file11b682549b2b6d/sample1_matched_reads_dedup.fastq.gz -> /tmp/Rtmp271HBk/file11b682549b2b6d/sample1_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/Rtmp271HBk/file11b682549b2b6d/sample2_matched_reads_dedup.fastq.gz -> /tmp/Rtmp271HBk/file11b682549b2b6d/sample2_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/Rtmp271HBk/file11b682549b2b6d/sample3_matched_reads_dedup.fastq.gz -> /tmp/Rtmp271HBk/file11b682549b2b6d/sample3_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

-- Running step: transcript_quantification @ Sat Oct  4 00:05:51 2025 ----------
2025-10-04T04:05:51.223863Z  INFO oarfish: setting user-provided filter parameters.
2025-10-04T04:05:51.224230Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp271HBk/file11b682549b2b6d/sampleA_realign2transcript.bam, contains 5 reference sequences.
2025-10-04T04:05:51.224252Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-04T04:05:51.224255Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-04T04:05:51.224310Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-04T04:05:51.224315Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-04T04:05:51.230247Z  INFO oarfish::single_cell: Processed 100 cells.
2025-10-04T04:05:51.508433Z  INFO oarfish: setting user-provided filter parameters.
2025-10-04T04:05:51.508921Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp271HBk/file11b682549b2b6d/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-10-04T04:05:51.508929Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-04T04:05:51.508932Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-04T04:05:51.508992Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-04T04:05:51.508997Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-04T04:05:51.785812Z  INFO oarfish: setting user-provided filter parameters.
2025-10-04T04:05:51.786729Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp271HBk/file11b682549b2b6d/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-10-04T04:05:51.786740Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-04T04:05:51.786743Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-04T04:05:51.786800Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-04T04:05:51.786805Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-04T04:05:52.063426Z  INFO oarfish: setting user-provided filter parameters.
2025-10-04T04:05:52.063933Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp271HBk/file11b682549b2b6d/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-10-04T04:05:52.063941Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-04T04:05:52.063944Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-04T04:05:52.063998Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-04T04:05:52.064003Z  INFO oarfish: parsed reference information for 5 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp271HBk/file11b68210e24c0d/config_file_1160834.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 @ Sat Oct  4 00:05:52 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmp271HBk/file11b68210e24c0d/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp271HBk/file11b68210e24c0d/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmp271HBk/file11b68210e24c0d/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmp271HBk/file11b68210e24c0d/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/Rtmp271HBk/file11b68210e24c0d/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp271HBk/file11b68210e24c0d/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/Rtmp271HBk/file11b68210e24c0d/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp271HBk/file11b68210e24c0d/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/Rtmp271HBk/file11b68210e24c0d/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp271HBk/file11b68210e24c0d/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 @ Sat Oct  4 00:05:53 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmp271HBk/file11b68210e24c0d/sampleA_matched_reads.fastq.gz ->/tmp/Rtmp271HBk/file11b68210e24c0d/sampleA_align2genome.bam
/tmp/Rtmp271HBk/file11b68210e24c0d/sample1_matched_reads.fastq.gz ->/tmp/Rtmp271HBk/file11b68210e24c0d/sample1_align2genome.bam
/tmp/Rtmp271HBk/file11b68210e24c0d/sample2_matched_reads.fastq.gz ->/tmp/Rtmp271HBk/file11b68210e24c0d/sample2_align2genome.bam
/tmp/Rtmp271HBk/file11b68210e24c0d/sample3_matched_reads.fastq.gz ->/tmp/Rtmp271HBk/file11b68210e24c0d/sample3_align2genome.bam
-- Running step: gene_quantification @ Sat Oct  4 00:06:12 2025 ----------------
00:06:12 Sat Oct 04 2025 quantify genes 
Using BAM(s): '/tmp/Rtmp271HBk/file11b68210e24c0d/sampleA_align2genome.bam',
'/tmp/Rtmp271HBk/file11b68210e24c0d/sample1_align2genome.bam',
'/tmp/Rtmp271HBk/file11b68210e24c0d/sample2_align2genome.bam', and
'/tmp/Rtmp271HBk/file11b68210e24c0d/sample3_align2genome.bam'
parsing /tmp/Rtmp271HBk/file11b68210e24c0d/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 21.68gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 346556.50Read/s]
parsing /tmp/Rtmp271HBk/file11b68210e24c0d/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 45.13gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1271155.29Read/s]
parsing /tmp/Rtmp271HBk/file11b68210e24c0d/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 48.81gene_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, 1124598.88Read/s]
parsing /tmp/Rtmp271HBk/file11b68210e24c0d/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 34.40gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 670659.42Read/s]
-- Running step: isoform_identification @ Sat Oct  4 00:06:13 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

  |                                                                            
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  |                                                                            
  |==================                                                    |  25%
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  |===================================                                   |  50%
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  |====================================================                  |  75%
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  |======================================================================| 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 @ Sat Oct  4 00:06:39 2025 -------------------
Checking for fastq file(s) /tmp/Rtmp271HBk/file11b68210e24c0d/fastq, /tmp/Rtmp271HBk/file11b68210e24c0d/fastq/sample1.fq.gz, /tmp/Rtmp271HBk/file11b68210e24c0d/fastq/sample2.fq.gz, /tmp/Rtmp271HBk/file11b68210e24c0d/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp271HBk/file11b68210e24c0d/sampleA_matched_reads.fastq.gz, /tmp/Rtmp271HBk/file11b68210e24c0d/sample1_matched_reads.fastq.gz, /tmp/Rtmp271HBk/file11b68210e24c0d/sample2_matched_reads.fastq.gz, /tmp/Rtmp271HBk/file11b68210e24c0d/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp271HBk/file11b68210e24c0d/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmp271HBk/file11b68210e24c0d/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmp271HBk/file11b68210e24c0d/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmp271HBk/file11b68210e24c0d/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/Rtmp271HBk/file11b68210e24c0d/sampleA_matched_reads_dedup.fastq.gz ->/tmp/Rtmp271HBk/file11b68210e24c0d/sampleA_realign2transcript.bam
/tmp/Rtmp271HBk/file11b68210e24c0d/sample1_matched_reads_dedup.fastq.gz ->/tmp/Rtmp271HBk/file11b68210e24c0d/sample1_realign2transcript.bam
/tmp/Rtmp271HBk/file11b68210e24c0d/sample2_matched_reads_dedup.fastq.gz ->/tmp/Rtmp271HBk/file11b68210e24c0d/sample2_realign2transcript.bam
/tmp/Rtmp271HBk/file11b68210e24c0d/sample3_matched_reads_dedup.fastq.gz ->/tmp/Rtmp271HBk/file11b68210e24c0d/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Sat Oct  4 00:07:00 2025 ----------
2025-10-04T04:07:00.300273Z  INFO oarfish: setting user-provided filter parameters.
2025-10-04T04:07:00.300772Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp271HBk/file11b68210e24c0d/sampleA_realign2transcript.bam, contains 5 reference sequences.
2025-10-04T04:07:00.300782Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-04T04:07:00.300786Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-04T04:07:00.300840Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-04T04:07:00.300846Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-04T04:07:00.307144Z  INFO oarfish::single_cell: Processed 100 cells.
2025-10-04T04:07:00.643562Z  INFO oarfish: setting user-provided filter parameters.
2025-10-04T04:07:00.644044Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp271HBk/file11b68210e24c0d/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-10-04T04:07:00.644055Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-04T04:07:00.644058Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-04T04:07:00.644122Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-04T04:07:00.644130Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-04T04:07:01.008454Z  INFO oarfish: setting user-provided filter parameters.
2025-10-04T04:07:01.008837Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp271HBk/file11b68210e24c0d/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-10-04T04:07:01.008848Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-04T04:07:01.008852Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-04T04:07:01.008909Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-04T04:07:01.008915Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-04T04:07:01.316625Z  INFO oarfish: setting user-provided filter parameters.
2025-10-04T04:07:01.317014Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp271HBk/file11b68210e24c0d/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-10-04T04:07:01.317025Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-04T04:07:01.317028Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-04T04:07:01.317086Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-04T04:07:01.317103Z  INFO oarfish: parsed reference information for 5 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp271HBk/file11b682233a1dce/config_file_1160834.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 @ Sat Oct  4 00:07:01 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/Rtmp271HBk/file11b682233a1dce/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp271HBk/file11b682233a1dce/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmp271HBk/file11b682233a1dce/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmp271HBk/file11b682233a1dce/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/Rtmp271HBk/file11b682233a1dce/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp271HBk/file11b682233a1dce/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/Rtmp271HBk/file11b682233a1dce/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp271HBk/file11b682233a1dce/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/Rtmp271HBk/file11b682233a1dce/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp271HBk/file11b682233a1dce/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 @ Sat Oct  4 00:07:02 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmp271HBk/file11b682233a1dce/sampleA_matched_reads.fastq.gz -> /tmp/Rtmp271HBk/file11b682233a1dce/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/Rtmp271HBk/file11b682233a1dce/sample1_matched_reads.fastq.gz -> /tmp/Rtmp271HBk/file11b682233a1dce/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/Rtmp271HBk/file11b682233a1dce/sample2_matched_reads.fastq.gz -> /tmp/Rtmp271HBk/file11b682233a1dce/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/Rtmp271HBk/file11b682233a1dce/sample3_matched_reads.fastq.gz -> /tmp/Rtmp271HBk/file11b682233a1dce/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 @ Sat Oct  4 00:07:04 2025 ----------------
00:07:04 Sat Oct 04 2025 quantify genes 
Using BAM(s): '/tmp/Rtmp271HBk/file11b682233a1dce/sampleA_align2genome.bam',
'/tmp/Rtmp271HBk/file11b682233a1dce/sample1_align2genome.bam',
'/tmp/Rtmp271HBk/file11b682233a1dce/sample2_align2genome.bam', and
'/tmp/Rtmp271HBk/file11b682233a1dce/sample3_align2genome.bam'
parsing /tmp/Rtmp271HBk/file11b682233a1dce/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 21.13gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 423821.19Read/s]
parsing /tmp/Rtmp271HBk/file11b682233a1dce/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 52.75gene_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, 1228992.03Read/s]
parsing /tmp/Rtmp271HBk/file11b682233a1dce/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 50.41gene_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, 1175401.86Read/s]
parsing /tmp/Rtmp271HBk/file11b682233a1dce/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 36.00gene_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, 791617.09Read/s]
-- Running step: isoform_identification @ Sat Oct  4 00:07:05 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

  |                                                                            
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  |                                                                            
  |==================                                                    |  25%
  |                                                                            
  |===================================                                   |  50%
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  |====================================================                  |  75%
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  |======================================================================| 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 @ Sat Oct  4 00:07:29 2025 -------------------
Checking for fastq file(s) /tmp/Rtmp271HBk/file11b682233a1dce/fastq, /tmp/Rtmp271HBk/file11b682233a1dce/fastq/sample1.fq.gz, /tmp/Rtmp271HBk/file11b682233a1dce/fastq/sample2.fq.gz, /tmp/Rtmp271HBk/file11b682233a1dce/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp271HBk/file11b682233a1dce/sampleA_matched_reads.fastq.gz, /tmp/Rtmp271HBk/file11b682233a1dce/sample1_matched_reads.fastq.gz, /tmp/Rtmp271HBk/file11b682233a1dce/sample2_matched_reads.fastq.gz, /tmp/Rtmp271HBk/file11b682233a1dce/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp271HBk/file11b682233a1dce/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmp271HBk/file11b682233a1dce/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmp271HBk/file11b682233a1dce/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmp271HBk/file11b682233a1dce/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/Rtmp271HBk/file11b682233a1dce/sampleA_matched_reads_dedup.fastq.gz -> /tmp/Rtmp271HBk/file11b682233a1dce/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/Rtmp271HBk/file11b682233a1dce/sample1_matched_reads_dedup.fastq.gz -> /tmp/Rtmp271HBk/file11b682233a1dce/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/Rtmp271HBk/file11b682233a1dce/sample2_matched_reads_dedup.fastq.gz -> /tmp/Rtmp271HBk/file11b682233a1dce/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/Rtmp271HBk/file11b682233a1dce/sample3_matched_reads_dedup.fastq.gz -> /tmp/Rtmp271HBk/file11b682233a1dce/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 @ Sat Oct  4 00:07:30 2025 ----------
00:07:30 Sat Oct 04 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/Rtmp271HBk/file11b682233a1dce/sampleA_realign2transcript.bam...
parsing /tmp/Rtmp271HBk/file11b682233a1dce/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmp271HBk/file11b682233a1dce/sampleA_realign2transcript.bamdone
parsing /tmp/Rtmp271HBk/file11b682233a1dce/sample1_realign2transcript.bam...
parsing /tmp/Rtmp271HBk/file11b682233a1dce/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmp271HBk/file11b682233a1dce/sample1_realign2transcript.bamdone
parsing /tmp/Rtmp271HBk/file11b682233a1dce/sample2_realign2transcript.bam...
parsing /tmp/Rtmp271HBk/file11b682233a1dce/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmp271HBk/file11b682233a1dce/sample2_realign2transcript.bamdone
parsing /tmp/Rtmp271HBk/file11b682233a1dce/sample3_realign2transcript.bam...
parsing /tmp/Rtmp271HBk/file11b682233a1dce/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmp271HBk/file11b682233a1dce/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp271HBk/file11b6826cb83f54/config_file_1160834.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 @ Sat Oct  4 00:07:31 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmp271HBk/file11b6826cb83f54/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp271HBk/file11b6826cb83f54/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmp271HBk/file11b6826cb83f54/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmp271HBk/file11b6826cb83f54/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/Rtmp271HBk/file11b6826cb83f54/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp271HBk/file11b6826cb83f54/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/Rtmp271HBk/file11b6826cb83f54/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp271HBk/file11b6826cb83f54/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/Rtmp271HBk/file11b6826cb83f54/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp271HBk/file11b6826cb83f54/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 @ Sat Oct  4 00:07:32 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmp271HBk/file11b6826cb83f54/sampleA_matched_reads.fastq.gz ->/tmp/Rtmp271HBk/file11b6826cb83f54/sampleA_align2genome.bam
/tmp/Rtmp271HBk/file11b6826cb83f54/sample1_matched_reads.fastq.gz ->/tmp/Rtmp271HBk/file11b6826cb83f54/sample1_align2genome.bam
/tmp/Rtmp271HBk/file11b6826cb83f54/sample2_matched_reads.fastq.gz ->/tmp/Rtmp271HBk/file11b6826cb83f54/sample2_align2genome.bam
/tmp/Rtmp271HBk/file11b6826cb83f54/sample3_matched_reads.fastq.gz ->/tmp/Rtmp271HBk/file11b6826cb83f54/sample3_align2genome.bam
-- Running step: gene_quantification @ Sat Oct  4 00:07:51 2025 ----------------
00:07:51 Sat Oct 04 2025 quantify genes 
Using BAM(s): '/tmp/Rtmp271HBk/file11b6826cb83f54/sampleA_align2genome.bam',
'/tmp/Rtmp271HBk/file11b6826cb83f54/sample1_align2genome.bam',
'/tmp/Rtmp271HBk/file11b6826cb83f54/sample2_align2genome.bam', and
'/tmp/Rtmp271HBk/file11b6826cb83f54/sample3_align2genome.bam'
	Counter({'counted_reads': 347, 'not_enough_coverage': 9, 'unmapped': 2})
	Counter({'counted_reads': 89, 'not_enough_coverage': 2})
	Counter({'counted_reads': 92, 'not_enough_coverage': 3})
	Counter({'counted_reads': 168, 'not_enough_coverage': 6, 'unmapped': 2})
parsing /tmp/Rtmp271HBk/file11b6826cb83f54/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 21.49gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 329854.98Read/s]
parsing /tmp/Rtmp271HBk/file11b6826cb83f54/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 39.77gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1096722.10Read/s]
parsing /tmp/Rtmp271HBk/file11b6826cb83f54/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 46.06gene_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, 1150006.58Read/s]
parsing /tmp/Rtmp271HBk/file11b6826cb83f54/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 35.45gene_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, 717220.25Read/s]
-- Running step: isoform_identification @ Sat Oct  4 00:07:52 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 @ Sat Oct  4 00:08:16 2025 -------------------
Checking for fastq file(s) /tmp/Rtmp271HBk/file11b6826cb83f54/fastq, /tmp/Rtmp271HBk/file11b6826cb83f54/fastq/sample1.fq.gz, /tmp/Rtmp271HBk/file11b6826cb83f54/fastq/sample2.fq.gz, /tmp/Rtmp271HBk/file11b6826cb83f54/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp271HBk/file11b6826cb83f54/sampleA_matched_reads.fastq.gz, /tmp/Rtmp271HBk/file11b6826cb83f54/sample1_matched_reads.fastq.gz, /tmp/Rtmp271HBk/file11b6826cb83f54/sample2_matched_reads.fastq.gz, /tmp/Rtmp271HBk/file11b6826cb83f54/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp271HBk/file11b6826cb83f54/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmp271HBk/file11b6826cb83f54/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmp271HBk/file11b6826cb83f54/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmp271HBk/file11b6826cb83f54/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/Rtmp271HBk/file11b6826cb83f54/sampleA_matched_reads_dedup.fastq.gz ->/tmp/Rtmp271HBk/file11b6826cb83f54/sampleA_realign2transcript.bam
/tmp/Rtmp271HBk/file11b6826cb83f54/sample1_matched_reads_dedup.fastq.gz ->/tmp/Rtmp271HBk/file11b6826cb83f54/sample1_realign2transcript.bam
/tmp/Rtmp271HBk/file11b6826cb83f54/sample2_matched_reads_dedup.fastq.gz ->/tmp/Rtmp271HBk/file11b6826cb83f54/sample2_realign2transcript.bam
/tmp/Rtmp271HBk/file11b6826cb83f54/sample3_matched_reads_dedup.fastq.gz ->/tmp/Rtmp271HBk/file11b6826cb83f54/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Sat Oct  4 00:08:35 2025 ----------
00:08:35 Sat Oct 04 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/Rtmp271HBk/file11b6826cb83f54/sampleA_realign2transcript.bam...
parsing /tmp/Rtmp271HBk/file11b6826cb83f54/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmp271HBk/file11b6826cb83f54/sampleA_realign2transcript.bamdone
parsing /tmp/Rtmp271HBk/file11b6826cb83f54/sample1_realign2transcript.bam...
parsing /tmp/Rtmp271HBk/file11b6826cb83f54/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmp271HBk/file11b6826cb83f54/sample1_realign2transcript.bamdone
parsing /tmp/Rtmp271HBk/file11b6826cb83f54/sample2_realign2transcript.bam...
parsing /tmp/Rtmp271HBk/file11b6826cb83f54/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmp271HBk/file11b6826cb83f54/sample2_realign2transcript.bamdone
parsing /tmp/Rtmp271HBk/file11b6826cb83f54/sample3_realign2transcript.bam...
parsing /tmp/Rtmp271HBk/file11b6826cb83f54/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmp271HBk/file11b6826cb83f54/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp271HBk/file11b68253b3b908/config_file_1160834.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 @ Sat Oct  4 00:08:37 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmp271HBk/file11b68253b3b908/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp271HBk/file11b68253b3b908/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmp271HBk/file11b68253b3b908/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmp271HBk/file11b68253b3b908/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/Rtmp271HBk/file11b68253b3b908/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp271HBk/file11b68253b3b908/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/Rtmp271HBk/file11b68253b3b908/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp271HBk/file11b68253b3b908/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/Rtmp271HBk/file11b68253b3b908/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp271HBk/file11b68253b3b908/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 @ Sat Oct  4 00:08:38 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmp271HBk/file11b68253b3b908/sampleA_matched_reads.fastq.gz -> /tmp/Rtmp271HBk/file11b68253b3b908/sampleA_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmp271HBk/file11b68253b3b908/sample1_matched_reads.fastq.gz -> /tmp/Rtmp271HBk/file11b68253b3b908/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmp271HBk/file11b68253b3b908/sample2_matched_reads.fastq.gz -> /tmp/Rtmp271HBk/file11b68253b3b908/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmp271HBk/file11b68253b3b908/sample3_matched_reads.fastq.gz -> /tmp/Rtmp271HBk/file11b68253b3b908/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Sat Oct  4 00:08:39 2025 ----------------
00:08:39 Sat Oct 04 2025 quantify genes 
Using BAM(s): '/tmp/Rtmp271HBk/file11b68253b3b908/sampleA_align2genome.bam',
'/tmp/Rtmp271HBk/file11b68253b3b908/sample1_align2genome.bam',
'/tmp/Rtmp271HBk/file11b68253b3b908/sample2_align2genome.bam', and
'/tmp/Rtmp271HBk/file11b68253b3b908/sample3_align2genome.bam'
	Counter({'counted_reads': 347, 'not_enough_coverage': 9, 'unmapped': 2})
	Counter({'counted_reads': 89, 'not_enough_coverage': 2})
	Counter({'counted_reads': 92, 'not_enough_coverage': 3})
	Counter({'counted_reads': 168, 'not_enough_coverage': 6, 'unmapped': 2})
parsing /tmp/Rtmp271HBk/file11b68253b3b908/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 22.11gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 356913.44Read/s]
parsing /tmp/Rtmp271HBk/file11b68253b3b908/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 49.51gene_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, 1282975.65Read/s]
parsing /tmp/Rtmp271HBk/file11b68253b3b908/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 43.71gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1211386.32Read/s]
parsing /tmp/Rtmp271HBk/file11b68253b3b908/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 32.29gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 681424.49Read/s]
-- Running step: isoform_identification @ Sat Oct  4 00:08:40 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sat Oct  4 00:08:41 2025 -------------------
Checking for fastq file(s) /tmp/Rtmp271HBk/file11b68253b3b908/fastq, /tmp/Rtmp271HBk/file11b68253b3b908/fastq/sample1.fq.gz, /tmp/Rtmp271HBk/file11b68253b3b908/fastq/sample2.fq.gz, /tmp/Rtmp271HBk/file11b68253b3b908/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp271HBk/file11b68253b3b908/sampleA_matched_reads.fastq.gz, /tmp/Rtmp271HBk/file11b68253b3b908/sample1_matched_reads.fastq.gz, /tmp/Rtmp271HBk/file11b68253b3b908/sample2_matched_reads.fastq.gz, /tmp/Rtmp271HBk/file11b68253b3b908/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp271HBk/file11b68253b3b908/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmp271HBk/file11b68253b3b908/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmp271HBk/file11b68253b3b908/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmp271HBk/file11b68253b3b908/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/Rtmp271HBk/file11b68253b3b908/sampleA_matched_reads_dedup.fastq.gz -> /tmp/Rtmp271HBk/file11b68253b3b908/sampleA_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/Rtmp271HBk/file11b68253b3b908/sample1_matched_reads_dedup.fastq.gz -> /tmp/Rtmp271HBk/file11b68253b3b908/sample1_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/Rtmp271HBk/file11b68253b3b908/sample2_matched_reads_dedup.fastq.gz -> /tmp/Rtmp271HBk/file11b68253b3b908/sample2_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/Rtmp271HBk/file11b68253b3b908/sample3_matched_reads_dedup.fastq.gz -> /tmp/Rtmp271HBk/file11b68253b3b908/sample3_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

-- Running step: transcript_quantification @ Sat Oct  4 00:08:42 2025 ----------
2025-10-04T04:08:42.991575Z  INFO oarfish: setting user-provided filter parameters.
2025-10-04T04:08:42.992017Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp271HBk/file11b68253b3b908/sampleA_realign2transcript.bam, contains 14 reference sequences.
2025-10-04T04:08:42.992026Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-04T04:08:42.992029Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-04T04:08:42.992112Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-04T04:08:42.992132Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-10-04T04:08:43.003969Z  INFO oarfish::single_cell: Processed 100 cells.
2025-10-04T04:08:43.544507Z  INFO oarfish: setting user-provided filter parameters.
2025-10-04T04:08:43.544903Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp271HBk/file11b68253b3b908/sample1_realign2transcript.bam, contains 14 reference sequences.
2025-10-04T04:08:43.544913Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-04T04:08:43.544916Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-04T04:08:43.545004Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-04T04:08:43.545012Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-10-04T04:08:44.108425Z  INFO oarfish: setting user-provided filter parameters.
2025-10-04T04:08:44.108778Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp271HBk/file11b68253b3b908/sample2_realign2transcript.bam, contains 14 reference sequences.
2025-10-04T04:08:44.108786Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-04T04:08:44.108789Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-04T04:08:44.108875Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-04T04:08:44.108883Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-10-04T04:08:44.662355Z  INFO oarfish: setting user-provided filter parameters.
2025-10-04T04:08:44.662738Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp271HBk/file11b68253b3b908/sample3_realign2transcript.bam, contains 14 reference sequences.
2025-10-04T04:08:44.662746Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-04T04:08:44.662749Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-04T04:08:44.662828Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-04T04:08:44.662835Z  INFO oarfish: parsed reference information for 14 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp271HBk/file11b6824e78538f/config_file_1160834.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 @ Sat Oct  4 00:08:45 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/Rtmp271HBk/file11b6824e78538f/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp271HBk/file11b6824e78538f/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmp271HBk/file11b6824e78538f/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmp271HBk/file11b6824e78538f/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/Rtmp271HBk/file11b6824e78538f/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp271HBk/file11b6824e78538f/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/Rtmp271HBk/file11b6824e78538f/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp271HBk/file11b6824e78538f/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/Rtmp271HBk/file11b6824e78538f/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp271HBk/file11b6824e78538f/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 @ Sat Oct  4 00:08:46 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmp271HBk/file11b6824e78538f/sampleA_matched_reads.fastq.gz ->/tmp/Rtmp271HBk/file11b6824e78538f/sampleA_align2genome.bam
/tmp/Rtmp271HBk/file11b6824e78538f/sample1_matched_reads.fastq.gz ->/tmp/Rtmp271HBk/file11b6824e78538f/sample1_align2genome.bam
/tmp/Rtmp271HBk/file11b6824e78538f/sample2_matched_reads.fastq.gz ->/tmp/Rtmp271HBk/file11b6824e78538f/sample2_align2genome.bam
/tmp/Rtmp271HBk/file11b6824e78538f/sample3_matched_reads.fastq.gz ->/tmp/Rtmp271HBk/file11b6824e78538f/sample3_align2genome.bam
-- Running step: gene_quantification @ Sat Oct  4 00:09:04 2025 ----------------
00:09:04 Sat Oct 04 2025 quantify genes 
Using BAM(s): '/tmp/Rtmp271HBk/file11b6824e78538f/sampleA_align2genome.bam',
'/tmp/Rtmp271HBk/file11b6824e78538f/sample1_align2genome.bam',
'/tmp/Rtmp271HBk/file11b6824e78538f/sample2_align2genome.bam', and
'/tmp/Rtmp271HBk/file11b6824e78538f/sample3_align2genome.bam'
parsing /tmp/Rtmp271HBk/file11b6824e78538f/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 16.98gene_group/s]
/home/biocbuild/bbs-3.22-bioc/R/site-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, 380981.72Read/s]
parsing /tmp/Rtmp271HBk/file11b6824e78538f/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 52.15gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1155965.16Read/s]
parsing /tmp/Rtmp271HBk/file11b6824e78538f/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 49.76gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1258945.85Read/s]
parsing /tmp/Rtmp271HBk/file11b6824e78538f/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 34.66gene_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, 735429.93Read/s]
-- Running step: isoform_identification @ Sat Oct  4 00:09:05 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sat Oct  4 00:09:05 2025 -------------------
Checking for fastq file(s) /tmp/Rtmp271HBk/file11b6824e78538f/fastq, /tmp/Rtmp271HBk/file11b6824e78538f/fastq/sample1.fq.gz, /tmp/Rtmp271HBk/file11b6824e78538f/fastq/sample2.fq.gz, /tmp/Rtmp271HBk/file11b6824e78538f/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp271HBk/file11b6824e78538f/sampleA_matched_reads.fastq.gz, /tmp/Rtmp271HBk/file11b6824e78538f/sample1_matched_reads.fastq.gz, /tmp/Rtmp271HBk/file11b6824e78538f/sample2_matched_reads.fastq.gz, /tmp/Rtmp271HBk/file11b6824e78538f/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp271HBk/file11b6824e78538f/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmp271HBk/file11b6824e78538f/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmp271HBk/file11b6824e78538f/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmp271HBk/file11b6824e78538f/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/Rtmp271HBk/file11b6824e78538f/sampleA_matched_reads_dedup.fastq.gz ->/tmp/Rtmp271HBk/file11b6824e78538f/sampleA_realign2transcript.bam
/tmp/Rtmp271HBk/file11b6824e78538f/sample1_matched_reads_dedup.fastq.gz ->/tmp/Rtmp271HBk/file11b6824e78538f/sample1_realign2transcript.bam
/tmp/Rtmp271HBk/file11b6824e78538f/sample2_matched_reads_dedup.fastq.gz ->/tmp/Rtmp271HBk/file11b6824e78538f/sample2_realign2transcript.bam
/tmp/Rtmp271HBk/file11b6824e78538f/sample3_matched_reads_dedup.fastq.gz ->/tmp/Rtmp271HBk/file11b6824e78538f/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Sat Oct  4 00:09:25 2025 ----------
2025-10-04T04:09:25.671391Z  INFO oarfish: setting user-provided filter parameters.
2025-10-04T04:09:25.671812Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp271HBk/file11b6824e78538f/sampleA_realign2transcript.bam, contains 14 reference sequences.
2025-10-04T04:09:25.671821Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-04T04:09:25.671825Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-04T04:09:25.671911Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-04T04:09:25.671919Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-10-04T04:09:25.684053Z  INFO oarfish::single_cell: Processed 100 cells.
2025-10-04T04:09:26.332667Z  INFO oarfish: setting user-provided filter parameters.
2025-10-04T04:09:26.333244Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp271HBk/file11b6824e78538f/sample1_realign2transcript.bam, contains 14 reference sequences.
2025-10-04T04:09:26.333255Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-04T04:09:26.333258Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-04T04:09:26.333334Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-04T04:09:26.333342Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-10-04T04:09:26.986377Z  INFO oarfish: setting user-provided filter parameters.
2025-10-04T04:09:26.986850Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp271HBk/file11b6824e78538f/sample2_realign2transcript.bam, contains 14 reference sequences.
2025-10-04T04:09:26.986859Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-04T04:09:26.986862Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-04T04:09:26.986955Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-04T04:09:26.986962Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-10-04T04:09:27.515554Z  INFO oarfish: setting user-provided filter parameters.
2025-10-04T04:09:27.515920Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp271HBk/file11b6824e78538f/sample3_realign2transcript.bam, contains 14 reference sequences.
2025-10-04T04:09:27.515929Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-04T04:09:27.515931Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-04T04:09:27.516011Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-04T04:09:27.516018Z  INFO oarfish: parsed reference information for 14 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp271HBk/file11b6821d204723/config_file_1160834.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 @ Sat Oct  4 00:09:28 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/Rtmp271HBk/file11b6821d204723/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp271HBk/file11b6821d204723/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmp271HBk/file11b6821d204723/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmp271HBk/file11b6821d204723/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/Rtmp271HBk/file11b6821d204723/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp271HBk/file11b6821d204723/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/Rtmp271HBk/file11b6821d204723/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp271HBk/file11b6821d204723/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/Rtmp271HBk/file11b6821d204723/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp271HBk/file11b6821d204723/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 @ Sat Oct  4 00:09:29 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmp271HBk/file11b6821d204723/sampleA_matched_reads.fastq.gz -> /tmp/Rtmp271HBk/file11b6821d204723/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/Rtmp271HBk/file11b6821d204723/sample1_matched_reads.fastq.gz -> /tmp/Rtmp271HBk/file11b6821d204723/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/Rtmp271HBk/file11b6821d204723/sample2_matched_reads.fastq.gz -> /tmp/Rtmp271HBk/file11b6821d204723/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/Rtmp271HBk/file11b6821d204723/sample3_matched_reads.fastq.gz -> /tmp/Rtmp271HBk/file11b6821d204723/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 @ Sat Oct  4 00:09:30 2025 ----------------
00:09:30 Sat Oct 04 2025 quantify genes 
Using BAM(s): '/tmp/Rtmp271HBk/file11b6821d204723/sampleA_align2genome.bam',
'/tmp/Rtmp271HBk/file11b6821d204723/sample1_align2genome.bam',
'/tmp/Rtmp271HBk/file11b6821d204723/sample2_align2genome.bam', and
'/tmp/Rtmp271HBk/file11b6821d204723/sample3_align2genome.bam'
parsing /tmp/Rtmp271HBk/file11b6821d204723/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 22.45gene_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, 360026.09Read/s]
parsing /tmp/Rtmp271HBk/file11b6821d204723/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  7.01gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  7.00gene_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, 1330005.07Read/s]
parsing /tmp/Rtmp271HBk/file11b6821d204723/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 51.23gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1171855.16Read/s]
parsing /tmp/Rtmp271HBk/file11b6821d204723/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 36.13gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 661603.89Read/s]
-- Running step: isoform_identification @ Sat Oct  4 00:09:31 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sat Oct  4 00:09:32 2025 -------------------
Checking for fastq file(s) /tmp/Rtmp271HBk/file11b6821d204723/fastq, /tmp/Rtmp271HBk/file11b6821d204723/fastq/sample1.fq.gz, /tmp/Rtmp271HBk/file11b6821d204723/fastq/sample2.fq.gz, /tmp/Rtmp271HBk/file11b6821d204723/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp271HBk/file11b6821d204723/sampleA_matched_reads.fastq.gz, /tmp/Rtmp271HBk/file11b6821d204723/sample1_matched_reads.fastq.gz, /tmp/Rtmp271HBk/file11b6821d204723/sample2_matched_reads.fastq.gz, /tmp/Rtmp271HBk/file11b6821d204723/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp271HBk/file11b6821d204723/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmp271HBk/file11b6821d204723/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmp271HBk/file11b6821d204723/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmp271HBk/file11b6821d204723/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/Rtmp271HBk/file11b6821d204723/sampleA_matched_reads_dedup.fastq.gz -> /tmp/Rtmp271HBk/file11b6821d204723/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/Rtmp271HBk/file11b6821d204723/sample1_matched_reads_dedup.fastq.gz -> /tmp/Rtmp271HBk/file11b6821d204723/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/Rtmp271HBk/file11b6821d204723/sample2_matched_reads_dedup.fastq.gz -> /tmp/Rtmp271HBk/file11b6821d204723/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/Rtmp271HBk/file11b6821d204723/sample3_matched_reads_dedup.fastq.gz -> /tmp/Rtmp271HBk/file11b6821d204723/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 @ Sat Oct  4 00:09:33 2025 ----------
00:09:33 Sat Oct 04 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/Rtmp271HBk/file11b6821d204723/sampleA_realign2transcript.bam...
parsing /tmp/Rtmp271HBk/file11b6821d204723/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmp271HBk/file11b6821d204723/sampleA_realign2transcript.bamdone
parsing /tmp/Rtmp271HBk/file11b6821d204723/sample1_realign2transcript.bam...
parsing /tmp/Rtmp271HBk/file11b6821d204723/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmp271HBk/file11b6821d204723/sample1_realign2transcript.bamdone
parsing /tmp/Rtmp271HBk/file11b6821d204723/sample2_realign2transcript.bam...
parsing /tmp/Rtmp271HBk/file11b6821d204723/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmp271HBk/file11b6821d204723/sample2_realign2transcript.bamdone
parsing /tmp/Rtmp271HBk/file11b6821d204723/sample3_realign2transcript.bam...
parsing /tmp/Rtmp271HBk/file11b6821d204723/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmp271HBk/file11b6821d204723/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp271HBk/file11b6822da5eb3e/config_file_1160834.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 @ Sat Oct  4 00:09:36 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmp271HBk/file11b6822da5eb3e/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp271HBk/file11b6822da5eb3e/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmp271HBk/file11b6822da5eb3e/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmp271HBk/file11b6822da5eb3e/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/Rtmp271HBk/file11b6822da5eb3e/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp271HBk/file11b6822da5eb3e/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/Rtmp271HBk/file11b6822da5eb3e/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp271HBk/file11b6822da5eb3e/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/Rtmp271HBk/file11b6822da5eb3e/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp271HBk/file11b6822da5eb3e/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 @ Sat Oct  4 00:09:36 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmp271HBk/file11b6822da5eb3e/sampleA_matched_reads.fastq.gz ->/tmp/Rtmp271HBk/file11b6822da5eb3e/sampleA_align2genome.bam
/tmp/Rtmp271HBk/file11b6822da5eb3e/sample1_matched_reads.fastq.gz ->/tmp/Rtmp271HBk/file11b6822da5eb3e/sample1_align2genome.bam
/tmp/Rtmp271HBk/file11b6822da5eb3e/sample2_matched_reads.fastq.gz ->/tmp/Rtmp271HBk/file11b6822da5eb3e/sample2_align2genome.bam
/tmp/Rtmp271HBk/file11b6822da5eb3e/sample3_matched_reads.fastq.gz ->/tmp/Rtmp271HBk/file11b6822da5eb3e/sample3_align2genome.bam
-- Running step: gene_quantification @ Sat Oct  4 00:09:55 2025 ----------------
00:09:55 Sat Oct 04 2025 quantify genes 
Using BAM(s): '/tmp/Rtmp271HBk/file11b6822da5eb3e/sampleA_align2genome.bam',
'/tmp/Rtmp271HBk/file11b6822da5eb3e/sample1_align2genome.bam',
'/tmp/Rtmp271HBk/file11b6822da5eb3e/sample2_align2genome.bam', and
'/tmp/Rtmp271HBk/file11b6822da5eb3e/sample3_align2genome.bam'
	Counter({'counted_reads': 358})
	Counter({'counted_reads': 91})
	Counter({'counted_reads': 95})
	Counter({'counted_reads': 176})
parsing /tmp/Rtmp271HBk/file11b6822da5eb3e/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 20.07gene_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, 388160.21Read/s]
parsing /tmp/Rtmp271HBk/file11b6822da5eb3e/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 51.29gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1301931.96Read/s]
parsing /tmp/Rtmp271HBk/file11b6822da5eb3e/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 51.96gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1276882.61Read/s]
parsing /tmp/Rtmp271HBk/file11b6822da5eb3e/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 36.41gene_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, 760278.42Read/s]
-- Running step: isoform_identification @ Sat Oct  4 00:09:56 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sat Oct  4 00:09:57 2025 -------------------
Checking for fastq file(s) /tmp/Rtmp271HBk/file11b6822da5eb3e/fastq, /tmp/Rtmp271HBk/file11b6822da5eb3e/fastq/sample1.fq.gz, /tmp/Rtmp271HBk/file11b6822da5eb3e/fastq/sample2.fq.gz, /tmp/Rtmp271HBk/file11b6822da5eb3e/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp271HBk/file11b6822da5eb3e/sampleA_matched_reads.fastq.gz, /tmp/Rtmp271HBk/file11b6822da5eb3e/sample1_matched_reads.fastq.gz, /tmp/Rtmp271HBk/file11b6822da5eb3e/sample2_matched_reads.fastq.gz, /tmp/Rtmp271HBk/file11b6822da5eb3e/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp271HBk/file11b6822da5eb3e/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmp271HBk/file11b6822da5eb3e/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmp271HBk/file11b6822da5eb3e/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmp271HBk/file11b6822da5eb3e/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/Rtmp271HBk/file11b6822da5eb3e/sampleA_matched_reads_dedup.fastq.gz ->/tmp/Rtmp271HBk/file11b6822da5eb3e/sampleA_realign2transcript.bam
/tmp/Rtmp271HBk/file11b6822da5eb3e/sample1_matched_reads_dedup.fastq.gz ->/tmp/Rtmp271HBk/file11b6822da5eb3e/sample1_realign2transcript.bam
/tmp/Rtmp271HBk/file11b6822da5eb3e/sample2_matched_reads_dedup.fastq.gz ->/tmp/Rtmp271HBk/file11b6822da5eb3e/sample2_realign2transcript.bam
/tmp/Rtmp271HBk/file11b6822da5eb3e/sample3_matched_reads_dedup.fastq.gz ->/tmp/Rtmp271HBk/file11b6822da5eb3e/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Sat Oct  4 00:10:15 2025 ----------
00:10:15 Sat Oct 04 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/Rtmp271HBk/file11b6822da5eb3e/sampleA_realign2transcript.bam...
parsing /tmp/Rtmp271HBk/file11b6822da5eb3e/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmp271HBk/file11b6822da5eb3e/sampleA_realign2transcript.bamdone
parsing /tmp/Rtmp271HBk/file11b6822da5eb3e/sample1_realign2transcript.bam...
parsing /tmp/Rtmp271HBk/file11b6822da5eb3e/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmp271HBk/file11b6822da5eb3e/sample1_realign2transcript.bamdone
parsing /tmp/Rtmp271HBk/file11b6822da5eb3e/sample2_realign2transcript.bam...
parsing /tmp/Rtmp271HBk/file11b6822da5eb3e/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmp271HBk/file11b6822da5eb3e/sample2_realign2transcript.bamdone
parsing /tmp/Rtmp271HBk/file11b6822da5eb3e/sample3_realign2transcript.bam...
parsing /tmp/Rtmp271HBk/file11b6822da5eb3e/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmp271HBk/file11b6822da5eb3e/sample3_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': 358})
	Counter({'counted_reads': 91})
	Counter({'counted_reads': 95})
	Counter({'counted_reads': 176})
> 
> proc.time()
    user   system  elapsed 
1694.556   44.722  758.023 

Example timings

FLAMES.Rcheck/FLAMES-Ex.timings

nameusersystemelapsed
BulkPipeline4.1990.1873.698
MultiSampleSCPipeline 9.981 0.66311.150
SingleCellPipeline2.8460.1471.839
add_gene_counts0.2760.0010.276
annotation_to_fasta0.1840.0030.188
blaze 4.41518.65412.735
bulk_long_pipeline 2.33113.356 2.484
combine_sce0.7070.0510.759
config-set0.1540.0170.170
config0.1560.0150.170
controllers-set0.3650.0290.398
controllers0.2240.0100.235
convolution_filter0.0010.0000.001
create_config0.0090.0010.011
create_sce_from_dir3.5932.5753.834
create_se_from_dir2.6260.1432.767
cutadapt0.1110.0200.130
example_pipeline0.3250.0110.337
experiment2.1450.0832.223
filter_annotation0.0430.0020.044
filter_coverage0.9850.0281.015
find_barcode1.7290.2061.941
find_bin0.0080.0000.008
find_variants20.436 0.06619.889
get_coverage0.9860.0391.028
index_genome0.1530.0070.162
mutation_positions1.4530.0001.453
plot_coverage2.6650.0522.720
plot_demultiplex2.5180.1122.661
plot_demultiplex_raw1.5830.0431.630
plot_durations2.4920.0602.551
plot_isoform_heatmap7.1130.1087.220
plot_isoform_reduced_dim23.863 0.19324.057
plot_isoforms3.1650.0013.166
resume_FLAMES2.2870.0692.354
run_FLAMES2.1890.0712.256
run_step1.0200.0331.055
sc_DTU_analysis7.1102.1087.027
sc_gene_entropy1.6710.1441.975
sc_genotype2.9490.6452.535
sc_impute_transcript0.5610.0120.573
sc_long_multisample_pipeline8.1267.0338.446
sc_long_pipeline3.2232.2202.938
sc_mutations2.9450.5092.890
sc_plot_genotype11.609 0.70411.142
show-FLAMESPipeline0.2930.0060.299
steps-set0.4420.0320.473
steps0.1420.0130.156
weight_transcripts0.0260.0020.029