Back to Multiple platform build/check report for BioC 3.22:   simplified   long
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This page was generated on 2025-10-18 12:03 -0400 (Sat, 18 Oct 2025).

HostnameOSArch (*)R versionInstalled pkgs
nebbiolo2Linux (Ubuntu 24.04.3 LTS)x86_644.5.1 Patched (2025-08-23 r88802) -- "Great Square Root" 4887
lconwaymacOS 12.7.6 Montereyx86_644.5.1 Patched (2025-09-10 r88807) -- "Great Square Root" 4677
kjohnson3macOS 13.7.7 Venturaarm644.5.1 Patched (2025-09-10 r88807) -- "Great Square Root" 4622
taishanLinux (openEuler 24.03 LTS)aarch644.5.0 (2025-04-11) -- "How About a Twenty-Six" 4632
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 740/2353HostnameOS / ArchINSTALLBUILDCHECKBUILD BIN
FLAMES 2.3.5  (landing page)
Changqing Wang
Snapshot Date: 2025-10-17 13:45 -0400 (Fri, 17 Oct 2025)
git_url: https://git.bioconductor.org/packages/FLAMES
git_branch: devel
git_last_commit: 5771592
git_last_commit_date: 2025-09-12 02:43:50 -0400 (Fri, 12 Sep 2025)
nebbiolo2Linux (Ubuntu 24.04.3 LTS) / x86_64  OK    OK    OK  UNNEEDED, same version is already published
lconwaymacOS 12.7.6 Monterey / x86_64  OK    OK    OK    OK  UNNEEDED, same version is already published
kjohnson3macOS 13.7.7 Ventura / arm64  OK    OK    OK    OK  UNNEEDED, same version is already published
taishanLinux (openEuler 24.03 LTS) / aarch64  ERROR    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: 2025-10-17 23:49:37 -0400 (Fri, 17 Oct 2025)
EndedAt: 2025-10-18 00:10:43 -0400 (Sat, 18 Oct 2025)
EllapsedTime: 1265.3 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     22.864  0.284  23.146
blaze                         4.336 16.939  11.813
find_variants                19.130  0.098  18.616
sc_long_multisample_pipeline  8.100  6.576   8.165
bulk_long_pipeline            2.359 12.037   2.441
sc_plot_genotype             10.591  0.753  10.197
MultiSampleSCPipeline         9.797  0.641  10.799
sc_DTU_analysis               6.744  1.767   6.518
plot_isoform_heatmap          6.781  0.115   6.896
create_sce_from_dir           3.390  2.193   3.511
* 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/Rtmpb249bZ/file3cbb945cfd09b9/config_file_3980180.json 
Writing configuration parameters to:  /tmp/Rtmpb249bZ/file3cbb945cfd09b9/config_file_3980180.json 
Writing configuration parameters to:  /tmp/Rtmpb249bZ/file3cbb945cfd09b9/config_file_3980180.json 
Writing configuration parameters to:  /tmp/Rtmpb249bZ/file3cbb942244c5d7/config_file_3980180.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/Rtmpb249bZ/file3cbb944fcb3448/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/Rtmpb249bZ/file3cbb9476afe396/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/Rtmpb249bZ/file3cbb9476afe396/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpb249bZ/file3cbb947326b39e/musc_rps24_1.fastq
Searching for barcodes...
Processing file: /tmp/Rtmpb249bZ/file3cbb947326b39e/musc_rps24_2.fastq
Searching for barcodes...
Processing file: /tmp/Rtmpb249bZ/file3cbb947326b39e/musc_rps24_3.fastq
Searching for barcodes...
Processing file: /tmp/Rtmpb249bZ/file3cbb947326b39e/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/Rtmpb249bZ/file3cbb9438311c2b/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/Rtmpb249bZ/file3cbb94720c31b5/config_file_3980180.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 17 23:58:12 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/Rtmpb249bZ/file3cbb94720c31b5/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/Rtmpb249bZ/file3cbb94720c31b5/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/Rtmpb249bZ/file3cbb94720c31b5/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Fri Oct 17 23:58:13 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

  |                                                                            
  |                                                                      |   0%[23:58:19] 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.

[23:58:19] 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.

[23:58:20] WARNING: src/learner.cc:553: 
  If you are loading a serialized model (like pickle in Python, RDS in R) generated by
  older XGBoost, please export the model by calling `Booster.save_model` from that version
  first, then load it back in current version. See:

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

  for more details about differences between saving model and serializing.

[23:58:20] WARNING: src/learner.cc:553: 
  If you are loading a serialized model (like pickle in Python, RDS in R) generated by
  older XGBoost, please export the model by calling `Booster.save_model` from that version
  first, then load it back in current version. See:

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

  for more details about differences between saving model and serializing.

[23:58:21] 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.

[23:58:21] 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 17 23:58:37 2025 -------------------
Realigning sample sample1 -> /tmp/Rtmpb249bZ/file3cbb94720c31b5/sample1_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample2 -> /tmp/Rtmpb249bZ/file3cbb94720c31b5/sample2_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample3 -> /tmp/Rtmpb249bZ/file3cbb94720c31b5/sample3_realign2transcript.bam
Skipped sorting BAM files.

-- Running step: transcript_quantification @ Fri Oct 17 23:58:37 2025 ----------
2025-10-18T03:58:37.836020Z  INFO oarfish: setting user-provided filter parameters.
2025-10-18T03:58:37.836540Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpb249bZ/file3cbb94720c31b5/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-10-18T03:58:37.836554Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-18T03:58:37.836558Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-18T03:58:37.836620Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-18T03:58:37.836626Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-18T03:58:37.838282Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-18T03:58:37.838412Z  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    │
╰─────────────────────────────────┴───────╯

2025-10-18T03:58:37.838436Z  INFO oarfish::bulk: Total number of alignment records : 116
2025-10-18T03:58:37.838450Z  INFO oarfish::bulk: number of aligned reads : 96
2025-10-18T03:58:37.838452Z  INFO oarfish::bulk: number of unique alignments : 86
2025-10-18T03:58:37.839089Z  INFO oarfish: oarfish completed successfully.
2025-10-18T03:58:37.846632Z  INFO oarfish: setting user-provided filter parameters.
2025-10-18T03:58:37.847048Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpb249bZ/file3cbb94720c31b5/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-10-18T03:58:37.847056Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-18T03:58:37.847060Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-18T03:58:37.847125Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-18T03:58:37.847133Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-18T03:58:37.848688Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-18T03:58:37.848805Z  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    │
╰─────────────────────────────────┴───────╯

2025-10-18T03:58:37.848832Z  INFO oarfish::bulk: Total number of alignment records : 114
2025-10-18T03:58:37.848834Z  INFO oarfish::bulk: number of aligned reads : 95
2025-10-18T03:58:37.848837Z  INFO oarfish::bulk: number of unique alignments : 82
2025-10-18T03:58:37.849429Z  INFO oarfish: oarfish completed successfully.
2025-10-18T03:58:37.856547Z  INFO oarfish: setting user-provided filter parameters.
2025-10-18T03:58:37.857003Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpb249bZ/file3cbb94720c31b5/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-10-18T03:58:37.857010Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-18T03:58:37.857013Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-18T03:58:37.857069Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-18T03:58:37.857074Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-18T03:58:37.860269Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2025-10-18T03:58:37.860465Z  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   │
╰─────────────────────────────────┴───────╯

2025-10-18T03:58:37.860510Z  INFO oarfish::bulk: Total number of alignment records : 239
2025-10-18T03:58:37.860513Z  INFO oarfish::bulk: number of aligned reads : 179
2025-10-18T03:58:37.860515Z  INFO oarfish::bulk: number of unique alignments : 143
2025-10-18T03:58:37.861209Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpb249bZ/file3cbb94124e61e1/config_file_3980180.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 17 23:58:38 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/Rtmpb249bZ/file3cbb94124e61e1/sample1_align2genome.bam
sample2 ->/tmp/Rtmpb249bZ/file3cbb94124e61e1/sample2_align2genome.bam
sample3 ->/tmp/Rtmpb249bZ/file3cbb94124e61e1/sample3_align2genome.bam
-- Running step: isoform_identification @ Fri Oct 17 23:58:56 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 4 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Fri Oct 17 23:59:16 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/Rtmpb249bZ/file3cbb94124e61e1/sample1_realign2transcript.bam
sample2 ->/tmp/Rtmpb249bZ/file3cbb94124e61e1/sample2_realign2transcript.bam
sample3 ->/tmp/Rtmpb249bZ/file3cbb94124e61e1/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Fri Oct 17 23:59:34 2025 ----------
2025-10-18T03:59:34.941172Z  INFO oarfish: setting user-provided filter parameters.
2025-10-18T03:59:34.941642Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpb249bZ/file3cbb94124e61e1/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-10-18T03:59:34.941666Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-18T03:59:34.941669Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-18T03:59:34.941728Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-18T03:59:34.941734Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-18T03:59:34.943286Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-18T03:59:34.943417Z  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    │
╰─────────────────────────────────┴───────╯

2025-10-18T03:59:34.943439Z  INFO oarfish::bulk: Total number of alignment records : 116
2025-10-18T03:59:34.943442Z  INFO oarfish::bulk: number of aligned reads : 96
2025-10-18T03:59:34.943444Z  INFO oarfish::bulk: number of unique alignments : 86
2025-10-18T03:59:34.944062Z  INFO oarfish: oarfish completed successfully.
2025-10-18T03:59:34.955304Z  INFO oarfish: setting user-provided filter parameters.
2025-10-18T03:59:34.955711Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpb249bZ/file3cbb94124e61e1/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-10-18T03:59:34.955720Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-18T03:59:34.955724Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-18T03:59:34.955790Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-18T03:59:34.955796Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-18T03:59:34.957386Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-18T03:59:34.957531Z  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    │
╰─────────────────────────────────┴───────╯

2025-10-18T03:59:34.957558Z  INFO oarfish::bulk: Total number of alignment records : 114
2025-10-18T03:59:34.957571Z  INFO oarfish::bulk: number of aligned reads : 95
2025-10-18T03:59:34.957573Z  INFO oarfish::bulk: number of unique alignments : 82
2025-10-18T03:59:34.958195Z  INFO oarfish: oarfish completed successfully.
2025-10-18T03:59:34.969282Z  INFO oarfish: setting user-provided filter parameters.
2025-10-18T03:59:34.969680Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpb249bZ/file3cbb94124e61e1/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-10-18T03:59:34.969691Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-18T03:59:34.969694Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-18T03:59:34.969757Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-18T03:59:34.969764Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-18T03:59:34.972595Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2025-10-18T03:59:34.972802Z  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   │
╰─────────────────────────────────┴───────╯

2025-10-18T03:59:34.972834Z  INFO oarfish::bulk: Total number of alignment records : 239
2025-10-18T03:59:34.972837Z  INFO oarfish::bulk: number of aligned reads : 179
2025-10-18T03:59:34.972839Z  INFO oarfish::bulk: number of unique alignments : 143
2025-10-18T03:59:34.973562Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpb249bZ/file3cbb942956ff12/config_file_3980180.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 17 23:59:35 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/Rtmpb249bZ/file3cbb942956ff12/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/Rtmpb249bZ/file3cbb942956ff12/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/Rtmpb249bZ/file3cbb942956ff12/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Fri Oct 17 23:59:36 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 17 23:59:54 2025 -------------------
Realigning sample sample1 -> /tmp/Rtmpb249bZ/file3cbb942956ff12/sample1_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample2 -> /tmp/Rtmpb249bZ/file3cbb942956ff12/sample2_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample3 -> /tmp/Rtmpb249bZ/file3cbb942956ff12/sample3_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Fri Oct 17 23:59:54 2025 ----------
23:59:54 Fri Oct 17 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/Rtmpb249bZ/file3cbb945854d178/config_file_3980180.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 17 23:59:55 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/Rtmpb249bZ/file3cbb945854d178/sample1_align2genome.bam
sample2 ->/tmp/Rtmpb249bZ/file3cbb945854d178/sample2_align2genome.bam
sample3 ->/tmp/Rtmpb249bZ/file3cbb945854d178/sample3_align2genome.bam
-- Running step: isoform_identification @ Sat Oct 18 00:00:14 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 18 00:00:32 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/Rtmpb249bZ/file3cbb945854d178/sample1_realign2transcript.bam
sample2 ->/tmp/Rtmpb249bZ/file3cbb945854d178/sample2_realign2transcript.bam
sample3 ->/tmp/Rtmpb249bZ/file3cbb945854d178/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Sat Oct 18 00:00:51 2025 ----------
00:00:51 Sat Oct 18 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Inputs:  ['/tmp/Rtmpb249bZ/file3cbb942956ff12/sample1_realign2transcript.bam', '/tmp/Rtmpb249bZ/file3cbb942956ff12/sample2_realign2transcript.bam', '/tmp/Rtmpb249bZ/file3cbb942956ff12/sample3_realign2transcript.bam'] /tmp/Rtmpb249bZ/file3cbb942956ff12/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/Rtmpb249bZ/file3cbb94867ae33/config_file_3980180.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 18 00:00:52 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/Rtmpb249bZ/file3cbb94867ae33/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/Rtmpb249bZ/file3cbb94867ae33/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/Rtmpb249bZ/file3cbb94867ae33/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Sat Oct 18 00:00:53 2025 -------------
Inputs:  ['/tmp/Rtmpb249bZ/file3cbb945854d178/sample1_realign2transcript.bam', '/tmp/Rtmpb249bZ/file3cbb945854d178/sample2_realign2transcript.bam', '/tmp/Rtmpb249bZ/file3cbb945854d178/sample3_realign2transcript.bam'] /tmp/Rtmpb249bZ/file3cbb945854d178/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 18 00:00:53 2025 -------------------
Realigning sample sample1 -> /tmp/Rtmpb249bZ/file3cbb94867ae33/sample1_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample2 -> /tmp/Rtmpb249bZ/file3cbb94867ae33/sample2_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample3 -> /tmp/Rtmpb249bZ/file3cbb94867ae33/sample3_realign2transcript.bam
Skipped sorting BAM files.

-- Running step: transcript_quantification @ Sat Oct 18 00:00:54 2025 ----------
2025-10-18T04:00:54.778644Z  INFO oarfish: setting user-provided filter parameters.
2025-10-18T04:00:54.779010Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpb249bZ/file3cbb94867ae33/sample1_realign2transcript.bam, contains 10 reference sequences.
2025-10-18T04:00:54.779021Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-18T04:00:54.779025Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-18T04:00:54.779090Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-18T04:00:54.779107Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-18T04:00:54.781699Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-18T04:00:54.781837Z  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    │
╰─────────────────────────────────┴───────╯

2025-10-18T04:00:54.781863Z  INFO oarfish::bulk: Total number of alignment records : 125
2025-10-18T04:00:54.781865Z  INFO oarfish::bulk: number of aligned reads : 98
2025-10-18T04:00:54.781868Z  INFO oarfish::bulk: number of unique alignments : 86
2025-10-18T04:00:54.782505Z  INFO oarfish: oarfish completed successfully.
2025-10-18T04:00:54.790290Z  INFO oarfish: setting user-provided filter parameters.
2025-10-18T04:00:54.790630Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpb249bZ/file3cbb94867ae33/sample2_realign2transcript.bam, contains 10 reference sequences.
2025-10-18T04:00:54.790638Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-18T04:00:54.790641Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-18T04:00:54.790706Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-18T04:00:54.790712Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-18T04:00:54.793431Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-18T04:00:54.793566Z  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    │
╰─────────────────────────────────┴───────╯

2025-10-18T04:00:54.793595Z  INFO oarfish::bulk: Total number of alignment records : 136
2025-10-18T04:00:54.793597Z  INFO oarfish::bulk: number of aligned reads : 97
2025-10-18T04:00:54.793600Z  INFO oarfish::bulk: number of unique alignments : 79
2025-10-18T04:00:54.794229Z  INFO oarfish: oarfish completed successfully.
2025-10-18T04:00:54.801732Z  INFO oarfish: setting user-provided filter parameters.
2025-10-18T04:00:54.802194Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpb249bZ/file3cbb94867ae33/sample3_realign2transcript.bam, contains 10 reference sequences.
2025-10-18T04:00:54.802205Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-18T04:00:54.802208Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-18T04:00:54.802276Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-18T04:00:54.802282Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-18T04:00:54.806476Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-18T04:00:54.806642Z  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   │
╰─────────────────────────────────┴───────╯

2025-10-18T04:00:54.806669Z  INFO oarfish::bulk: Total number of alignment records : 272
2025-10-18T04:00:54.806671Z  INFO oarfish::bulk: number of aligned reads : 187
2025-10-18T04:00:54.806673Z  INFO oarfish::bulk: number of unique alignments : 140
2025-10-18T04:00:54.807400Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpb249bZ/file3cbb947c96f3e2/config_file_3980180.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 18 00:00:55 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/Rtmpb249bZ/file3cbb947c96f3e2/sample1_align2genome.bam
sample2 ->/tmp/Rtmpb249bZ/file3cbb947c96f3e2/sample2_align2genome.bam
sample3 ->/tmp/Rtmpb249bZ/file3cbb947c96f3e2/sample3_align2genome.bam
-- Running step: isoform_identification @ Sat Oct 18 00:01:13 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sat Oct 18 00:01:14 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/Rtmpb249bZ/file3cbb947c96f3e2/sample1_realign2transcript.bam
sample2 ->/tmp/Rtmpb249bZ/file3cbb947c96f3e2/sample2_realign2transcript.bam
sample3 ->/tmp/Rtmpb249bZ/file3cbb947c96f3e2/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Sat Oct 18 00:01:32 2025 ----------
2025-10-18T04:01:32.294465Z  INFO oarfish: setting user-provided filter parameters.
2025-10-18T04:01:32.294852Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpb249bZ/file3cbb947c96f3e2/sample1_realign2transcript.bam, contains 10 reference sequences.
2025-10-18T04:01:32.294863Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-18T04:01:32.294879Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-18T04:01:32.294951Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-18T04:01:32.294958Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-18T04:01:32.297614Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-18T04:01:32.297759Z  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    │
╰─────────────────────────────────┴───────╯

2025-10-18T04:01:32.297781Z  INFO oarfish::bulk: Total number of alignment records : 125
2025-10-18T04:01:32.297784Z  INFO oarfish::bulk: number of aligned reads : 98
2025-10-18T04:01:32.297786Z  INFO oarfish::bulk: number of unique alignments : 86
2025-10-18T04:01:32.298416Z  INFO oarfish: oarfish completed successfully.
2025-10-18T04:01:32.309824Z  INFO oarfish: setting user-provided filter parameters.
2025-10-18T04:01:32.310296Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpb249bZ/file3cbb947c96f3e2/sample2_realign2transcript.bam, contains 10 reference sequences.
2025-10-18T04:01:32.310307Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-18T04:01:32.310309Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-18T04:01:32.310377Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-18T04:01:32.310383Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-18T04:01:32.313190Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-18T04:01:32.313317Z  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    │
╰─────────────────────────────────┴───────╯

2025-10-18T04:01:32.313341Z  INFO oarfish::bulk: Total number of alignment records : 136
2025-10-18T04:01:32.313344Z  INFO oarfish::bulk: number of aligned reads : 97
2025-10-18T04:01:32.313356Z  INFO oarfish::bulk: number of unique alignments : 79
2025-10-18T04:01:32.313932Z  INFO oarfish: oarfish completed successfully.
2025-10-18T04:01:32.325180Z  INFO oarfish: setting user-provided filter parameters.
2025-10-18T04:01:32.325550Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpb249bZ/file3cbb947c96f3e2/sample3_realign2transcript.bam, contains 10 reference sequences.
2025-10-18T04:01:32.325559Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-18T04:01:32.325561Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-18T04:01:32.325638Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-18T04:01:32.325645Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-18T04:01:32.329912Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-18T04:01:32.330084Z  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   │
╰─────────────────────────────────┴───────╯

2025-10-18T04:01:32.330111Z  INFO oarfish::bulk: Total number of alignment records : 272
2025-10-18T04:01:32.330113Z  INFO oarfish::bulk: number of aligned reads : 187
2025-10-18T04:01:32.330131Z  INFO oarfish::bulk: number of unique alignments : 140
2025-10-18T04:01:32.330801Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpb249bZ/file3cbb9453619365/config_file_3980180.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 18 00:01:32 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/Rtmpb249bZ/file3cbb9453619365/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/Rtmpb249bZ/file3cbb9453619365/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/Rtmpb249bZ/file3cbb9453619365/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Sat Oct 18 00:01:33 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sat Oct 18 00:01:33 2025 -------------------
Realigning sample sample1 -> /tmp/Rtmpb249bZ/file3cbb9453619365/sample1_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample2 -> /tmp/Rtmpb249bZ/file3cbb9453619365/sample2_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample3 -> /tmp/Rtmpb249bZ/file3cbb9453619365/sample3_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Sat Oct 18 00:01:34 2025 ----------
00:01:34 Sat Oct 18 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/Rtmpb249bZ/file3cbb943212ad65/config_file_3980180.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 18 00:01:35 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/Rtmpb249bZ/file3cbb943212ad65/sample1_align2genome.bam
sample2 ->/tmp/Rtmpb249bZ/file3cbb943212ad65/sample2_align2genome.bam
sample3 ->/tmp/Rtmpb249bZ/file3cbb943212ad65/sample3_align2genome.bam
-- Running step: isoform_identification @ Sat Oct 18 00:01:54 2025 -------------
Inputs:  ['/tmp/Rtmpb249bZ/file3cbb9453619365/sample1_realign2transcript.bam', '/tmp/Rtmpb249bZ/file3cbb9453619365/sample2_realign2transcript.bam', '/tmp/Rtmpb249bZ/file3cbb9453619365/sample3_realign2transcript.bam'] /tmp/Rtmpb249bZ/file3cbb9453619365/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 18 00:01:54 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/Rtmpb249bZ/file3cbb943212ad65/sample1_realign2transcript.bam
sample2 ->/tmp/Rtmpb249bZ/file3cbb943212ad65/sample2_realign2transcript.bam
sample3 ->/tmp/Rtmpb249bZ/file3cbb943212ad65/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Sat Oct 18 00:02:12 2025 ----------
00:02:12 Sat Oct 18 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/Rtmpb249bZ/file3cbb94267a1fb7/config_file_3980180.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 18 00:02:13 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpb249bZ/file3cbb94267a1fb7/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 18 00:02:14 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmpb249bZ/file3cbb94267a1fb7/matched_reads.fastq.gz -> /tmp/Rtmpb249bZ/file3cbb94267a1fb7/align2genome.bam
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: isoform_identification @ Sat Oct 18 00:02:14 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 18 00:02:23 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/Rtmpb249bZ/file3cbb94267a1fb7/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpb249bZ/file3cbb94267a1fb7/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/Rtmpb249bZ/file3cbb94267a1fb7/matched_reads.fastq.gz -> /tmp/Rtmpb249bZ/file3cbb94267a1fb7/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 18 00:02:24 2025 ----------
2025-10-18T04:02:24.074561Z  INFO oarfish: setting user-provided filter parameters.
2025-10-18T04:02:24.075092Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpb249bZ/file3cbb94267a1fb7/realign2transcript.bam, contains 5 reference sequences.
2025-10-18T04:02:24.075104Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-18T04:02:24.075107Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-18T04:02:24.075172Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-18T04:02:24.075181Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-18T04:02:24.081381Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpb249bZ/file3cbb9419970aa4/config_file_3980180.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 18 00:02:24 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpb249bZ/file3cbb9419970aa4/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 18 00:02:24 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmpb249bZ/file3cbb9419970aa4/matched_reads.fastq.gz ->/tmp/Rtmpb249bZ/file3cbb9419970aa4/align2genome.bam
-- Running step: isoform_identification @ Sat Oct 18 00:02:42 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 18 00:02:52 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/Rtmpb249bZ/file3cbb9419970aa4/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpb249bZ/file3cbb9419970aa4/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/Rtmpb249bZ/file3cbb9419970aa4/matched_reads.fastq.gz ->/tmp/Rtmpb249bZ/file3cbb9419970aa4/realign2transcript.bam
-- Running step: transcript_quantification @ Sat Oct 18 00:03:09 2025 ----------
2025-10-18T04:03:09.172374Z  INFO oarfish: setting user-provided filter parameters.
2025-10-18T04:03:09.172828Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpb249bZ/file3cbb9419970aa4/realign2transcript.bam, contains 5 reference sequences.
2025-10-18T04:03:09.172841Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-18T04:03:09.172845Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-18T04:03:09.172907Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-18T04:03:09.172913Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-18T04:03:09.179400Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpb249bZ/file3cbb941f8a421a/config_file_3980180.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 18 00:03:09 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpb249bZ/file3cbb941f8a421a/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 18 00:03:09 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmpb249bZ/file3cbb941f8a421a/matched_reads.fastq.gz -> /tmp/Rtmpb249bZ/file3cbb941f8a421a/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 18 00:03:10 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 18 00:03:19 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/Rtmpb249bZ/file3cbb941f8a421a/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpb249bZ/file3cbb941f8a421a/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/Rtmpb249bZ/file3cbb941f8a421a/matched_reads.fastq.gz -> /tmp/Rtmpb249bZ/file3cbb941f8a421a/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 18 00:03:19 2025 ----------
00:03:19 Sat Oct 18 2025 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Inputs:  ['/tmp/Rtmpb249bZ/file3cbb943212ad65/sample1_realign2transcript.bam', '/tmp/Rtmpb249bZ/file3cbb943212ad65/sample2_realign2transcript.bam', '/tmp/Rtmpb249bZ/file3cbb943212ad65/sample3_realign2transcript.bam'] /tmp/Rtmpb249bZ/file3cbb943212ad65/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/Rtmpb249bZ/file3cbb944836099c/config_file_3980180.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 18 00:03:20 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/Rtmpb249bZ/file3cbb944836099c/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 18 00:03:20 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmpb249bZ/file3cbb944836099c/matched_reads.fastq.gz ->/tmp/Rtmpb249bZ/file3cbb944836099c/align2genome.bam
-- Running step: isoform_identification @ Sat Oct 18 00:03:38 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---
Detected 2 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Sat Oct 18 00:03:47 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/Rtmpb249bZ/file3cbb944836099c/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpb249bZ/file3cbb944836099c/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/Rtmpb249bZ/file3cbb944836099c/matched_reads.fastq.gz ->/tmp/Rtmpb249bZ/file3cbb944836099c/realign2transcript.bam
-- Running step: transcript_quantification @ Sat Oct 18 00:04:04 2025 ----------
00:04:04 Sat Oct 18 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/Rtmpb249bZ/file3cbb94b41a3e0/config_file_3980180.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 18 00:04:05 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/Rtmpb249bZ/file3cbb94b41a3e0/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 18 00:04:05 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmpb249bZ/file3cbb94b41a3e0/matched_reads.fastq.gz -> /tmp/Rtmpb249bZ/file3cbb94b41a3e0/align2genome.bam
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: isoform_identification @ Sat Oct 18 00:04:06 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 18 00:04:06 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/Rtmpb249bZ/file3cbb94b41a3e0/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpb249bZ/file3cbb94b41a3e0/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/Rtmpb249bZ/file3cbb94b41a3e0/matched_reads.fastq.gz -> /tmp/Rtmpb249bZ/file3cbb94b41a3e0/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 18 00:04:06 2025 ----------
2025-10-18T04:04:06.785155Z  INFO oarfish: setting user-provided filter parameters.
2025-10-18T04:04:06.785611Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpb249bZ/file3cbb94b41a3e0/realign2transcript.bam, contains 10 reference sequences.
2025-10-18T04:04:06.785625Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-18T04:04:06.785627Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-18T04:04:06.785700Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-18T04:04:06.785708Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-18T04:04:06.796031Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpb249bZ/file3cbb945d5f5c68/config_file_3980180.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 18 00:04:07 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/Rtmpb249bZ/file3cbb945d5f5c68/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 18 00:04:07 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmpb249bZ/file3cbb945d5f5c68/matched_reads.fastq.gz ->/tmp/Rtmpb249bZ/file3cbb945d5f5c68/align2genome.bam
-- Running step: isoform_identification @ Sat Oct 18 00:04:24 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sat Oct 18 00:04:24 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/Rtmpb249bZ/file3cbb945d5f5c68/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpb249bZ/file3cbb945d5f5c68/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/Rtmpb249bZ/file3cbb945d5f5c68/matched_reads.fastq.gz ->/tmp/Rtmpb249bZ/file3cbb945d5f5c68/realign2transcript.bam
-- Running step: transcript_quantification @ Sat Oct 18 00:04:41 2025 ----------
2025-10-18T04:04:41.801620Z  INFO oarfish: setting user-provided filter parameters.
2025-10-18T04:04:41.802254Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpb249bZ/file3cbb945d5f5c68/realign2transcript.bam, contains 10 reference sequences.
2025-10-18T04:04:41.802265Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-18T04:04:41.802269Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-18T04:04:41.802345Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-18T04:04:41.802352Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-18T04:04:41.813863Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpb249bZ/file3cbb942ce73de2/config_file_3980180.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 18 00:04:42 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpb249bZ/file3cbb942ce73de2/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 18 00:04:42 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmpb249bZ/file3cbb942ce73de2/matched_reads.fastq.gz -> /tmp/Rtmpb249bZ/file3cbb942ce73de2/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 18 00:04:43 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sat Oct 18 00:04:43 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/Rtmpb249bZ/file3cbb942ce73de2/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpb249bZ/file3cbb942ce73de2/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/Rtmpb249bZ/file3cbb942ce73de2/matched_reads.fastq.gz -> /tmp/Rtmpb249bZ/file3cbb942ce73de2/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 18 00:04:43 2025 ----------
00:04:43 Sat Oct 18 2025 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpb249bZ/file3cbb947ef775c3/config_file_3980180.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 18 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/Rtmpb249bZ/file3cbb947ef775c3/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 18 00:04:45 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmpb249bZ/file3cbb947ef775c3/matched_reads.fastq.gz ->/tmp/Rtmpb249bZ/file3cbb947ef775c3/align2genome.bam
-- Running step: isoform_identification @ Sat Oct 18 00:05:03 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 18 00:05:03 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/Rtmpb249bZ/file3cbb947ef775c3/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpb249bZ/file3cbb947ef775c3/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/Rtmpb249bZ/file3cbb947ef775c3/matched_reads.fastq.gz ->/tmp/Rtmpb249bZ/file3cbb947ef775c3/realign2transcript.bam
-- Running step: transcript_quantification @ Sat Oct 18 00:05:21 2025 ----------
00:05:21 Sat Oct 18 2025 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpb249bZ/file3cbb942baab1c7/config_file_3980180.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 18 00:05:22 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/Rtmpb249bZ/file3cbb942baab1c7/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpb249bZ/file3cbb942baab1c7/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpb249bZ/file3cbb942baab1c7/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpb249bZ/file3cbb942baab1c7/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/Rtmpb249bZ/file3cbb942baab1c7/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpb249bZ/file3cbb942baab1c7/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/Rtmpb249bZ/file3cbb942baab1c7/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpb249bZ/file3cbb942baab1c7/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/Rtmpb249bZ/file3cbb942baab1c7/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpb249bZ/file3cbb942baab1c7/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 18 00:05:23 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmpb249bZ/file3cbb942baab1c7/sampleA_matched_reads.fastq.gz -> /tmp/Rtmpb249bZ/file3cbb942baab1c7/sampleA_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmpb249bZ/file3cbb942baab1c7/sample1_matched_reads.fastq.gz -> /tmp/Rtmpb249bZ/file3cbb942baab1c7/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmpb249bZ/file3cbb942baab1c7/sample2_matched_reads.fastq.gz -> /tmp/Rtmpb249bZ/file3cbb942baab1c7/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmpb249bZ/file3cbb942baab1c7/sample3_matched_reads.fastq.gz -> /tmp/Rtmpb249bZ/file3cbb942baab1c7/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Sat Oct 18 00:05:24 2025 ----------------
00:05:24 Sat Oct 18 2025 quantify genes 
Using BAM(s): '/tmp/Rtmpb249bZ/file3cbb942baab1c7/sampleA_align2genome.bam',
'/tmp/Rtmpb249bZ/file3cbb942baab1c7/sample1_align2genome.bam',
'/tmp/Rtmpb249bZ/file3cbb942baab1c7/sample2_align2genome.bam', and
'/tmp/Rtmpb249bZ/file3cbb942baab1c7/sample3_align2genome.bam'
	Counter({'counted_reads': 368, 'unmapped': 4})
parsing /tmp/Rtmpb249bZ/file3cbb942baab1c7/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 19.09gene_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, 400831.80Read/s]
parsing /tmp/Rtmpb249bZ/file3cbb942baab1c7/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 39.47gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1147992.12Read/s]
parsing /tmp/Rtmpb249bZ/file3cbb942baab1c7/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 48.31gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1005924.79Read/s]
parsing /tmp/Rtmpb249bZ/file3cbb942baab1c7/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 31.18gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 676893.68Read/s]
-- Running step: isoform_identification @ Sat Oct 18 00:05:26 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 18 00:05:51 2025 -------------------
Checking for fastq file(s) /tmp/Rtmpb249bZ/file3cbb942baab1c7/fastq, /tmp/Rtmpb249bZ/file3cbb942baab1c7/fastq/sample1.fq.gz, /tmp/Rtmpb249bZ/file3cbb942baab1c7/fastq/sample2.fq.gz, /tmp/Rtmpb249bZ/file3cbb942baab1c7/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpb249bZ/file3cbb942baab1c7/sampleA_matched_reads.fastq.gz, /tmp/Rtmpb249bZ/file3cbb942baab1c7/sample1_matched_reads.fastq.gz, /tmp/Rtmpb249bZ/file3cbb942baab1c7/sample2_matched_reads.fastq.gz, /tmp/Rtmpb249bZ/file3cbb942baab1c7/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpb249bZ/file3cbb942baab1c7/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmpb249bZ/file3cbb942baab1c7/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmpb249bZ/file3cbb942baab1c7/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmpb249bZ/file3cbb942baab1c7/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/Rtmpb249bZ/file3cbb942baab1c7/sampleA_matched_reads_dedup.fastq.gz -> /tmp/Rtmpb249bZ/file3cbb942baab1c7/sampleA_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/Rtmpb249bZ/file3cbb942baab1c7/sample1_matched_reads_dedup.fastq.gz -> /tmp/Rtmpb249bZ/file3cbb942baab1c7/sample1_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/Rtmpb249bZ/file3cbb942baab1c7/sample2_matched_reads_dedup.fastq.gz -> /tmp/Rtmpb249bZ/file3cbb942baab1c7/sample2_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/Rtmpb249bZ/file3cbb942baab1c7/sample3_matched_reads_dedup.fastq.gz -> /tmp/Rtmpb249bZ/file3cbb942baab1c7/sample3_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

-- Running step: transcript_quantification @ Sat Oct 18 00:05:51 2025 ----------
2025-10-18T04:05:51.899356Z  INFO oarfish: setting user-provided filter parameters.
2025-10-18T04:05:51.899743Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpb249bZ/file3cbb942baab1c7/sampleA_realign2transcript.bam, contains 5 reference sequences.
2025-10-18T04:05:51.899765Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-18T04:05:51.899769Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-18T04:05:51.899829Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-18T04:05:51.899834Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-18T04:05:51.905576Z  INFO oarfish::single_cell: Processed 100 cells.
2025-10-18T04:05:52.216181Z  INFO oarfish: setting user-provided filter parameters.
2025-10-18T04:05:52.216563Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpb249bZ/file3cbb942baab1c7/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-10-18T04:05:52.216571Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-18T04:05:52.216574Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-18T04:05:52.216629Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-18T04:05:52.216635Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-18T04:05:52.572043Z  INFO oarfish: setting user-provided filter parameters.
2025-10-18T04:05:52.572395Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpb249bZ/file3cbb942baab1c7/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-10-18T04:05:52.572406Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-18T04:05:52.572409Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-18T04:05:52.572458Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-18T04:05:52.572463Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-18T04:05:52.866364Z  INFO oarfish: setting user-provided filter parameters.
2025-10-18T04:05:52.866776Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpb249bZ/file3cbb942baab1c7/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-10-18T04:05:52.866785Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-18T04:05:52.866788Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-18T04:05:52.866841Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-18T04:05:52.866846Z  INFO oarfish: parsed reference information for 5 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpb249bZ/file3cbb94937ed07/config_file_3980180.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 18 00:05:53 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpb249bZ/file3cbb94937ed07/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpb249bZ/file3cbb94937ed07/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpb249bZ/file3cbb94937ed07/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpb249bZ/file3cbb94937ed07/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/Rtmpb249bZ/file3cbb94937ed07/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpb249bZ/file3cbb94937ed07/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/Rtmpb249bZ/file3cbb94937ed07/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpb249bZ/file3cbb94937ed07/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/Rtmpb249bZ/file3cbb94937ed07/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpb249bZ/file3cbb94937ed07/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 18 00:05:54 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmpb249bZ/file3cbb94937ed07/sampleA_matched_reads.fastq.gz ->/tmp/Rtmpb249bZ/file3cbb94937ed07/sampleA_align2genome.bam
/tmp/Rtmpb249bZ/file3cbb94937ed07/sample1_matched_reads.fastq.gz ->/tmp/Rtmpb249bZ/file3cbb94937ed07/sample1_align2genome.bam
/tmp/Rtmpb249bZ/file3cbb94937ed07/sample2_matched_reads.fastq.gz ->/tmp/Rtmpb249bZ/file3cbb94937ed07/sample2_align2genome.bam
/tmp/Rtmpb249bZ/file3cbb94937ed07/sample3_matched_reads.fastq.gz ->/tmp/Rtmpb249bZ/file3cbb94937ed07/sample3_align2genome.bam
-- Running step: gene_quantification @ Sat Oct 18 00:06:13 2025 ----------------
00:06:13 Sat Oct 18 2025 quantify genes 
Using BAM(s): '/tmp/Rtmpb249bZ/file3cbb94937ed07/sampleA_align2genome.bam',
'/tmp/Rtmpb249bZ/file3cbb94937ed07/sample1_align2genome.bam',
'/tmp/Rtmpb249bZ/file3cbb94937ed07/sample2_align2genome.bam', and
'/tmp/Rtmpb249bZ/file3cbb94937ed07/sample3_align2genome.bam'
parsing /tmp/Rtmpb249bZ/file3cbb94937ed07/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 19.47gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 325948.40Read/s]
parsing /tmp/Rtmpb249bZ/file3cbb94937ed07/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 47.96gene_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, 1233618.82Read/s]
parsing /tmp/Rtmpb249bZ/file3cbb94937ed07/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 46.61gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1082233.46Read/s]
parsing /tmp/Rtmpb249bZ/file3cbb94937ed07/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 24.99gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 650400.69Read/s]
-- Running step: isoform_identification @ Sat Oct 18 00:06:14 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

  |                                                                            
  |                                                                      |   0%
  |                                                                            
  |==================                                                    |  25%
  |                                                                            
  |===================================                                   |  50%
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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 18 00:06:37 2025 -------------------
Checking for fastq file(s) /tmp/Rtmpb249bZ/file3cbb94937ed07/fastq, /tmp/Rtmpb249bZ/file3cbb94937ed07/fastq/sample1.fq.gz, /tmp/Rtmpb249bZ/file3cbb94937ed07/fastq/sample2.fq.gz, /tmp/Rtmpb249bZ/file3cbb94937ed07/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpb249bZ/file3cbb94937ed07/sampleA_matched_reads.fastq.gz, /tmp/Rtmpb249bZ/file3cbb94937ed07/sample1_matched_reads.fastq.gz, /tmp/Rtmpb249bZ/file3cbb94937ed07/sample2_matched_reads.fastq.gz, /tmp/Rtmpb249bZ/file3cbb94937ed07/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpb249bZ/file3cbb94937ed07/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmpb249bZ/file3cbb94937ed07/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmpb249bZ/file3cbb94937ed07/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmpb249bZ/file3cbb94937ed07/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/Rtmpb249bZ/file3cbb94937ed07/sampleA_matched_reads_dedup.fastq.gz ->/tmp/Rtmpb249bZ/file3cbb94937ed07/sampleA_realign2transcript.bam
/tmp/Rtmpb249bZ/file3cbb94937ed07/sample1_matched_reads_dedup.fastq.gz ->/tmp/Rtmpb249bZ/file3cbb94937ed07/sample1_realign2transcript.bam
/tmp/Rtmpb249bZ/file3cbb94937ed07/sample2_matched_reads_dedup.fastq.gz ->/tmp/Rtmpb249bZ/file3cbb94937ed07/sample2_realign2transcript.bam
/tmp/Rtmpb249bZ/file3cbb94937ed07/sample3_matched_reads_dedup.fastq.gz ->/tmp/Rtmpb249bZ/file3cbb94937ed07/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Sat Oct 18 00:06:54 2025 ----------
2025-10-18T04:06:54.559775Z  INFO oarfish: setting user-provided filter parameters.
2025-10-18T04:06:54.560173Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpb249bZ/file3cbb94937ed07/sampleA_realign2transcript.bam, contains 5 reference sequences.
2025-10-18T04:06:54.560185Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-18T04:06:54.560188Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-18T04:06:54.560255Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-18T04:06:54.560260Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-18T04:06:54.566054Z  INFO oarfish::single_cell: Processed 100 cells.
2025-10-18T04:06:54.884288Z  INFO oarfish: setting user-provided filter parameters.
2025-10-18T04:06:54.884774Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpb249bZ/file3cbb94937ed07/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-10-18T04:06:54.884783Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-18T04:06:54.884786Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-18T04:06:54.884856Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-18T04:06:54.884862Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-18T04:06:55.193949Z  INFO oarfish: setting user-provided filter parameters.
2025-10-18T04:06:55.194413Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpb249bZ/file3cbb94937ed07/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-10-18T04:06:55.194425Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-18T04:06:55.194429Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-18T04:06:55.194492Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-18T04:06:55.194498Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-18T04:06:55.518654Z  INFO oarfish: setting user-provided filter parameters.
2025-10-18T04:06:55.519037Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpb249bZ/file3cbb94937ed07/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-10-18T04:06:55.519048Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-18T04:06:55.519052Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-18T04:06:55.519126Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-18T04:06:55.519135Z  INFO oarfish: parsed reference information for 5 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpb249bZ/file3cbb941a0b2db4/config_file_3980180.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 18 00:06:56 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpb249bZ/file3cbb941a0b2db4/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpb249bZ/file3cbb941a0b2db4/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpb249bZ/file3cbb941a0b2db4/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpb249bZ/file3cbb941a0b2db4/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/Rtmpb249bZ/file3cbb941a0b2db4/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpb249bZ/file3cbb941a0b2db4/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/Rtmpb249bZ/file3cbb941a0b2db4/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpb249bZ/file3cbb941a0b2db4/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/Rtmpb249bZ/file3cbb941a0b2db4/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpb249bZ/file3cbb941a0b2db4/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 18 00:06:56 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmpb249bZ/file3cbb941a0b2db4/sampleA_matched_reads.fastq.gz -> /tmp/Rtmpb249bZ/file3cbb941a0b2db4/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/Rtmpb249bZ/file3cbb941a0b2db4/sample1_matched_reads.fastq.gz -> /tmp/Rtmpb249bZ/file3cbb941a0b2db4/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/Rtmpb249bZ/file3cbb941a0b2db4/sample2_matched_reads.fastq.gz -> /tmp/Rtmpb249bZ/file3cbb941a0b2db4/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/Rtmpb249bZ/file3cbb941a0b2db4/sample3_matched_reads.fastq.gz -> /tmp/Rtmpb249bZ/file3cbb941a0b2db4/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 18 00:06:58 2025 ----------------
00:06:58 Sat Oct 18 2025 quantify genes 
Using BAM(s): '/tmp/Rtmpb249bZ/file3cbb941a0b2db4/sampleA_align2genome.bam',
'/tmp/Rtmpb249bZ/file3cbb941a0b2db4/sample1_align2genome.bam',
'/tmp/Rtmpb249bZ/file3cbb941a0b2db4/sample2_align2genome.bam', and
'/tmp/Rtmpb249bZ/file3cbb941a0b2db4/sample3_align2genome.bam'
parsing /tmp/Rtmpb249bZ/file3cbb941a0b2db4/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 21.40gene_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, 432991.70Read/s]
parsing /tmp/Rtmpb249bZ/file3cbb941a0b2db4/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 52.09gene_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, 1397356.08Read/s]
parsing /tmp/Rtmpb249bZ/file3cbb941a0b2db4/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 50.14gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1332879.12Read/s]
parsing /tmp/Rtmpb249bZ/file3cbb941a0b2db4/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 35.35gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 704830.27Read/s]
-- Running step: isoform_identification @ Sat Oct 18 00:06:59 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

  |                                                                            
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  |                                                                            
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  |                                                                            
  |===================================                                   |  50%
  |                                                                            
  |====================================================                  |  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 18 00:07:22 2025 -------------------
Checking for fastq file(s) /tmp/Rtmpb249bZ/file3cbb941a0b2db4/fastq, /tmp/Rtmpb249bZ/file3cbb941a0b2db4/fastq/sample1.fq.gz, /tmp/Rtmpb249bZ/file3cbb941a0b2db4/fastq/sample2.fq.gz, /tmp/Rtmpb249bZ/file3cbb941a0b2db4/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpb249bZ/file3cbb941a0b2db4/sampleA_matched_reads.fastq.gz, /tmp/Rtmpb249bZ/file3cbb941a0b2db4/sample1_matched_reads.fastq.gz, /tmp/Rtmpb249bZ/file3cbb941a0b2db4/sample2_matched_reads.fastq.gz, /tmp/Rtmpb249bZ/file3cbb941a0b2db4/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpb249bZ/file3cbb941a0b2db4/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmpb249bZ/file3cbb941a0b2db4/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmpb249bZ/file3cbb941a0b2db4/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmpb249bZ/file3cbb941a0b2db4/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/Rtmpb249bZ/file3cbb941a0b2db4/sampleA_matched_reads_dedup.fastq.gz -> /tmp/Rtmpb249bZ/file3cbb941a0b2db4/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/Rtmpb249bZ/file3cbb941a0b2db4/sample1_matched_reads_dedup.fastq.gz -> /tmp/Rtmpb249bZ/file3cbb941a0b2db4/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/Rtmpb249bZ/file3cbb941a0b2db4/sample2_matched_reads_dedup.fastq.gz -> /tmp/Rtmpb249bZ/file3cbb941a0b2db4/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/Rtmpb249bZ/file3cbb941a0b2db4/sample3_matched_reads_dedup.fastq.gz -> /tmp/Rtmpb249bZ/file3cbb941a0b2db4/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 18 00:07:23 2025 ----------
00:07:23 Sat Oct 18 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/Rtmpb249bZ/file3cbb941a0b2db4/sampleA_realign2transcript.bam...
parsing /tmp/Rtmpb249bZ/file3cbb941a0b2db4/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpb249bZ/file3cbb941a0b2db4/sampleA_realign2transcript.bamdone
parsing /tmp/Rtmpb249bZ/file3cbb941a0b2db4/sample1_realign2transcript.bam...
parsing /tmp/Rtmpb249bZ/file3cbb941a0b2db4/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpb249bZ/file3cbb941a0b2db4/sample1_realign2transcript.bamdone
parsing /tmp/Rtmpb249bZ/file3cbb941a0b2db4/sample2_realign2transcript.bam...
parsing /tmp/Rtmpb249bZ/file3cbb941a0b2db4/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpb249bZ/file3cbb941a0b2db4/sample2_realign2transcript.bamdone
parsing /tmp/Rtmpb249bZ/file3cbb941a0b2db4/sample3_realign2transcript.bam...
parsing /tmp/Rtmpb249bZ/file3cbb941a0b2db4/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpb249bZ/file3cbb941a0b2db4/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpb249bZ/file3cbb94654aac45/config_file_3980180.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 18 00:07:25 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpb249bZ/file3cbb94654aac45/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpb249bZ/file3cbb94654aac45/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpb249bZ/file3cbb94654aac45/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpb249bZ/file3cbb94654aac45/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/Rtmpb249bZ/file3cbb94654aac45/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpb249bZ/file3cbb94654aac45/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/Rtmpb249bZ/file3cbb94654aac45/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpb249bZ/file3cbb94654aac45/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/Rtmpb249bZ/file3cbb94654aac45/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpb249bZ/file3cbb94654aac45/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 18 00:07:25 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmpb249bZ/file3cbb94654aac45/sampleA_matched_reads.fastq.gz ->/tmp/Rtmpb249bZ/file3cbb94654aac45/sampleA_align2genome.bam
/tmp/Rtmpb249bZ/file3cbb94654aac45/sample1_matched_reads.fastq.gz ->/tmp/Rtmpb249bZ/file3cbb94654aac45/sample1_align2genome.bam
/tmp/Rtmpb249bZ/file3cbb94654aac45/sample2_matched_reads.fastq.gz ->/tmp/Rtmpb249bZ/file3cbb94654aac45/sample2_align2genome.bam
/tmp/Rtmpb249bZ/file3cbb94654aac45/sample3_matched_reads.fastq.gz ->/tmp/Rtmpb249bZ/file3cbb94654aac45/sample3_align2genome.bam
-- Running step: gene_quantification @ Sat Oct 18 00:07:44 2025 ----------------
00:07:44 Sat Oct 18 2025 quantify genes 
Using BAM(s): '/tmp/Rtmpb249bZ/file3cbb94654aac45/sampleA_align2genome.bam',
'/tmp/Rtmpb249bZ/file3cbb94654aac45/sample1_align2genome.bam',
'/tmp/Rtmpb249bZ/file3cbb94654aac45/sample2_align2genome.bam', and
'/tmp/Rtmpb249bZ/file3cbb94654aac45/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/Rtmpb249bZ/file3cbb94654aac45/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 22.17gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 400755.21Read/s]
parsing /tmp/Rtmpb249bZ/file3cbb94654aac45/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 38.66gene_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, 1372302.05Read/s]
parsing /tmp/Rtmpb249bZ/file3cbb94654aac45/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 47.38gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1306799.60Read/s]
parsing /tmp/Rtmpb249bZ/file3cbb94654aac45/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 36.04gene_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, 740833.69Read/s]
-- Running step: isoform_identification @ Sat Oct 18 00:07:45 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 18 00:08:06 2025 -------------------
Checking for fastq file(s) /tmp/Rtmpb249bZ/file3cbb94654aac45/fastq, /tmp/Rtmpb249bZ/file3cbb94654aac45/fastq/sample1.fq.gz, /tmp/Rtmpb249bZ/file3cbb94654aac45/fastq/sample2.fq.gz, /tmp/Rtmpb249bZ/file3cbb94654aac45/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpb249bZ/file3cbb94654aac45/sampleA_matched_reads.fastq.gz, /tmp/Rtmpb249bZ/file3cbb94654aac45/sample1_matched_reads.fastq.gz, /tmp/Rtmpb249bZ/file3cbb94654aac45/sample2_matched_reads.fastq.gz, /tmp/Rtmpb249bZ/file3cbb94654aac45/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpb249bZ/file3cbb94654aac45/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmpb249bZ/file3cbb94654aac45/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmpb249bZ/file3cbb94654aac45/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmpb249bZ/file3cbb94654aac45/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/Rtmpb249bZ/file3cbb94654aac45/sampleA_matched_reads_dedup.fastq.gz ->/tmp/Rtmpb249bZ/file3cbb94654aac45/sampleA_realign2transcript.bam
/tmp/Rtmpb249bZ/file3cbb94654aac45/sample1_matched_reads_dedup.fastq.gz ->/tmp/Rtmpb249bZ/file3cbb94654aac45/sample1_realign2transcript.bam
/tmp/Rtmpb249bZ/file3cbb94654aac45/sample2_matched_reads_dedup.fastq.gz ->/tmp/Rtmpb249bZ/file3cbb94654aac45/sample2_realign2transcript.bam
/tmp/Rtmpb249bZ/file3cbb94654aac45/sample3_matched_reads_dedup.fastq.gz ->/tmp/Rtmpb249bZ/file3cbb94654aac45/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Sat Oct 18 00:08:23 2025 ----------
00:08:23 Sat Oct 18 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/Rtmpb249bZ/file3cbb94654aac45/sampleA_realign2transcript.bam...
parsing /tmp/Rtmpb249bZ/file3cbb94654aac45/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpb249bZ/file3cbb94654aac45/sampleA_realign2transcript.bamdone
parsing /tmp/Rtmpb249bZ/file3cbb94654aac45/sample1_realign2transcript.bam...
parsing /tmp/Rtmpb249bZ/file3cbb94654aac45/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpb249bZ/file3cbb94654aac45/sample1_realign2transcript.bamdone
parsing /tmp/Rtmpb249bZ/file3cbb94654aac45/sample2_realign2transcript.bam...
parsing /tmp/Rtmpb249bZ/file3cbb94654aac45/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpb249bZ/file3cbb94654aac45/sample2_realign2transcript.bamdone
parsing /tmp/Rtmpb249bZ/file3cbb94654aac45/sample3_realign2transcript.bam...
parsing /tmp/Rtmpb249bZ/file3cbb94654aac45/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpb249bZ/file3cbb94654aac45/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpb249bZ/file3cbb9456388636/config_file_3980180.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 18 00:08:25 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpb249bZ/file3cbb9456388636/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpb249bZ/file3cbb9456388636/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpb249bZ/file3cbb9456388636/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpb249bZ/file3cbb9456388636/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/Rtmpb249bZ/file3cbb9456388636/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpb249bZ/file3cbb9456388636/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/Rtmpb249bZ/file3cbb9456388636/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpb249bZ/file3cbb9456388636/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/Rtmpb249bZ/file3cbb9456388636/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpb249bZ/file3cbb9456388636/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 18 00:08:26 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmpb249bZ/file3cbb9456388636/sampleA_matched_reads.fastq.gz -> /tmp/Rtmpb249bZ/file3cbb9456388636/sampleA_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmpb249bZ/file3cbb9456388636/sample1_matched_reads.fastq.gz -> /tmp/Rtmpb249bZ/file3cbb9456388636/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmpb249bZ/file3cbb9456388636/sample2_matched_reads.fastq.gz -> /tmp/Rtmpb249bZ/file3cbb9456388636/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmpb249bZ/file3cbb9456388636/sample3_matched_reads.fastq.gz -> /tmp/Rtmpb249bZ/file3cbb9456388636/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Sat Oct 18 00:08:27 2025 ----------------
00:08:27 Sat Oct 18 2025 quantify genes 
Using BAM(s): '/tmp/Rtmpb249bZ/file3cbb9456388636/sampleA_align2genome.bam',
'/tmp/Rtmpb249bZ/file3cbb9456388636/sample1_align2genome.bam',
'/tmp/Rtmpb249bZ/file3cbb9456388636/sample2_align2genome.bam', and
'/tmp/Rtmpb249bZ/file3cbb9456388636/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/Rtmpb249bZ/file3cbb9456388636/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 22.17gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 389964.67Read/s]
parsing /tmp/Rtmpb249bZ/file3cbb9456388636/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 49.89gene_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, 1458989.84Read/s]
parsing /tmp/Rtmpb249bZ/file3cbb9456388636/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 51.70gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1305985.80Read/s]
parsing /tmp/Rtmpb249bZ/file3cbb9456388636/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 36.10gene_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, 751829.07Read/s]
-- Running step: isoform_identification @ Sat Oct 18 00:08:28 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sat Oct 18 00:08:29 2025 -------------------
Checking for fastq file(s) /tmp/Rtmpb249bZ/file3cbb9456388636/fastq, /tmp/Rtmpb249bZ/file3cbb9456388636/fastq/sample1.fq.gz, /tmp/Rtmpb249bZ/file3cbb9456388636/fastq/sample2.fq.gz, /tmp/Rtmpb249bZ/file3cbb9456388636/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpb249bZ/file3cbb9456388636/sampleA_matched_reads.fastq.gz, /tmp/Rtmpb249bZ/file3cbb9456388636/sample1_matched_reads.fastq.gz, /tmp/Rtmpb249bZ/file3cbb9456388636/sample2_matched_reads.fastq.gz, /tmp/Rtmpb249bZ/file3cbb9456388636/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpb249bZ/file3cbb9456388636/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmpb249bZ/file3cbb9456388636/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmpb249bZ/file3cbb9456388636/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmpb249bZ/file3cbb9456388636/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/Rtmpb249bZ/file3cbb9456388636/sampleA_matched_reads_dedup.fastq.gz -> /tmp/Rtmpb249bZ/file3cbb9456388636/sampleA_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/Rtmpb249bZ/file3cbb9456388636/sample1_matched_reads_dedup.fastq.gz -> /tmp/Rtmpb249bZ/file3cbb9456388636/sample1_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/Rtmpb249bZ/file3cbb9456388636/sample2_matched_reads_dedup.fastq.gz -> /tmp/Rtmpb249bZ/file3cbb9456388636/sample2_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/Rtmpb249bZ/file3cbb9456388636/sample3_matched_reads_dedup.fastq.gz -> /tmp/Rtmpb249bZ/file3cbb9456388636/sample3_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

-- Running step: transcript_quantification @ Sat Oct 18 00:08:31 2025 ----------
2025-10-18T04:08:31.023556Z  INFO oarfish: setting user-provided filter parameters.
2025-10-18T04:08:31.024057Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpb249bZ/file3cbb9456388636/sampleA_realign2transcript.bam, contains 14 reference sequences.
2025-10-18T04:08:31.024066Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-18T04:08:31.024069Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-18T04:08:31.024179Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-18T04:08:31.024191Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-10-18T04:08:31.035720Z  INFO oarfish::single_cell: Processed 100 cells.
2025-10-18T04:08:31.559547Z  INFO oarfish: setting user-provided filter parameters.
2025-10-18T04:08:31.560028Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpb249bZ/file3cbb9456388636/sample1_realign2transcript.bam, contains 14 reference sequences.
2025-10-18T04:08:31.560039Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-18T04:08:31.560043Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-18T04:08:31.560137Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-18T04:08:31.560151Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-10-18T04:08:32.077423Z  INFO oarfish: setting user-provided filter parameters.
2025-10-18T04:08:32.077888Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpb249bZ/file3cbb9456388636/sample2_realign2transcript.bam, contains 14 reference sequences.
2025-10-18T04:08:32.077896Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-18T04:08:32.077899Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-18T04:08:32.077988Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-18T04:08:32.077996Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-10-18T04:08:32.606569Z  INFO oarfish: setting user-provided filter parameters.
2025-10-18T04:08:32.606975Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpb249bZ/file3cbb9456388636/sample3_realign2transcript.bam, contains 14 reference sequences.
2025-10-18T04:08:32.606984Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-18T04:08:32.606987Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-18T04:08:32.607075Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-18T04:08:32.607083Z  INFO oarfish: parsed reference information for 14 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpb249bZ/file3cbb9420d9a004/config_file_3980180.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 18 00:08:33 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/Rtmpb249bZ/file3cbb9420d9a004/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpb249bZ/file3cbb9420d9a004/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpb249bZ/file3cbb9420d9a004/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpb249bZ/file3cbb9420d9a004/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/Rtmpb249bZ/file3cbb9420d9a004/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpb249bZ/file3cbb9420d9a004/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/Rtmpb249bZ/file3cbb9420d9a004/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpb249bZ/file3cbb9420d9a004/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/Rtmpb249bZ/file3cbb9420d9a004/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpb249bZ/file3cbb9420d9a004/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 18 00:08:34 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmpb249bZ/file3cbb9420d9a004/sampleA_matched_reads.fastq.gz ->/tmp/Rtmpb249bZ/file3cbb9420d9a004/sampleA_align2genome.bam
/tmp/Rtmpb249bZ/file3cbb9420d9a004/sample1_matched_reads.fastq.gz ->/tmp/Rtmpb249bZ/file3cbb9420d9a004/sample1_align2genome.bam
/tmp/Rtmpb249bZ/file3cbb9420d9a004/sample2_matched_reads.fastq.gz ->/tmp/Rtmpb249bZ/file3cbb9420d9a004/sample2_align2genome.bam
/tmp/Rtmpb249bZ/file3cbb9420d9a004/sample3_matched_reads.fastq.gz ->/tmp/Rtmpb249bZ/file3cbb9420d9a004/sample3_align2genome.bam
-- Running step: gene_quantification @ Sat Oct 18 00:08:51 2025 ----------------
00:08:51 Sat Oct 18 2025 quantify genes 
Using BAM(s): '/tmp/Rtmpb249bZ/file3cbb9420d9a004/sampleA_align2genome.bam',
'/tmp/Rtmpb249bZ/file3cbb9420d9a004/sample1_align2genome.bam',
'/tmp/Rtmpb249bZ/file3cbb9420d9a004/sample2_align2genome.bam', and
'/tmp/Rtmpb249bZ/file3cbb9420d9a004/sample3_align2genome.bam'
parsing /tmp/Rtmpb249bZ/file3cbb9420d9a004/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 22.96gene_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, 385817.94Read/s]
parsing /tmp/Rtmpb249bZ/file3cbb9420d9a004/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 53.30gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1303550.47Read/s]
parsing /tmp/Rtmpb249bZ/file3cbb9420d9a004/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 52.13gene_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, 1375362.01Read/s]
parsing /tmp/Rtmpb249bZ/file3cbb9420d9a004/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 34.37gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 746051.94Read/s]
-- Running step: isoform_identification @ Sat Oct 18 00:08:52 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sat Oct 18 00:08:53 2025 -------------------
Checking for fastq file(s) /tmp/Rtmpb249bZ/file3cbb9420d9a004/fastq, /tmp/Rtmpb249bZ/file3cbb9420d9a004/fastq/sample1.fq.gz, /tmp/Rtmpb249bZ/file3cbb9420d9a004/fastq/sample2.fq.gz, /tmp/Rtmpb249bZ/file3cbb9420d9a004/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpb249bZ/file3cbb9420d9a004/sampleA_matched_reads.fastq.gz, /tmp/Rtmpb249bZ/file3cbb9420d9a004/sample1_matched_reads.fastq.gz, /tmp/Rtmpb249bZ/file3cbb9420d9a004/sample2_matched_reads.fastq.gz, /tmp/Rtmpb249bZ/file3cbb9420d9a004/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpb249bZ/file3cbb9420d9a004/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmpb249bZ/file3cbb9420d9a004/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmpb249bZ/file3cbb9420d9a004/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmpb249bZ/file3cbb9420d9a004/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/Rtmpb249bZ/file3cbb9420d9a004/sampleA_matched_reads_dedup.fastq.gz ->/tmp/Rtmpb249bZ/file3cbb9420d9a004/sampleA_realign2transcript.bam
/tmp/Rtmpb249bZ/file3cbb9420d9a004/sample1_matched_reads_dedup.fastq.gz ->/tmp/Rtmpb249bZ/file3cbb9420d9a004/sample1_realign2transcript.bam
/tmp/Rtmpb249bZ/file3cbb9420d9a004/sample2_matched_reads_dedup.fastq.gz ->/tmp/Rtmpb249bZ/file3cbb9420d9a004/sample2_realign2transcript.bam
/tmp/Rtmpb249bZ/file3cbb9420d9a004/sample3_matched_reads_dedup.fastq.gz ->/tmp/Rtmpb249bZ/file3cbb9420d9a004/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Sat Oct 18 00:09:11 2025 ----------
2025-10-18T04:09:11.737100Z  INFO oarfish: setting user-provided filter parameters.
2025-10-18T04:09:11.737615Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpb249bZ/file3cbb9420d9a004/sampleA_realign2transcript.bam, contains 14 reference sequences.
2025-10-18T04:09:11.737636Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-18T04:09:11.737639Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-18T04:09:11.737729Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-18T04:09:11.737737Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-10-18T04:09:11.749698Z  INFO oarfish::single_cell: Processed 100 cells.
2025-10-18T04:09:12.391325Z  INFO oarfish: setting user-provided filter parameters.
2025-10-18T04:09:12.391885Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpb249bZ/file3cbb9420d9a004/sample1_realign2transcript.bam, contains 14 reference sequences.
2025-10-18T04:09:12.391893Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-18T04:09:12.391897Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-18T04:09:12.391981Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-18T04:09:12.391989Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-10-18T04:09:13.007546Z  INFO oarfish: setting user-provided filter parameters.
2025-10-18T04:09:13.008087Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpb249bZ/file3cbb9420d9a004/sample2_realign2transcript.bam, contains 14 reference sequences.
2025-10-18T04:09:13.008096Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-18T04:09:13.008099Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-18T04:09:13.008196Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-18T04:09:13.008207Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-10-18T04:09:13.597992Z  INFO oarfish: setting user-provided filter parameters.
2025-10-18T04:09:13.598465Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpb249bZ/file3cbb9420d9a004/sample3_realign2transcript.bam, contains 14 reference sequences.
2025-10-18T04:09:13.598476Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-18T04:09:13.598480Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-18T04:09:13.598561Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-18T04:09:13.598570Z  INFO oarfish: parsed reference information for 14 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpb249bZ/file3cbb945f284e49/config_file_3980180.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 18 00:09:14 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpb249bZ/file3cbb945f284e49/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpb249bZ/file3cbb945f284e49/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpb249bZ/file3cbb945f284e49/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpb249bZ/file3cbb945f284e49/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/Rtmpb249bZ/file3cbb945f284e49/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpb249bZ/file3cbb945f284e49/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/Rtmpb249bZ/file3cbb945f284e49/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpb249bZ/file3cbb945f284e49/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/Rtmpb249bZ/file3cbb945f284e49/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpb249bZ/file3cbb945f284e49/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 18 00:09:15 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmpb249bZ/file3cbb945f284e49/sampleA_matched_reads.fastq.gz -> /tmp/Rtmpb249bZ/file3cbb945f284e49/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/Rtmpb249bZ/file3cbb945f284e49/sample1_matched_reads.fastq.gz -> /tmp/Rtmpb249bZ/file3cbb945f284e49/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/Rtmpb249bZ/file3cbb945f284e49/sample2_matched_reads.fastq.gz -> /tmp/Rtmpb249bZ/file3cbb945f284e49/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/Rtmpb249bZ/file3cbb945f284e49/sample3_matched_reads.fastq.gz -> /tmp/Rtmpb249bZ/file3cbb945f284e49/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 18 00:09:16 2025 ----------------
00:09:16 Sat Oct 18 2025 quantify genes 
Using BAM(s): '/tmp/Rtmpb249bZ/file3cbb945f284e49/sampleA_align2genome.bam',
'/tmp/Rtmpb249bZ/file3cbb945f284e49/sample1_align2genome.bam',
'/tmp/Rtmpb249bZ/file3cbb945f284e49/sample2_align2genome.bam', and
'/tmp/Rtmpb249bZ/file3cbb945f284e49/sample3_align2genome.bam'
parsing /tmp/Rtmpb249bZ/file3cbb945f284e49/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 22.55gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 412500.39Read/s]
parsing /tmp/Rtmpb249bZ/file3cbb945f284e49/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  7.89gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  7.87gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1366757.04Read/s]
parsing /tmp/Rtmpb249bZ/file3cbb945f284e49/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 51.28gene_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, 1296940.01Read/s]
parsing /tmp/Rtmpb249bZ/file3cbb945f284e49/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 36.64gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 724905.63Read/s]
-- Running step: isoform_identification @ Sat Oct 18 00:09:17 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sat Oct 18 00:09:18 2025 -------------------
Checking for fastq file(s) /tmp/Rtmpb249bZ/file3cbb945f284e49/fastq, /tmp/Rtmpb249bZ/file3cbb945f284e49/fastq/sample1.fq.gz, /tmp/Rtmpb249bZ/file3cbb945f284e49/fastq/sample2.fq.gz, /tmp/Rtmpb249bZ/file3cbb945f284e49/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpb249bZ/file3cbb945f284e49/sampleA_matched_reads.fastq.gz, /tmp/Rtmpb249bZ/file3cbb945f284e49/sample1_matched_reads.fastq.gz, /tmp/Rtmpb249bZ/file3cbb945f284e49/sample2_matched_reads.fastq.gz, /tmp/Rtmpb249bZ/file3cbb945f284e49/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpb249bZ/file3cbb945f284e49/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmpb249bZ/file3cbb945f284e49/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmpb249bZ/file3cbb945f284e49/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmpb249bZ/file3cbb945f284e49/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/Rtmpb249bZ/file3cbb945f284e49/sampleA_matched_reads_dedup.fastq.gz -> /tmp/Rtmpb249bZ/file3cbb945f284e49/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/Rtmpb249bZ/file3cbb945f284e49/sample1_matched_reads_dedup.fastq.gz -> /tmp/Rtmpb249bZ/file3cbb945f284e49/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/Rtmpb249bZ/file3cbb945f284e49/sample2_matched_reads_dedup.fastq.gz -> /tmp/Rtmpb249bZ/file3cbb945f284e49/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/Rtmpb249bZ/file3cbb945f284e49/sample3_matched_reads_dedup.fastq.gz -> /tmp/Rtmpb249bZ/file3cbb945f284e49/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 18 00:09:19 2025 ----------
00:09:19 Sat Oct 18 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/Rtmpb249bZ/file3cbb945f284e49/sampleA_realign2transcript.bam...
parsing /tmp/Rtmpb249bZ/file3cbb945f284e49/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpb249bZ/file3cbb945f284e49/sampleA_realign2transcript.bamdone
parsing /tmp/Rtmpb249bZ/file3cbb945f284e49/sample1_realign2transcript.bam...
parsing /tmp/Rtmpb249bZ/file3cbb945f284e49/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpb249bZ/file3cbb945f284e49/sample1_realign2transcript.bamdone
parsing /tmp/Rtmpb249bZ/file3cbb945f284e49/sample2_realign2transcript.bam...
parsing /tmp/Rtmpb249bZ/file3cbb945f284e49/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpb249bZ/file3cbb945f284e49/sample2_realign2transcript.bamdone
parsing /tmp/Rtmpb249bZ/file3cbb945f284e49/sample3_realign2transcript.bam...
parsing /tmp/Rtmpb249bZ/file3cbb945f284e49/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpb249bZ/file3cbb945f284e49/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpb249bZ/file3cbb94732f6a92/config_file_3980180.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 18 00:09:22 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/Rtmpb249bZ/file3cbb94732f6a92/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpb249bZ/file3cbb94732f6a92/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpb249bZ/file3cbb94732f6a92/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpb249bZ/file3cbb94732f6a92/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/Rtmpb249bZ/file3cbb94732f6a92/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpb249bZ/file3cbb94732f6a92/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/Rtmpb249bZ/file3cbb94732f6a92/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpb249bZ/file3cbb94732f6a92/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/Rtmpb249bZ/file3cbb94732f6a92/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpb249bZ/file3cbb94732f6a92/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 18 00:09:22 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmpb249bZ/file3cbb94732f6a92/sampleA_matched_reads.fastq.gz ->/tmp/Rtmpb249bZ/file3cbb94732f6a92/sampleA_align2genome.bam
/tmp/Rtmpb249bZ/file3cbb94732f6a92/sample1_matched_reads.fastq.gz ->/tmp/Rtmpb249bZ/file3cbb94732f6a92/sample1_align2genome.bam
/tmp/Rtmpb249bZ/file3cbb94732f6a92/sample2_matched_reads.fastq.gz ->/tmp/Rtmpb249bZ/file3cbb94732f6a92/sample2_align2genome.bam
/tmp/Rtmpb249bZ/file3cbb94732f6a92/sample3_matched_reads.fastq.gz ->/tmp/Rtmpb249bZ/file3cbb94732f6a92/sample3_align2genome.bam
-- Running step: gene_quantification @ Sat Oct 18 00:09:41 2025 ----------------
00:09:41 Sat Oct 18 2025 quantify genes 
Using BAM(s): '/tmp/Rtmpb249bZ/file3cbb94732f6a92/sampleA_align2genome.bam',
'/tmp/Rtmpb249bZ/file3cbb94732f6a92/sample1_align2genome.bam',
'/tmp/Rtmpb249bZ/file3cbb94732f6a92/sample2_align2genome.bam', and
'/tmp/Rtmpb249bZ/file3cbb94732f6a92/sample3_align2genome.bam'
	Counter({'counted_reads': 358})
	Counter({'counted_reads': 91})
	Counter({'counted_reads': 95})
	Counter({'counted_reads': 176})
parsing /tmp/Rtmpb249bZ/file3cbb94732f6a92/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 18.32gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 349956.95Read/s]
parsing /tmp/Rtmpb249bZ/file3cbb94732f6a92/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 47.23gene_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, 1246227.72Read/s]
parsing /tmp/Rtmpb249bZ/file3cbb94732f6a92/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 49.84gene_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, 1150763.83Read/s]
parsing /tmp/Rtmpb249bZ/file3cbb94732f6a92/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 34.65gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 739997.18Read/s]
-- Running step: isoform_identification @ Sat Oct 18 00:09:42 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sat Oct 18 00:09:42 2025 -------------------
Checking for fastq file(s) /tmp/Rtmpb249bZ/file3cbb94732f6a92/fastq, /tmp/Rtmpb249bZ/file3cbb94732f6a92/fastq/sample1.fq.gz, /tmp/Rtmpb249bZ/file3cbb94732f6a92/fastq/sample2.fq.gz, /tmp/Rtmpb249bZ/file3cbb94732f6a92/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpb249bZ/file3cbb94732f6a92/sampleA_matched_reads.fastq.gz, /tmp/Rtmpb249bZ/file3cbb94732f6a92/sample1_matched_reads.fastq.gz, /tmp/Rtmpb249bZ/file3cbb94732f6a92/sample2_matched_reads.fastq.gz, /tmp/Rtmpb249bZ/file3cbb94732f6a92/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpb249bZ/file3cbb94732f6a92/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmpb249bZ/file3cbb94732f6a92/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmpb249bZ/file3cbb94732f6a92/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmpb249bZ/file3cbb94732f6a92/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/Rtmpb249bZ/file3cbb94732f6a92/sampleA_matched_reads_dedup.fastq.gz ->/tmp/Rtmpb249bZ/file3cbb94732f6a92/sampleA_realign2transcript.bam
/tmp/Rtmpb249bZ/file3cbb94732f6a92/sample1_matched_reads_dedup.fastq.gz ->/tmp/Rtmpb249bZ/file3cbb94732f6a92/sample1_realign2transcript.bam
/tmp/Rtmpb249bZ/file3cbb94732f6a92/sample2_matched_reads_dedup.fastq.gz ->/tmp/Rtmpb249bZ/file3cbb94732f6a92/sample2_realign2transcript.bam
/tmp/Rtmpb249bZ/file3cbb94732f6a92/sample3_matched_reads_dedup.fastq.gz ->/tmp/Rtmpb249bZ/file3cbb94732f6a92/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Sat Oct 18 00:10:00 2025 ----------
00:10:00 Sat Oct 18 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/Rtmpb249bZ/file3cbb94732f6a92/sampleA_realign2transcript.bam...
parsing /tmp/Rtmpb249bZ/file3cbb94732f6a92/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpb249bZ/file3cbb94732f6a92/sampleA_realign2transcript.bamdone
parsing /tmp/Rtmpb249bZ/file3cbb94732f6a92/sample1_realign2transcript.bam...
parsing /tmp/Rtmpb249bZ/file3cbb94732f6a92/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpb249bZ/file3cbb94732f6a92/sample1_realign2transcript.bamdone
parsing /tmp/Rtmpb249bZ/file3cbb94732f6a92/sample2_realign2transcript.bam...
parsing /tmp/Rtmpb249bZ/file3cbb94732f6a92/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpb249bZ/file3cbb94732f6a92/sample2_realign2transcript.bamdone
parsing /tmp/Rtmpb249bZ/file3cbb94732f6a92/sample3_realign2transcript.bam...
parsing /tmp/Rtmpb249bZ/file3cbb94732f6a92/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpb249bZ/file3cbb94732f6a92/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 
704.477  43.711 729.938 

Example timings

FLAMES.Rcheck/FLAMES-Ex.timings

nameusersystemelapsed
BulkPipeline3.4150.2043.483
MultiSampleSCPipeline 9.797 0.64110.799
SingleCellPipeline2.7470.1151.703
add_gene_counts0.2680.0050.273
annotation_to_fasta0.1920.0090.201
blaze 4.33616.93911.813
bulk_long_pipeline 2.35912.037 2.441
combine_sce0.6770.1050.782
config-set0.1560.0070.164
config0.1470.0100.157
controllers-set0.3400.0240.367
controllers0.2040.0080.212
convolution_filter0.0000.0000.001
create_config0.0100.0000.011
create_sce_from_dir3.3902.1933.511
create_se_from_dir2.4840.1152.596
cutadapt0.1000.0140.113
example_pipeline0.3000.0090.309
experiment2.1240.0752.198
filter_annotation0.0400.0020.043
filter_coverage0.9690.0441.015
find_barcode1.4700.2001.677
find_bin0.0020.0020.004
find_variants19.130 0.09818.616
get_coverage0.9660.0371.005
index_genome0.1470.0070.154
mutation_positions1.3740.0681.441
plot_coverage2.6740.0432.719
plot_demultiplex2.5670.1592.756
plot_demultiplex_raw1.6230.0421.669
plot_durations2.3380.0822.420
plot_isoform_heatmap6.7810.1156.896
plot_isoform_reduced_dim22.864 0.28423.146
plot_isoforms3.2420.0023.244
resume_FLAMES2.2970.0772.373
run_FLAMES2.1110.0772.185
run_step1.0040.0381.043
sc_DTU_analysis6.7441.7676.518
sc_gene_entropy1.5130.1401.819
sc_genotype3.0600.6102.616
sc_impute_transcript0.5830.0010.583
sc_long_multisample_pipeline8.1006.5768.165
sc_long_pipeline3.0831.5872.708
sc_mutations2.7970.5742.804
sc_plot_genotype10.591 0.75310.197
show-FLAMESPipeline0.2950.0060.301
steps-set0.4330.0220.455
steps0.1370.0160.153
weight_transcripts0.0250.0030.028