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###
### 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_2.4.2.tar.gz
###
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* using log directory ‘/home/biocbuild/bbs-3.22-bioc/meat/FLAMES.Rcheck’
* using R version 4.5.2 (2025-10-31)
* 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 ‘2.4.2’
* 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 ‘outdir’
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 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 ‘outdir’
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 global
  function definition for ‘setNames’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘j’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘genome_bam’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘minimap2’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘samtools’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘threads’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘outdir’
plot_durations,FLAMES.Pipeline: no visible binding for global variable
  ‘step’
plot_durations,FLAMES.Pipeline: no visible binding for global variable
  ‘duration’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘j’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘transcriptome_assembly’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘transcriptome_bam’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘minimap2’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘samtools’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘outdir’
resume_FLAMES,FLAMES.Pipeline : <anonymous>: no visible global function
  definition for ‘capture.output’
run_FLAMES,FLAMES.Pipeline : <anonymous>: no visible global function
  definition for ‘capture.output’
Undefined global functions or variables:
  BarcodeEditDist CellBarcode Count FSM_match FlankEditDist Freq Reads
  Sample Type UMI UMI_count adj.p.value allele allele_count as
  bam_index barcode barcode_rank barcodes_file capture.output
  cell_total_reads chisq.test count counts_no_ins cumpct demultiplexed
  reads demultiplexed_fastq download.file duration everything
  expect_cell_number expr fastq filter_res genome_bam genotype head
  imageX imageY in_tissue input j length_bin max_length min_length
  minimap2 multi-matching reads mutation_index n_reads na.omit name new
  nucleotide outdir output p.value packageVersion pct pos
  read1_with_adapter read_counts ref samtools scale_alpha_continuous
  scale_colour_gradient setNames single match reads starts_with step
  test threads total total reads total_counts tr_length transcript
  transcriptome_assembly transcriptome_bam undemultiplexted reads unzip
  value which_label x y
Consider adding
  importFrom("base", "match", "single")
  importFrom("methods", "as", "new")
  importFrom("stats", "chisq.test", "na.omit", "setNames", "step")
  importFrom("utils", "capture.output", "download.file", "head",
             "packageVersion", "unzip")
to your NAMESPACE file (and ensure that your DESCRIPTION Imports field
contains 'methods').
* checking Rd files ... OK
* checking Rd metadata ... OK
* checking Rd cross-references ... OK
* checking for missing documentation entries ... OK
* checking for code/documentation mismatches ... OK
* checking Rd \usage sections ... OK
* checking Rd contents ... OK
* checking for unstated dependencies in examples ... OK
* checking contents of ‘data’ directory ... OK
* checking data for non-ASCII characters ... OK
* checking data for ASCII and uncompressed saves ... OK
* checking line endings in shell scripts ... OK
* checking line endings in C/C++/Fortran sources/headers ... OK
* checking line endings in Makefiles ... OK
* checking compilation flags in Makevars ... OK
* checking for GNU extensions in Makefiles ... INFO
GNU make is a SystemRequirements.
* checking for portable use of $(BLAS_LIBS) and $(LAPACK_LIBS) ... OK
* checking use of PKG_*FLAGS in Makefiles ... OK
* checking include directives in Makefiles ... OK
* checking compiled code ... NOTE
Note: information on .o files is not available
File ‘/home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/libs/FLAMES.so’:
  Found ‘abort’, possibly from ‘abort’ (C)
  Found ‘exit’, possibly from ‘exit’ (C)
  Found ‘stderr’, possibly from ‘stderr’ (C)
  Found ‘stdout’, possibly from ‘stdout’ (C)

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

See ‘Writing portable packages’ in the ‘Writing R Extensions’ manual.
* checking files in ‘vignettes’ ... OK
* checking examples ... OK
Examples with CPU (user + system) or elapsed time > 5s
                               user system elapsed
plot_isoform_reduced_dim     23.789  1.490  25.277
blaze                         4.711 18.268  12.917
find_variants                20.139  0.057  19.589
sc_long_multisample_pipeline  8.044  6.776   8.204
bulk_long_pipeline            2.316 12.163   2.435
sc_plot_genotype             11.109  0.997  10.950
MultiSampleSCPipeline         9.837  0.538  10.782
sc_DTU_analysis               7.312  2.050   7.360
plot_isoform_heatmap          7.389  0.720   8.109
create_sce_from_dir           3.457  2.304   3.631
* 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.

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###
### Running command:
###
###   /home/biocbuild/bbs-3.22-bioc/R/bin/R CMD INSTALL FLAMES
###
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* installing to library ‘/home/biocbuild/bbs-3.22-bioc/R/site-library’
* installing *source* package ‘FLAMES’ ...
** this is package ‘FLAMES’ version ‘2.4.2’
** 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)

R version 4.5.2 (2025-10-31) -- "[Not] Part in a Rumble"
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.

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Type 'contributors()' for more information and
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Type 'demo()' for some demos, 'help()' for on-line help, or
'help.start()' for an HTML browser interface to help.
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> # 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/RtmpNbUBPg/file7589624d0c149/config_file_481430.json 
Writing configuration parameters to:  /tmp/RtmpNbUBPg/file7589624d0c149/config_file_481430.json 
Writing configuration parameters to:  /tmp/RtmpNbUBPg/file7589624d0c149/config_file_481430.json 
Writing configuration parameters to:  /tmp/RtmpNbUBPg/file7589613b7af25/config_file_481430.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/RtmpNbUBPg/file7589617563a52/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/RtmpNbUBPg/file758963446641d/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/RtmpNbUBPg/file758963446641d/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpNbUBPg/file758963c1e1ba1/musc_rps24_1.fastq
Searching for barcodes...
Processing file: /tmp/RtmpNbUBPg/file758963c1e1ba1/musc_rps24_2.fastq
Searching for barcodes...
Processing file: /tmp/RtmpNbUBPg/file758963c1e1ba1/musc_rps24_3.fastq
Searching for barcodes...
Processing file: /tmp/RtmpNbUBPg/file758963c1e1ba1/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/RtmpNbUBPg/file758961822994c/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/RtmpNbUBPg/file758964fb7ef9f/config_file_481430.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 @ Thu Feb 26 23:42:47 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpNbUBPg/file758964fb7ef9f/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpNbUBPg/file758964fb7ef9f/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpNbUBPg/file758964fb7ef9f/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Thu Feb 26 23:42:48 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 4 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Thu Feb 26 23:43:12 2026 -------------------
Realigning sample sample1 -> /tmp/RtmpNbUBPg/file758964fb7ef9f/sample1_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample2 -> /tmp/RtmpNbUBPg/file758964fb7ef9f/sample2_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample3 -> /tmp/RtmpNbUBPg/file758964fb7ef9f/sample3_realign2transcript.bam
Skipped sorting BAM files.

-- Running step: transcript_quantification @ Thu Feb 26 23:43:12 2026 ----------
2026-02-27T04:43:13.012018Z  INFO oarfish: setting user-provided filter parameters.
2026-02-27T04:43:13.012490Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpNbUBPg/file758964fb7ef9f/sample1_realign2transcript.bam, contains 5 reference sequences.
2026-02-27T04:43:13.012501Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-02-27T04:43:13.012528Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-02-27T04:43:13.012579Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-02-27T04:43:13.012585Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-02-27T04:43:13.014274Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-02-27T04:43:13.014393Z  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    │
╰─────────────────────────────────┴───────╯

2026-02-27T04:43:13.014416Z  INFO oarfish::bulk: Total number of alignment records : 116
2026-02-27T04:43:13.014419Z  INFO oarfish::bulk: number of aligned reads : 96
2026-02-27T04:43:13.014421Z  INFO oarfish::bulk: number of unique alignments : 86
2026-02-27T04:43:13.014993Z  INFO oarfish: oarfish completed successfully.
2026-02-27T04:43:13.022136Z  INFO oarfish: setting user-provided filter parameters.
2026-02-27T04:43:13.022466Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpNbUBPg/file758964fb7ef9f/sample2_realign2transcript.bam, contains 5 reference sequences.
2026-02-27T04:43:13.022474Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-02-27T04:43:13.022476Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-02-27T04:43:13.022538Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-02-27T04:43:13.022545Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-02-27T04:43:13.024062Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-02-27T04:43:13.024174Z  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    │
╰─────────────────────────────────┴───────╯

2026-02-27T04:43:13.024197Z  INFO oarfish::bulk: Total number of alignment records : 114
2026-02-27T04:43:13.024199Z  INFO oarfish::bulk: number of aligned reads : 95
2026-02-27T04:43:13.024209Z  INFO oarfish::bulk: number of unique alignments : 82
2026-02-27T04:43:13.024771Z  INFO oarfish: oarfish completed successfully.
2026-02-27T04:43:13.031475Z  INFO oarfish: setting user-provided filter parameters.
2026-02-27T04:43:13.031818Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpNbUBPg/file758964fb7ef9f/sample3_realign2transcript.bam, contains 5 reference sequences.
2026-02-27T04:43:13.031828Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-02-27T04:43:13.031831Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-02-27T04:43:13.031883Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-02-27T04:43:13.031888Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-02-27T04:43:13.034610Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2026-02-27T04:43:13.034759Z  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   │
╰─────────────────────────────────┴───────╯

2026-02-27T04:43:13.034786Z  INFO oarfish::bulk: Total number of alignment records : 239
2026-02-27T04:43:13.034789Z  INFO oarfish::bulk: number of aligned reads : 179
2026-02-27T04:43:13.034791Z  INFO oarfish::bulk: number of unique alignments : 143
2026-02-27T04:43:13.035465Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpNbUBPg/file7589652641393/config_file_481430.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 @ Thu Feb 26 23:43:13 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpNbUBPg/file7589652641393/sample1_align2genome.bam
sample2 ->/tmp/RtmpNbUBPg/file7589652641393/sample2_align2genome.bam
sample3 ->/tmp/RtmpNbUBPg/file7589652641393/sample3_align2genome.bam
-- Running step: isoform_identification @ Thu Feb 26 23:43:34 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

  |                                                                            
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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 @ Thu Feb 26 23:43:53 2026 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpNbUBPg/file7589652641393/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpNbUBPg/file7589652641393/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpNbUBPg/file7589652641393/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Thu Feb 26 23:44:14 2026 ----------
2026-02-27T04:44:14.211858Z  INFO oarfish: setting user-provided filter parameters.
2026-02-27T04:44:14.212468Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpNbUBPg/file7589652641393/sample1_realign2transcript.bam, contains 5 reference sequences.
2026-02-27T04:44:14.212480Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-02-27T04:44:14.212484Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-02-27T04:44:14.212551Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-02-27T04:44:14.212559Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-02-27T04:44:14.214526Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-02-27T04:44:14.214674Z  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    │
╰─────────────────────────────────┴───────╯

2026-02-27T04:44:14.214703Z  INFO oarfish::bulk: Total number of alignment records : 116
2026-02-27T04:44:14.214706Z  INFO oarfish::bulk: number of aligned reads : 96
2026-02-27T04:44:14.214710Z  INFO oarfish::bulk: number of unique alignments : 86
2026-02-27T04:44:14.215351Z  INFO oarfish: oarfish completed successfully.
2026-02-27T04:44:14.225747Z  INFO oarfish: setting user-provided filter parameters.
2026-02-27T04:44:14.226136Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpNbUBPg/file7589652641393/sample2_realign2transcript.bam, contains 5 reference sequences.
2026-02-27T04:44:14.226148Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-02-27T04:44:14.226165Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-02-27T04:44:14.226229Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-02-27T04:44:14.226234Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-02-27T04:44:14.227795Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-02-27T04:44:14.227934Z  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    │
╰─────────────────────────────────┴───────╯

2026-02-27T04:44:14.227960Z  INFO oarfish::bulk: Total number of alignment records : 114
2026-02-27T04:44:14.227962Z  INFO oarfish::bulk: number of aligned reads : 95
2026-02-27T04:44:14.227965Z  INFO oarfish::bulk: number of unique alignments : 82
2026-02-27T04:44:14.228581Z  INFO oarfish: oarfish completed successfully.
2026-02-27T04:44:14.237850Z  INFO oarfish: setting user-provided filter parameters.
2026-02-27T04:44:14.238474Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpNbUBPg/file7589652641393/sample3_realign2transcript.bam, contains 5 reference sequences.
2026-02-27T04:44:14.238483Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-02-27T04:44:14.238486Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-02-27T04:44:14.238552Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-02-27T04:44:14.238563Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-02-27T04:44:14.241487Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2026-02-27T04:44:14.241675Z  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   │
╰─────────────────────────────────┴───────╯

2026-02-27T04:44:14.241715Z  INFO oarfish::bulk: Total number of alignment records : 239
2026-02-27T04:44:14.241718Z  INFO oarfish::bulk: number of aligned reads : 179
2026-02-27T04:44:14.241733Z  INFO oarfish::bulk: number of unique alignments : 143
2026-02-27T04:44:14.242445Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpNbUBPg/file758967380cc07/config_file_481430.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 @ Thu Feb 26 23:44:14 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpNbUBPg/file758967380cc07/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpNbUBPg/file758967380cc07/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpNbUBPg/file758967380cc07/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Thu Feb 26 23:44:15 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 4 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Thu Feb 26 23:44:33 2026 -------------------
Realigning sample sample1 -> /tmp/RtmpNbUBPg/file758967380cc07/sample1_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample2 -> /tmp/RtmpNbUBPg/file758967380cc07/sample2_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample3 -> /tmp/RtmpNbUBPg/file758967380cc07/sample3_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Thu Feb 26 23:44:33 2026 ----------
23:44:33 Thu Feb 26 2026 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/RtmpNbUBPg/file75896c471bac/config_file_481430.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 @ Thu Feb 26 23:44:34 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpNbUBPg/file75896c471bac/sample1_align2genome.bam
sample2 ->/tmp/RtmpNbUBPg/file75896c471bac/sample2_align2genome.bam
sample3 ->/tmp/RtmpNbUBPg/file75896c471bac/sample3_align2genome.bam
-- Running step: isoform_identification @ Thu Feb 26 23:44:55 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 4 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Thu Feb 26 23:45:13 2026 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpNbUBPg/file75896c471bac/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpNbUBPg/file75896c471bac/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpNbUBPg/file75896c471bac/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Thu Feb 26 23:45:32 2026 ----------
23:45:32 Thu Feb 26 2026 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Inputs:  ['/tmp/RtmpNbUBPg/file758967380cc07/sample1_realign2transcript.bam', '/tmp/RtmpNbUBPg/file758967380cc07/sample2_realign2transcript.bam', '/tmp/RtmpNbUBPg/file758967380cc07/sample3_realign2transcript.bam'] /tmp/RtmpNbUBPg/file758967380cc07/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/RtmpNbUBPg/file7589678412770/config_file_481430.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 @ Thu Feb 26 23:45:33 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpNbUBPg/file7589678412770/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpNbUBPg/file7589678412770/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpNbUBPg/file7589678412770/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Thu Feb 26 23:45:34 2026 -------------
Inputs:  ['/tmp/RtmpNbUBPg/file75896c471bac/sample1_realign2transcript.bam', '/tmp/RtmpNbUBPg/file75896c471bac/sample2_realign2transcript.bam', '/tmp/RtmpNbUBPg/file75896c471bac/sample3_realign2transcript.bam'] /tmp/RtmpNbUBPg/file75896c471bac/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 @ Thu Feb 26 23:45:34 2026 -------------------
Realigning sample sample1 -> /tmp/RtmpNbUBPg/file7589678412770/sample1_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample2 -> /tmp/RtmpNbUBPg/file7589678412770/sample2_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample3 -> /tmp/RtmpNbUBPg/file7589678412770/sample3_realign2transcript.bam
Skipped sorting BAM files.

-- Running step: transcript_quantification @ Thu Feb 26 23:45:35 2026 ----------
2026-02-27T04:45:35.509581Z  INFO oarfish: setting user-provided filter parameters.
2026-02-27T04:45:35.509928Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpNbUBPg/file7589678412770/sample1_realign2transcript.bam, contains 10 reference sequences.
2026-02-27T04:45:35.509936Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-02-27T04:45:35.509939Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-02-27T04:45:35.510005Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-02-27T04:45:35.510011Z  INFO oarfish: parsed reference information for 10 transcripts.
2026-02-27T04:45:35.512457Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-02-27T04:45:35.512592Z  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    │
╰─────────────────────────────────┴───────╯

2026-02-27T04:45:35.512614Z  INFO oarfish::bulk: Total number of alignment records : 125
2026-02-27T04:45:35.512616Z  INFO oarfish::bulk: number of aligned reads : 98
2026-02-27T04:45:35.512618Z  INFO oarfish::bulk: number of unique alignments : 86
2026-02-27T04:45:35.513185Z  INFO oarfish: oarfish completed successfully.
2026-02-27T04:45:35.520405Z  INFO oarfish: setting user-provided filter parameters.
2026-02-27T04:45:35.520746Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpNbUBPg/file7589678412770/sample2_realign2transcript.bam, contains 10 reference sequences.
2026-02-27T04:45:35.520755Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-02-27T04:45:35.520758Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-02-27T04:45:35.520824Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-02-27T04:45:35.520830Z  INFO oarfish: parsed reference information for 10 transcripts.
2026-02-27T04:45:35.523332Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-02-27T04:45:35.523449Z  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    │
╰─────────────────────────────────┴───────╯

2026-02-27T04:45:35.523482Z  INFO oarfish::bulk: Total number of alignment records : 136
2026-02-27T04:45:35.523484Z  INFO oarfish::bulk: number of aligned reads : 97
2026-02-27T04:45:35.523486Z  INFO oarfish::bulk: number of unique alignments : 79
2026-02-27T04:45:35.524048Z  INFO oarfish: oarfish completed successfully.
2026-02-27T04:45:35.531140Z  INFO oarfish: setting user-provided filter parameters.
2026-02-27T04:45:35.531491Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpNbUBPg/file7589678412770/sample3_realign2transcript.bam, contains 10 reference sequences.
2026-02-27T04:45:35.531499Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-02-27T04:45:35.531502Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-02-27T04:45:35.531572Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-02-27T04:45:35.531581Z  INFO oarfish: parsed reference information for 10 transcripts.
2026-02-27T04:45:35.535762Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-02-27T04:45:35.535922Z  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   │
╰─────────────────────────────────┴───────╯

2026-02-27T04:45:35.535951Z  INFO oarfish::bulk: Total number of alignment records : 272
2026-02-27T04:45:35.535953Z  INFO oarfish::bulk: number of aligned reads : 187
2026-02-27T04:45:35.535955Z  INFO oarfish::bulk: number of unique alignments : 140
2026-02-27T04:45:35.536664Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpNbUBPg/file758965560da8a/config_file_481430.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 @ Thu Feb 26 23:45:35 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpNbUBPg/file758965560da8a/sample1_align2genome.bam
sample2 ->/tmp/RtmpNbUBPg/file758965560da8a/sample2_align2genome.bam
sample3 ->/tmp/RtmpNbUBPg/file758965560da8a/sample3_align2genome.bam
-- Running step: isoform_identification @ Thu Feb 26 23:45:55 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Thu Feb 26 23:45:55 2026 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpNbUBPg/file758965560da8a/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpNbUBPg/file758965560da8a/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpNbUBPg/file758965560da8a/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Thu Feb 26 23:46:15 2026 ----------
2026-02-27T04:46:15.083394Z  INFO oarfish: setting user-provided filter parameters.
2026-02-27T04:46:15.083875Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpNbUBPg/file758965560da8a/sample1_realign2transcript.bam, contains 10 reference sequences.
2026-02-27T04:46:15.083886Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-02-27T04:46:15.083890Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-02-27T04:46:15.083960Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-02-27T04:46:15.083968Z  INFO oarfish: parsed reference information for 10 transcripts.
2026-02-27T04:46:15.086497Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-02-27T04:46:15.086634Z  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    │
╰─────────────────────────────────┴───────╯

2026-02-27T04:46:15.086657Z  INFO oarfish::bulk: Total number of alignment records : 125
2026-02-27T04:46:15.086661Z  INFO oarfish::bulk: number of aligned reads : 98
2026-02-27T04:46:15.086664Z  INFO oarfish::bulk: number of unique alignments : 86
2026-02-27T04:46:15.087292Z  INFO oarfish: oarfish completed successfully.
2026-02-27T04:46:15.094869Z  INFO oarfish: setting user-provided filter parameters.
2026-02-27T04:46:15.095246Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpNbUBPg/file758965560da8a/sample2_realign2transcript.bam, contains 10 reference sequences.
2026-02-27T04:46:15.095257Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-02-27T04:46:15.095261Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-02-27T04:46:15.095331Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-02-27T04:46:15.095350Z  INFO oarfish: parsed reference information for 10 transcripts.
2026-02-27T04:46:15.098072Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-02-27T04:46:15.098194Z  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    │
╰─────────────────────────────────┴───────╯

2026-02-27T04:46:15.098218Z  INFO oarfish::bulk: Total number of alignment records : 136
2026-02-27T04:46:15.098220Z  INFO oarfish::bulk: number of aligned reads : 97
2026-02-27T04:46:15.098222Z  INFO oarfish::bulk: number of unique alignments : 79
2026-02-27T04:46:15.098818Z  INFO oarfish: oarfish completed successfully.
2026-02-27T04:46:15.106010Z  INFO oarfish: setting user-provided filter parameters.
2026-02-27T04:46:15.106368Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpNbUBPg/file758965560da8a/sample3_realign2transcript.bam, contains 10 reference sequences.
2026-02-27T04:46:15.106375Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-02-27T04:46:15.106379Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-02-27T04:46:15.106441Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-02-27T04:46:15.106447Z  INFO oarfish: parsed reference information for 10 transcripts.
2026-02-27T04:46:15.110643Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-02-27T04:46:15.110814Z  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   │
╰─────────────────────────────────┴───────╯

2026-02-27T04:46:15.110843Z  INFO oarfish::bulk: Total number of alignment records : 272
2026-02-27T04:46:15.110845Z  INFO oarfish::bulk: number of aligned reads : 187
2026-02-27T04:46:15.110847Z  INFO oarfish::bulk: number of unique alignments : 140
2026-02-27T04:46:15.111538Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpNbUBPg/file75896634a8086/config_file_481430.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 @ Thu Feb 26 23:46:15 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpNbUBPg/file75896634a8086/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpNbUBPg/file75896634a8086/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpNbUBPg/file75896634a8086/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Thu Feb 26 23:46:16 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Thu Feb 26 23:46:16 2026 -------------------
Realigning sample sample1 -> /tmp/RtmpNbUBPg/file75896634a8086/sample1_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample2 -> /tmp/RtmpNbUBPg/file75896634a8086/sample2_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample3 -> /tmp/RtmpNbUBPg/file75896634a8086/sample3_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Thu Feb 26 23:46:17 2026 ----------
23:46:17 Thu Feb 26 2026 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/RtmpNbUBPg/file758964118efc3/config_file_481430.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 @ Thu Feb 26 23:46:18 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpNbUBPg/file758964118efc3/sample1_align2genome.bam
sample2 ->/tmp/RtmpNbUBPg/file758964118efc3/sample2_align2genome.bam
sample3 ->/tmp/RtmpNbUBPg/file758964118efc3/sample3_align2genome.bam
-- Running step: isoform_identification @ Thu Feb 26 23:46:37 2026 -------------
Inputs:  ['/tmp/RtmpNbUBPg/file75896634a8086/sample1_realign2transcript.bam', '/tmp/RtmpNbUBPg/file75896634a8086/sample2_realign2transcript.bam', '/tmp/RtmpNbUBPg/file75896634a8086/sample3_realign2transcript.bam'] /tmp/RtmpNbUBPg/file75896634a8086/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 @ Thu Feb 26 23:46:38 2026 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpNbUBPg/file758964118efc3/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpNbUBPg/file758964118efc3/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpNbUBPg/file758964118efc3/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Thu Feb 26 23:46:56 2026 ----------
23:46:56 Thu Feb 26 2026 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/RtmpNbUBPg/file758964a2c2859/config_file_481430.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 @ Thu Feb 26 23:46:57 2026 ----------------
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/RtmpNbUBPg/file758964a2c2859/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 @ Thu Feb 26 23:46:57 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpNbUBPg/file758964a2c2859/matched_reads.fastq.gz -> /tmp/RtmpNbUBPg/file758964a2c2859/align2genome.bam
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: isoform_identification @ Thu Feb 26 23:46:57 2026 -------------
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 @ Thu Feb 26 23:47:06 2026 -------------------
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/RtmpNbUBPg/file758964a2c2859/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpNbUBPg/file758964a2c2859/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/RtmpNbUBPg/file758964a2c2859/matched_reads.fastq.gz -> /tmp/RtmpNbUBPg/file758964a2c2859/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 @ Thu Feb 26 23:47:07 2026 ----------
2026-02-27T04:47:07.117735Z  INFO oarfish: setting user-provided filter parameters.
2026-02-27T04:47:07.118314Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpNbUBPg/file758964a2c2859/realign2transcript.bam, contains 5 reference sequences.
2026-02-27T04:47:07.118329Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-02-27T04:47:07.118333Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-02-27T04:47:07.118399Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-02-27T04:47:07.118406Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-02-27T04:47:07.124797Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpNbUBPg/file758961f2c917f/config_file_481430.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 @ Thu Feb 26 23:47:07 2026 ----------------
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/RtmpNbUBPg/file758961f2c917f/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 @ Thu Feb 26 23:47:07 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpNbUBPg/file758961f2c917f/matched_reads.fastq.gz ->/tmp/RtmpNbUBPg/file758961f2c917f/align2genome.bam
-- Running step: isoform_identification @ Thu Feb 26 23:47:25 2026 -------------
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 @ Thu Feb 26 23:47:34 2026 -------------------
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/RtmpNbUBPg/file758961f2c917f/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpNbUBPg/file758961f2c917f/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/RtmpNbUBPg/file758961f2c917f/matched_reads.fastq.gz ->/tmp/RtmpNbUBPg/file758961f2c917f/realign2transcript.bam
-- Running step: transcript_quantification @ Thu Feb 26 23:47:53 2026 ----------
2026-02-27T04:47:53.293555Z  INFO oarfish: setting user-provided filter parameters.
2026-02-27T04:47:53.293969Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpNbUBPg/file758961f2c917f/realign2transcript.bam, contains 5 reference sequences.
2026-02-27T04:47:53.293979Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-02-27T04:47:53.293982Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-02-27T04:47:53.294037Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-02-27T04:47:53.294045Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-02-27T04:47:53.300299Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpNbUBPg/file758962d0a5e52/config_file_481430.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 @ Thu Feb 26 23:47:53 2026 ----------------
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/RtmpNbUBPg/file758962d0a5e52/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 @ Thu Feb 26 23:47:54 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpNbUBPg/file758962d0a5e52/matched_reads.fastq.gz -> /tmp/RtmpNbUBPg/file758962d0a5e52/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 @ Thu Feb 26 23:47:54 2026 -------------
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 @ Thu Feb 26 23:48:03 2026 -------------------
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/RtmpNbUBPg/file758962d0a5e52/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpNbUBPg/file758962d0a5e52/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/RtmpNbUBPg/file758962d0a5e52/matched_reads.fastq.gz -> /tmp/RtmpNbUBPg/file758962d0a5e52/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 @ Thu Feb 26 23:48:03 2026 ----------
23:48:03 Thu Feb 26 2026 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Inputs:  ['/tmp/RtmpNbUBPg/file758964118efc3/sample1_realign2transcript.bam', '/tmp/RtmpNbUBPg/file758964118efc3/sample2_realign2transcript.bam', '/tmp/RtmpNbUBPg/file758964118efc3/sample3_realign2transcript.bam'] /tmp/RtmpNbUBPg/file758964118efc3/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/RtmpNbUBPg/file758966da4cb0a/config_file_481430.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 @ Thu Feb 26 23:48:04 2026 ----------------
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/RtmpNbUBPg/file758966da4cb0a/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 @ Thu Feb 26 23:48:04 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpNbUBPg/file758966da4cb0a/matched_reads.fastq.gz ->/tmp/RtmpNbUBPg/file758966da4cb0a/align2genome.bam
-- Running step: isoform_identification @ Thu Feb 26 23:48:22 2026 -------------
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 @ Thu Feb 26 23:48:31 2026 -------------------
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/RtmpNbUBPg/file758966da4cb0a/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpNbUBPg/file758966da4cb0a/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/RtmpNbUBPg/file758966da4cb0a/matched_reads.fastq.gz ->/tmp/RtmpNbUBPg/file758966da4cb0a/realign2transcript.bam
-- Running step: transcript_quantification @ Thu Feb 26 23:48:49 2026 ----------
23:48:49 Thu Feb 26 2026 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/RtmpNbUBPg/file7589669ba53fd/config_file_481430.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 @ Thu Feb 26 23:48:50 2026 ----------------
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/RtmpNbUBPg/file7589669ba53fd/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 @ Thu Feb 26 23:48:50 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpNbUBPg/file7589669ba53fd/matched_reads.fastq.gz -> /tmp/RtmpNbUBPg/file7589669ba53fd/align2genome.bam
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: isoform_identification @ Thu Feb 26 23:48:50 2026 -------------
	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 @ Thu Feb 26 23:48:51 2026 -------------------
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/RtmpNbUBPg/file7589669ba53fd/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpNbUBPg/file7589669ba53fd/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/RtmpNbUBPg/file7589669ba53fd/matched_reads.fastq.gz -> /tmp/RtmpNbUBPg/file7589669ba53fd/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 @ Thu Feb 26 23:48:51 2026 ----------
2026-02-27T04:48:51.494207Z  INFO oarfish: setting user-provided filter parameters.
2026-02-27T04:48:51.494860Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpNbUBPg/file7589669ba53fd/realign2transcript.bam, contains 10 reference sequences.
2026-02-27T04:48:51.494873Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-02-27T04:48:51.494877Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-02-27T04:48:51.494960Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-02-27T04:48:51.494968Z  INFO oarfish: parsed reference information for 10 transcripts.
2026-02-27T04:48:51.505094Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpNbUBPg/file758967aed9c45/config_file_481430.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 @ Thu Feb 26 23:48:52 2026 ----------------
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/RtmpNbUBPg/file758967aed9c45/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 @ Thu Feb 26 23:48:52 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpNbUBPg/file758967aed9c45/matched_reads.fastq.gz ->/tmp/RtmpNbUBPg/file758967aed9c45/align2genome.bam
-- Running step: isoform_identification @ Thu Feb 26 23:49:11 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Thu Feb 26 23:49:11 2026 -------------------
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/RtmpNbUBPg/file758967aed9c45/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpNbUBPg/file758967aed9c45/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/RtmpNbUBPg/file758967aed9c45/matched_reads.fastq.gz ->/tmp/RtmpNbUBPg/file758967aed9c45/realign2transcript.bam
-- Running step: transcript_quantification @ Thu Feb 26 23:49:30 2026 ----------
2026-02-27T04:49:30.934231Z  INFO oarfish: setting user-provided filter parameters.
2026-02-27T04:49:30.934815Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpNbUBPg/file758967aed9c45/realign2transcript.bam, contains 10 reference sequences.
2026-02-27T04:49:30.934828Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-02-27T04:49:30.934832Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-02-27T04:49:30.934903Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-02-27T04:49:30.934911Z  INFO oarfish: parsed reference information for 10 transcripts.
2026-02-27T04:49:30.945462Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpNbUBPg/file758965cb45e9e/config_file_481430.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 @ Thu Feb 26 23:49:31 2026 ----------------
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/RtmpNbUBPg/file758965cb45e9e/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 @ Thu Feb 26 23:49:31 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpNbUBPg/file758965cb45e9e/matched_reads.fastq.gz -> /tmp/RtmpNbUBPg/file758965cb45e9e/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 @ Thu Feb 26 23:49:32 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Thu Feb 26 23:49:32 2026 -------------------
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/RtmpNbUBPg/file758965cb45e9e/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpNbUBPg/file758965cb45e9e/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/RtmpNbUBPg/file758965cb45e9e/matched_reads.fastq.gz -> /tmp/RtmpNbUBPg/file758965cb45e9e/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 @ Thu Feb 26 23:49:32 2026 ----------
23:49:32 Thu Feb 26 2026 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpNbUBPg/file75896486cee3c/config_file_481430.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 @ Thu Feb 26 23:49:33 2026 ----------------
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/RtmpNbUBPg/file75896486cee3c/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 @ Thu Feb 26 23:49:34 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpNbUBPg/file75896486cee3c/matched_reads.fastq.gz ->/tmp/RtmpNbUBPg/file75896486cee3c/align2genome.bam
-- Running step: isoform_identification @ Thu Feb 26 23:49:52 2026 -------------
	Counter({'counted_reads': 368, 'unmapped': 4})
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Thu Feb 26 23:49:53 2026 -------------------
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/RtmpNbUBPg/file75896486cee3c/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpNbUBPg/file75896486cee3c/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/RtmpNbUBPg/file75896486cee3c/matched_reads.fastq.gz ->/tmp/RtmpNbUBPg/file75896486cee3c/realign2transcript.bam
-- Running step: transcript_quantification @ Thu Feb 26 23:50:11 2026 ----------
23:50:11 Thu Feb 26 2026 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpNbUBPg/file758966f5d64d2/config_file_481430.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 @ Thu Feb 26 23:50:12 2026 ----------------
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/RtmpNbUBPg/file758966f5d64d2/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpNbUBPg/file758966f5d64d2/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpNbUBPg/file758966f5d64d2/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpNbUBPg/file758966f5d64d2/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/RtmpNbUBPg/file758966f5d64d2/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpNbUBPg/file758966f5d64d2/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/RtmpNbUBPg/file758966f5d64d2/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpNbUBPg/file758966f5d64d2/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/RtmpNbUBPg/file758966f5d64d2/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpNbUBPg/file758966f5d64d2/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 @ Thu Feb 26 23:50:13 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpNbUBPg/file758966f5d64d2/sampleA_matched_reads.fastq.gz -> /tmp/RtmpNbUBPg/file758966f5d64d2/sampleA_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpNbUBPg/file758966f5d64d2/sample1_matched_reads.fastq.gz -> /tmp/RtmpNbUBPg/file758966f5d64d2/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpNbUBPg/file758966f5d64d2/sample2_matched_reads.fastq.gz -> /tmp/RtmpNbUBPg/file758966f5d64d2/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpNbUBPg/file758966f5d64d2/sample3_matched_reads.fastq.gz -> /tmp/RtmpNbUBPg/file758966f5d64d2/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Thu Feb 26 23:50:15 2026 ----------------
23:50:15 Thu Feb 26 2026 quantify genes 
Using BAM(s): '/tmp/RtmpNbUBPg/file758966f5d64d2/sampleA_align2genome.bam',
'/tmp/RtmpNbUBPg/file758966f5d64d2/sample1_align2genome.bam',
'/tmp/RtmpNbUBPg/file758966f5d64d2/sample2_align2genome.bam', and
'/tmp/RtmpNbUBPg/file758966f5d64d2/sample3_align2genome.bam'
	Counter({'counted_reads': 368, 'unmapped': 4})
parsing /tmp/RtmpNbUBPg/file758966f5d64d2/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 20.96gene_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, 445614.72Read/s]
parsing /tmp/RtmpNbUBPg/file758966f5d64d2/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 51.59gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1438968.03Read/s]
parsing /tmp/RtmpNbUBPg/file758966f5d64d2/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 50.77gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1359491.77Read/s]
parsing /tmp/RtmpNbUBPg/file758966f5d64d2/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 35.53gene_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, 726512.85Read/s]
-- Running step: isoform_identification @ Thu Feb 26 23:50:16 2026 -------------
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 @ Thu Feb 26 23:50:39 2026 -------------------
Checking for fastq file(s) /tmp/RtmpNbUBPg/file758966f5d64d2/fastq, /tmp/RtmpNbUBPg/file758966f5d64d2/fastq/sample1.fq.gz, /tmp/RtmpNbUBPg/file758966f5d64d2/fastq/sample2.fq.gz, /tmp/RtmpNbUBPg/file758966f5d64d2/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpNbUBPg/file758966f5d64d2/sampleA_matched_reads.fastq.gz, /tmp/RtmpNbUBPg/file758966f5d64d2/sample1_matched_reads.fastq.gz, /tmp/RtmpNbUBPg/file758966f5d64d2/sample2_matched_reads.fastq.gz, /tmp/RtmpNbUBPg/file758966f5d64d2/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpNbUBPg/file758966f5d64d2/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpNbUBPg/file758966f5d64d2/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpNbUBPg/file758966f5d64d2/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpNbUBPg/file758966f5d64d2/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpNbUBPg/file758966f5d64d2/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpNbUBPg/file758966f5d64d2/sampleA_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpNbUBPg/file758966f5d64d2/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpNbUBPg/file758966f5d64d2/sample1_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpNbUBPg/file758966f5d64d2/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpNbUBPg/file758966f5d64d2/sample2_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpNbUBPg/file758966f5d64d2/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpNbUBPg/file758966f5d64d2/sample3_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

-- Running step: transcript_quantification @ Thu Feb 26 23:50:40 2026 ----------
2026-02-27T04:50:40.088892Z  INFO oarfish: setting user-provided filter parameters.
2026-02-27T04:50:40.089240Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpNbUBPg/file758966f5d64d2/sampleA_realign2transcript.bam, contains 5 reference sequences.
2026-02-27T04:50:40.089248Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-02-27T04:50:40.089250Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-02-27T04:50:40.089306Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-02-27T04:50:40.089311Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-02-27T04:50:40.095031Z  INFO oarfish::single_cell: Processed 100 cells.
2026-02-27T04:50:40.382259Z  INFO oarfish: setting user-provided filter parameters.
2026-02-27T04:50:40.382611Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpNbUBPg/file758966f5d64d2/sample1_realign2transcript.bam, contains 5 reference sequences.
2026-02-27T04:50:40.382623Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-02-27T04:50:40.382627Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-02-27T04:50:40.382678Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-02-27T04:50:40.382683Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-02-27T04:50:40.694120Z  INFO oarfish: setting user-provided filter parameters.
2026-02-27T04:50:40.694906Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpNbUBPg/file758966f5d64d2/sample2_realign2transcript.bam, contains 5 reference sequences.
2026-02-27T04:50:40.694917Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-02-27T04:50:40.694920Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-02-27T04:50:40.694976Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-02-27T04:50:40.694982Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-02-27T04:50:40.974969Z  INFO oarfish: setting user-provided filter parameters.
2026-02-27T04:50:40.975412Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpNbUBPg/file758966f5d64d2/sample3_realign2transcript.bam, contains 5 reference sequences.
2026-02-27T04:50:40.975430Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-02-27T04:50:40.975433Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-02-27T04:50:40.975489Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-02-27T04:50:40.975494Z  INFO oarfish: parsed reference information for 5 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpNbUBPg/file758966867be1/config_file_481430.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 @ Thu Feb 26 23:50:41 2026 ----------------
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/RtmpNbUBPg/file758966867be1/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpNbUBPg/file758966867be1/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpNbUBPg/file758966867be1/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpNbUBPg/file758966867be1/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/RtmpNbUBPg/file758966867be1/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpNbUBPg/file758966867be1/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/RtmpNbUBPg/file758966867be1/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpNbUBPg/file758966867be1/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/RtmpNbUBPg/file758966867be1/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpNbUBPg/file758966867be1/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 @ Thu Feb 26 23:50:42 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpNbUBPg/file758966867be1/sampleA_matched_reads.fastq.gz ->/tmp/RtmpNbUBPg/file758966867be1/sampleA_align2genome.bam
/tmp/RtmpNbUBPg/file758966867be1/sample1_matched_reads.fastq.gz ->/tmp/RtmpNbUBPg/file758966867be1/sample1_align2genome.bam
/tmp/RtmpNbUBPg/file758966867be1/sample2_matched_reads.fastq.gz ->/tmp/RtmpNbUBPg/file758966867be1/sample2_align2genome.bam
/tmp/RtmpNbUBPg/file758966867be1/sample3_matched_reads.fastq.gz ->/tmp/RtmpNbUBPg/file758966867be1/sample3_align2genome.bam
-- Running step: gene_quantification @ Thu Feb 26 23:51:02 2026 ----------------
23:51:02 Thu Feb 26 2026 quantify genes 
Using BAM(s): '/tmp/RtmpNbUBPg/file758966867be1/sampleA_align2genome.bam',
'/tmp/RtmpNbUBPg/file758966867be1/sample1_align2genome.bam',
'/tmp/RtmpNbUBPg/file758966867be1/sample2_align2genome.bam', and
'/tmp/RtmpNbUBPg/file758966867be1/sample3_align2genome.bam'
parsing /tmp/RtmpNbUBPg/file758966867be1/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 16.18gene_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, 384262.68Read/s]
parsing /tmp/RtmpNbUBPg/file758966867be1/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 51.38gene_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, 1337981.37Read/s]
parsing /tmp/RtmpNbUBPg/file758966867be1/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 51.69gene_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, 1210966.62Read/s]
parsing /tmp/RtmpNbUBPg/file758966867be1/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 34.47gene_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, 793233.98Read/s]
-- Running step: isoform_identification @ Thu Feb 26 23:51:03 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

  |                                                                            
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  |                                                                            
  |==================                                                    |  25%
  |                                                                            
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  |                                                                            
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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 @ Thu Feb 26 23:51:25 2026 -------------------
Checking for fastq file(s) /tmp/RtmpNbUBPg/file758966867be1/fastq, /tmp/RtmpNbUBPg/file758966867be1/fastq/sample1.fq.gz, /tmp/RtmpNbUBPg/file758966867be1/fastq/sample2.fq.gz, /tmp/RtmpNbUBPg/file758966867be1/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpNbUBPg/file758966867be1/sampleA_matched_reads.fastq.gz, /tmp/RtmpNbUBPg/file758966867be1/sample1_matched_reads.fastq.gz, /tmp/RtmpNbUBPg/file758966867be1/sample2_matched_reads.fastq.gz, /tmp/RtmpNbUBPg/file758966867be1/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpNbUBPg/file758966867be1/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpNbUBPg/file758966867be1/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpNbUBPg/file758966867be1/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpNbUBPg/file758966867be1/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpNbUBPg/file758966867be1/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpNbUBPg/file758966867be1/sampleA_realign2transcript.bam
/tmp/RtmpNbUBPg/file758966867be1/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpNbUBPg/file758966867be1/sample1_realign2transcript.bam
/tmp/RtmpNbUBPg/file758966867be1/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpNbUBPg/file758966867be1/sample2_realign2transcript.bam
/tmp/RtmpNbUBPg/file758966867be1/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpNbUBPg/file758966867be1/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Thu Feb 26 23:51:44 2026 ----------
2026-02-27T04:51:44.268669Z  INFO oarfish: setting user-provided filter parameters.
2026-02-27T04:51:44.269352Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpNbUBPg/file758966867be1/sampleA_realign2transcript.bam, contains 5 reference sequences.
2026-02-27T04:51:44.269365Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-02-27T04:51:44.269368Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-02-27T04:51:44.269427Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-02-27T04:51:44.269434Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-02-27T04:51:44.275443Z  INFO oarfish::single_cell: Processed 100 cells.
2026-02-27T04:51:44.656993Z  INFO oarfish: setting user-provided filter parameters.
2026-02-27T04:51:44.657599Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpNbUBPg/file758966867be1/sample1_realign2transcript.bam, contains 5 reference sequences.
2026-02-27T04:51:44.657611Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-02-27T04:51:44.657615Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-02-27T04:51:44.657676Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-02-27T04:51:44.657683Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-02-27T04:51:45.017652Z  INFO oarfish: setting user-provided filter parameters.
2026-02-27T04:51:45.017997Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpNbUBPg/file758966867be1/sample2_realign2transcript.bam, contains 5 reference sequences.
2026-02-27T04:51:45.018018Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-02-27T04:51:45.018021Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-02-27T04:51:45.018076Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-02-27T04:51:45.018081Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-02-27T04:51:45.331155Z  INFO oarfish: setting user-provided filter parameters.
2026-02-27T04:51:45.331632Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpNbUBPg/file758966867be1/sample3_realign2transcript.bam, contains 5 reference sequences.
2026-02-27T04:51:45.331644Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-02-27T04:51:45.331646Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-02-27T04:51:45.331702Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-02-27T04:51:45.331708Z  INFO oarfish: parsed reference information for 5 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpNbUBPg/file758967a10217a/config_file_481430.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 @ Thu Feb 26 23:51:45 2026 ----------------
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/RtmpNbUBPg/file758967a10217a/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpNbUBPg/file758967a10217a/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpNbUBPg/file758967a10217a/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpNbUBPg/file758967a10217a/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/RtmpNbUBPg/file758967a10217a/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpNbUBPg/file758967a10217a/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/RtmpNbUBPg/file758967a10217a/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpNbUBPg/file758967a10217a/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/RtmpNbUBPg/file758967a10217a/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpNbUBPg/file758967a10217a/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 @ Thu Feb 26 23:51:46 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpNbUBPg/file758967a10217a/sampleA_matched_reads.fastq.gz -> /tmp/RtmpNbUBPg/file758967a10217a/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/RtmpNbUBPg/file758967a10217a/sample1_matched_reads.fastq.gz -> /tmp/RtmpNbUBPg/file758967a10217a/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/RtmpNbUBPg/file758967a10217a/sample2_matched_reads.fastq.gz -> /tmp/RtmpNbUBPg/file758967a10217a/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/RtmpNbUBPg/file758967a10217a/sample3_matched_reads.fastq.gz -> /tmp/RtmpNbUBPg/file758967a10217a/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 @ Thu Feb 26 23:51:48 2026 ----------------
23:51:48 Thu Feb 26 2026 quantify genes 
Using BAM(s): '/tmp/RtmpNbUBPg/file758967a10217a/sampleA_align2genome.bam',
'/tmp/RtmpNbUBPg/file758967a10217a/sample1_align2genome.bam',
'/tmp/RtmpNbUBPg/file758967a10217a/sample2_align2genome.bam', and
'/tmp/RtmpNbUBPg/file758967a10217a/sample3_align2genome.bam'
parsing /tmp/RtmpNbUBPg/file758967a10217a/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 22.10gene_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, 418376.09Read/s]
parsing /tmp/RtmpNbUBPg/file758967a10217a/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 53.18gene_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, 1362494.80Read/s]
parsing /tmp/RtmpNbUBPg/file758967a10217a/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 52.38gene_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, 1380795.36Read/s]
parsing /tmp/RtmpNbUBPg/file758967a10217a/sample3_align2genome.bam...
Assigning reads to genes...

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

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 722856.75Read/s]
-- Running step: isoform_identification @ Thu Feb 26 23:51:49 2026 -------------
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 @ Thu Feb 26 23:52:12 2026 -------------------
Checking for fastq file(s) /tmp/RtmpNbUBPg/file758967a10217a/fastq, /tmp/RtmpNbUBPg/file758967a10217a/fastq/sample1.fq.gz, /tmp/RtmpNbUBPg/file758967a10217a/fastq/sample2.fq.gz, /tmp/RtmpNbUBPg/file758967a10217a/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpNbUBPg/file758967a10217a/sampleA_matched_reads.fastq.gz, /tmp/RtmpNbUBPg/file758967a10217a/sample1_matched_reads.fastq.gz, /tmp/RtmpNbUBPg/file758967a10217a/sample2_matched_reads.fastq.gz, /tmp/RtmpNbUBPg/file758967a10217a/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpNbUBPg/file758967a10217a/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpNbUBPg/file758967a10217a/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpNbUBPg/file758967a10217a/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpNbUBPg/file758967a10217a/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpNbUBPg/file758967a10217a/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpNbUBPg/file758967a10217a/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/RtmpNbUBPg/file758967a10217a/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpNbUBPg/file758967a10217a/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/RtmpNbUBPg/file758967a10217a/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpNbUBPg/file758967a10217a/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/RtmpNbUBPg/file758967a10217a/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpNbUBPg/file758967a10217a/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 @ Thu Feb 26 23:52:13 2026 ----------
23:52:13 Thu Feb 26 2026 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpNbUBPg/file758967a10217a/sampleA_realign2transcript.bam...
parsing /tmp/RtmpNbUBPg/file758967a10217a/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpNbUBPg/file758967a10217a/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpNbUBPg/file758967a10217a/sample1_realign2transcript.bam...
parsing /tmp/RtmpNbUBPg/file758967a10217a/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpNbUBPg/file758967a10217a/sample1_realign2transcript.bamdone
parsing /tmp/RtmpNbUBPg/file758967a10217a/sample2_realign2transcript.bam...
parsing /tmp/RtmpNbUBPg/file758967a10217a/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpNbUBPg/file758967a10217a/sample2_realign2transcript.bamdone
parsing /tmp/RtmpNbUBPg/file758967a10217a/sample3_realign2transcript.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})
parsing /tmp/RtmpNbUBPg/file758967a10217a/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpNbUBPg/file758967a10217a/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpNbUBPg/file7589667eaa163/config_file_481430.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 @ Thu Feb 26 23:52:15 2026 ----------------
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/RtmpNbUBPg/file7589667eaa163/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpNbUBPg/file7589667eaa163/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpNbUBPg/file7589667eaa163/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpNbUBPg/file7589667eaa163/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/RtmpNbUBPg/file7589667eaa163/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpNbUBPg/file7589667eaa163/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/RtmpNbUBPg/file7589667eaa163/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpNbUBPg/file7589667eaa163/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/RtmpNbUBPg/file7589667eaa163/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpNbUBPg/file7589667eaa163/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 @ Thu Feb 26 23:52:15 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpNbUBPg/file7589667eaa163/sampleA_matched_reads.fastq.gz ->/tmp/RtmpNbUBPg/file7589667eaa163/sampleA_align2genome.bam
/tmp/RtmpNbUBPg/file7589667eaa163/sample1_matched_reads.fastq.gz ->/tmp/RtmpNbUBPg/file7589667eaa163/sample1_align2genome.bam
/tmp/RtmpNbUBPg/file7589667eaa163/sample2_matched_reads.fastq.gz ->/tmp/RtmpNbUBPg/file7589667eaa163/sample2_align2genome.bam
/tmp/RtmpNbUBPg/file7589667eaa163/sample3_matched_reads.fastq.gz ->/tmp/RtmpNbUBPg/file7589667eaa163/sample3_align2genome.bam
-- Running step: gene_quantification @ Thu Feb 26 23:52:34 2026 ----------------
23:52:34 Thu Feb 26 2026 quantify genes 
Using BAM(s): '/tmp/RtmpNbUBPg/file7589667eaa163/sampleA_align2genome.bam',
'/tmp/RtmpNbUBPg/file7589667eaa163/sample1_align2genome.bam',
'/tmp/RtmpNbUBPg/file7589667eaa163/sample2_align2genome.bam', and
'/tmp/RtmpNbUBPg/file7589667eaa163/sample3_align2genome.bam'
	Counter({'counted_reads': 168, 'not_enough_coverage': 6, 'unmapped': 2})
parsing /tmp/RtmpNbUBPg/file7589667eaa163/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 21.86gene_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, 423324.99Read/s]
parsing /tmp/RtmpNbUBPg/file7589667eaa163/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 46.53gene_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, 1376625.97Read/s]
parsing /tmp/RtmpNbUBPg/file7589667eaa163/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 52.31gene_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, 1287860.48Read/s]
parsing /tmp/RtmpNbUBPg/file7589667eaa163/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 35.26gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 735223.67Read/s]
-- Running step: isoform_identification @ Thu Feb 26 23:52:35 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 6 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Thu Feb 26 23:52:57 2026 -------------------
Checking for fastq file(s) /tmp/RtmpNbUBPg/file7589667eaa163/fastq, /tmp/RtmpNbUBPg/file7589667eaa163/fastq/sample1.fq.gz, /tmp/RtmpNbUBPg/file7589667eaa163/fastq/sample2.fq.gz, /tmp/RtmpNbUBPg/file7589667eaa163/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpNbUBPg/file7589667eaa163/sampleA_matched_reads.fastq.gz, /tmp/RtmpNbUBPg/file7589667eaa163/sample1_matched_reads.fastq.gz, /tmp/RtmpNbUBPg/file7589667eaa163/sample2_matched_reads.fastq.gz, /tmp/RtmpNbUBPg/file7589667eaa163/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpNbUBPg/file7589667eaa163/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpNbUBPg/file7589667eaa163/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpNbUBPg/file7589667eaa163/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpNbUBPg/file7589667eaa163/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpNbUBPg/file7589667eaa163/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpNbUBPg/file7589667eaa163/sampleA_realign2transcript.bam
/tmp/RtmpNbUBPg/file7589667eaa163/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpNbUBPg/file7589667eaa163/sample1_realign2transcript.bam
/tmp/RtmpNbUBPg/file7589667eaa163/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpNbUBPg/file7589667eaa163/sample2_realign2transcript.bam
/tmp/RtmpNbUBPg/file7589667eaa163/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpNbUBPg/file7589667eaa163/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Thu Feb 26 23:53:15 2026 ----------
23:53:15 Thu Feb 26 2026 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpNbUBPg/file7589667eaa163/sampleA_realign2transcript.bam...
parsing /tmp/RtmpNbUBPg/file7589667eaa163/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpNbUBPg/file7589667eaa163/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpNbUBPg/file7589667eaa163/sample1_realign2transcript.bam...
parsing /tmp/RtmpNbUBPg/file7589667eaa163/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpNbUBPg/file7589667eaa163/sample1_realign2transcript.bamdone
parsing /tmp/RtmpNbUBPg/file7589667eaa163/sample2_realign2transcript.bam...
parsing /tmp/RtmpNbUBPg/file7589667eaa163/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpNbUBPg/file7589667eaa163/sample2_realign2transcript.bamdone
parsing /tmp/RtmpNbUBPg/file7589667eaa163/sample3_realign2transcript.bam...
parsing /tmp/RtmpNbUBPg/file7589667eaa163/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpNbUBPg/file7589667eaa163/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpNbUBPg/file758962716c0a1/config_file_481430.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 @ Thu Feb 26 23:53:17 2026 ----------------
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/RtmpNbUBPg/file758962716c0a1/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpNbUBPg/file758962716c0a1/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpNbUBPg/file758962716c0a1/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpNbUBPg/file758962716c0a1/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/RtmpNbUBPg/file758962716c0a1/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpNbUBPg/file758962716c0a1/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/RtmpNbUBPg/file758962716c0a1/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpNbUBPg/file758962716c0a1/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/RtmpNbUBPg/file758962716c0a1/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpNbUBPg/file758962716c0a1/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 @ Thu Feb 26 23:53:18 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpNbUBPg/file758962716c0a1/sampleA_matched_reads.fastq.gz -> /tmp/RtmpNbUBPg/file758962716c0a1/sampleA_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpNbUBPg/file758962716c0a1/sample1_matched_reads.fastq.gz -> /tmp/RtmpNbUBPg/file758962716c0a1/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpNbUBPg/file758962716c0a1/sample2_matched_reads.fastq.gz -> /tmp/RtmpNbUBPg/file758962716c0a1/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpNbUBPg/file758962716c0a1/sample3_matched_reads.fastq.gz -> /tmp/RtmpNbUBPg/file758962716c0a1/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Thu Feb 26 23:53:20 2026 ----------------
23:53:20 Thu Feb 26 2026 quantify genes 
Using BAM(s): '/tmp/RtmpNbUBPg/file758962716c0a1/sampleA_align2genome.bam',
'/tmp/RtmpNbUBPg/file758962716c0a1/sample1_align2genome.bam',
'/tmp/RtmpNbUBPg/file758962716c0a1/sample2_align2genome.bam', and
'/tmp/RtmpNbUBPg/file758962716c0a1/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/RtmpNbUBPg/file758962716c0a1/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 23.37gene_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, 398576.86Read/s]
parsing /tmp/RtmpNbUBPg/file758962716c0a1/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 49.24gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1328656.87Read/s]
parsing /tmp/RtmpNbUBPg/file758962716c0a1/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 49.25gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1232169.21Read/s]
parsing /tmp/RtmpNbUBPg/file758962716c0a1/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 36.84gene_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, 720472.72Read/s]
-- Running step: isoform_identification @ Thu Feb 26 23:53:20 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Thu Feb 26 23:53:21 2026 -------------------
Checking for fastq file(s) /tmp/RtmpNbUBPg/file758962716c0a1/fastq, /tmp/RtmpNbUBPg/file758962716c0a1/fastq/sample1.fq.gz, /tmp/RtmpNbUBPg/file758962716c0a1/fastq/sample2.fq.gz, /tmp/RtmpNbUBPg/file758962716c0a1/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpNbUBPg/file758962716c0a1/sampleA_matched_reads.fastq.gz, /tmp/RtmpNbUBPg/file758962716c0a1/sample1_matched_reads.fastq.gz, /tmp/RtmpNbUBPg/file758962716c0a1/sample2_matched_reads.fastq.gz, /tmp/RtmpNbUBPg/file758962716c0a1/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpNbUBPg/file758962716c0a1/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpNbUBPg/file758962716c0a1/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpNbUBPg/file758962716c0a1/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpNbUBPg/file758962716c0a1/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpNbUBPg/file758962716c0a1/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpNbUBPg/file758962716c0a1/sampleA_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpNbUBPg/file758962716c0a1/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpNbUBPg/file758962716c0a1/sample1_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpNbUBPg/file758962716c0a1/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpNbUBPg/file758962716c0a1/sample2_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpNbUBPg/file758962716c0a1/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpNbUBPg/file758962716c0a1/sample3_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

-- Running step: transcript_quantification @ Thu Feb 26 23:53:23 2026 ----------
2026-02-27T04:53:23.165451Z  INFO oarfish: setting user-provided filter parameters.
2026-02-27T04:53:23.165925Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpNbUBPg/file758962716c0a1/sampleA_realign2transcript.bam, contains 14 reference sequences.
2026-02-27T04:53:23.165936Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-02-27T04:53:23.165938Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-02-27T04:53:23.166014Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-02-27T04:53:23.166022Z  INFO oarfish: parsed reference information for 14 transcripts.
2026-02-27T04:53:23.177372Z  INFO oarfish::single_cell: Processed 100 cells.
2026-02-27T04:53:23.689014Z  INFO oarfish: setting user-provided filter parameters.
2026-02-27T04:53:23.689471Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpNbUBPg/file758962716c0a1/sample1_realign2transcript.bam, contains 14 reference sequences.
2026-02-27T04:53:23.689480Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-02-27T04:53:23.689483Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-02-27T04:53:23.689582Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-02-27T04:53:23.689595Z  INFO oarfish: parsed reference information for 14 transcripts.
2026-02-27T04:53:24.228236Z  INFO oarfish: setting user-provided filter parameters.
2026-02-27T04:53:24.229286Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpNbUBPg/file758962716c0a1/sample2_realign2transcript.bam, contains 14 reference sequences.
2026-02-27T04:53:24.229394Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-02-27T04:53:24.229397Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-02-27T04:53:24.229482Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-02-27T04:53:24.229489Z  INFO oarfish: parsed reference information for 14 transcripts.
2026-02-27T04:53:24.791415Z  INFO oarfish: setting user-provided filter parameters.
2026-02-27T04:53:24.791863Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpNbUBPg/file758962716c0a1/sample3_realign2transcript.bam, contains 14 reference sequences.
2026-02-27T04:53:24.791886Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-02-27T04:53:24.791889Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-02-27T04:53:24.791971Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-02-27T04:53:24.791979Z  INFO oarfish: parsed reference information for 14 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpNbUBPg/file75896524a8e3f/config_file_481430.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 @ Thu Feb 26 23:53:25 2026 ----------------
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/RtmpNbUBPg/file75896524a8e3f/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpNbUBPg/file75896524a8e3f/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpNbUBPg/file75896524a8e3f/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpNbUBPg/file75896524a8e3f/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/RtmpNbUBPg/file75896524a8e3f/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpNbUBPg/file75896524a8e3f/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/RtmpNbUBPg/file75896524a8e3f/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpNbUBPg/file75896524a8e3f/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/RtmpNbUBPg/file75896524a8e3f/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpNbUBPg/file75896524a8e3f/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 @ Thu Feb 26 23:53:26 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpNbUBPg/file75896524a8e3f/sampleA_matched_reads.fastq.gz ->/tmp/RtmpNbUBPg/file75896524a8e3f/sampleA_align2genome.bam
/tmp/RtmpNbUBPg/file75896524a8e3f/sample1_matched_reads.fastq.gz ->/tmp/RtmpNbUBPg/file75896524a8e3f/sample1_align2genome.bam
/tmp/RtmpNbUBPg/file75896524a8e3f/sample2_matched_reads.fastq.gz ->/tmp/RtmpNbUBPg/file75896524a8e3f/sample2_align2genome.bam
/tmp/RtmpNbUBPg/file75896524a8e3f/sample3_matched_reads.fastq.gz ->/tmp/RtmpNbUBPg/file75896524a8e3f/sample3_align2genome.bam
-- Running step: gene_quantification @ Thu Feb 26 23:53:45 2026 ----------------
23:53:45 Thu Feb 26 2026 quantify genes 
Using BAM(s): '/tmp/RtmpNbUBPg/file75896524a8e3f/sampleA_align2genome.bam',
'/tmp/RtmpNbUBPg/file75896524a8e3f/sample1_align2genome.bam',
'/tmp/RtmpNbUBPg/file75896524a8e3f/sample2_align2genome.bam', and
'/tmp/RtmpNbUBPg/file75896524a8e3f/sample3_align2genome.bam'
parsing /tmp/RtmpNbUBPg/file75896524a8e3f/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 21.15gene_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, 344190.38Read/s]
parsing /tmp/RtmpNbUBPg/file75896524a8e3f/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 48.21gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1232458.86Read/s]
parsing /tmp/RtmpNbUBPg/file75896524a8e3f/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 51.50gene_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, 1053633.44Read/s]
parsing /tmp/RtmpNbUBPg/file75896524a8e3f/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 36.76gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 700077.45Read/s]
-- Running step: isoform_identification @ Thu Feb 26 23:53:46 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Thu Feb 26 23:53:47 2026 -------------------
Checking for fastq file(s) /tmp/RtmpNbUBPg/file75896524a8e3f/fastq, /tmp/RtmpNbUBPg/file75896524a8e3f/fastq/sample1.fq.gz, /tmp/RtmpNbUBPg/file75896524a8e3f/fastq/sample2.fq.gz, /tmp/RtmpNbUBPg/file75896524a8e3f/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpNbUBPg/file75896524a8e3f/sampleA_matched_reads.fastq.gz, /tmp/RtmpNbUBPg/file75896524a8e3f/sample1_matched_reads.fastq.gz, /tmp/RtmpNbUBPg/file75896524a8e3f/sample2_matched_reads.fastq.gz, /tmp/RtmpNbUBPg/file75896524a8e3f/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpNbUBPg/file75896524a8e3f/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpNbUBPg/file75896524a8e3f/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpNbUBPg/file75896524a8e3f/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpNbUBPg/file75896524a8e3f/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpNbUBPg/file75896524a8e3f/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpNbUBPg/file75896524a8e3f/sampleA_realign2transcript.bam
/tmp/RtmpNbUBPg/file75896524a8e3f/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpNbUBPg/file75896524a8e3f/sample1_realign2transcript.bam
/tmp/RtmpNbUBPg/file75896524a8e3f/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpNbUBPg/file75896524a8e3f/sample2_realign2transcript.bam
/tmp/RtmpNbUBPg/file75896524a8e3f/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpNbUBPg/file75896524a8e3f/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Thu Feb 26 23:54:07 2026 ----------
2026-02-27T04:54:07.380671Z  INFO oarfish: setting user-provided filter parameters.
2026-02-27T04:54:07.381166Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpNbUBPg/file75896524a8e3f/sampleA_realign2transcript.bam, contains 14 reference sequences.
2026-02-27T04:54:07.381177Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-02-27T04:54:07.381181Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-02-27T04:54:07.381265Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-02-27T04:54:07.381273Z  INFO oarfish: parsed reference information for 14 transcripts.
2026-02-27T04:54:07.393313Z  INFO oarfish::single_cell: Processed 100 cells.
2026-02-27T04:54:08.020651Z  INFO oarfish: setting user-provided filter parameters.
2026-02-27T04:54:08.021136Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpNbUBPg/file75896524a8e3f/sample1_realign2transcript.bam, contains 14 reference sequences.
2026-02-27T04:54:08.021147Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-02-27T04:54:08.021151Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-02-27T04:54:08.021233Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-02-27T04:54:08.021241Z  INFO oarfish: parsed reference information for 14 transcripts.
2026-02-27T04:54:08.584443Z  INFO oarfish: setting user-provided filter parameters.
2026-02-27T04:54:08.584823Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpNbUBPg/file75896524a8e3f/sample2_realign2transcript.bam, contains 14 reference sequences.
2026-02-27T04:54:08.584834Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-02-27T04:54:08.584837Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-02-27T04:54:08.584908Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-02-27T04:54:08.584917Z  INFO oarfish: parsed reference information for 14 transcripts.
2026-02-27T04:54:09.132243Z  INFO oarfish: setting user-provided filter parameters.
2026-02-27T04:54:09.132647Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpNbUBPg/file75896524a8e3f/sample3_realign2transcript.bam, contains 14 reference sequences.
2026-02-27T04:54:09.132658Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-02-27T04:54:09.132661Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-02-27T04:54:09.132742Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-02-27T04:54:09.132750Z  INFO oarfish: parsed reference information for 14 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpNbUBPg/file75896140c440/config_file_481430.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 @ Thu Feb 26 23:54:09 2026 ----------------
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/RtmpNbUBPg/file75896140c440/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpNbUBPg/file75896140c440/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpNbUBPg/file75896140c440/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpNbUBPg/file75896140c440/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/RtmpNbUBPg/file75896140c440/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpNbUBPg/file75896140c440/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/RtmpNbUBPg/file75896140c440/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpNbUBPg/file75896140c440/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/RtmpNbUBPg/file75896140c440/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpNbUBPg/file75896140c440/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 @ Thu Feb 26 23:54:10 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpNbUBPg/file75896140c440/sampleA_matched_reads.fastq.gz -> /tmp/RtmpNbUBPg/file75896140c440/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/RtmpNbUBPg/file75896140c440/sample1_matched_reads.fastq.gz -> /tmp/RtmpNbUBPg/file75896140c440/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/RtmpNbUBPg/file75896140c440/sample2_matched_reads.fastq.gz -> /tmp/RtmpNbUBPg/file75896140c440/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/RtmpNbUBPg/file75896140c440/sample3_matched_reads.fastq.gz -> /tmp/RtmpNbUBPg/file75896140c440/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 @ Thu Feb 26 23:54:12 2026 ----------------
23:54:12 Thu Feb 26 2026 quantify genes 
Using BAM(s): '/tmp/RtmpNbUBPg/file75896140c440/sampleA_align2genome.bam',
'/tmp/RtmpNbUBPg/file75896140c440/sample1_align2genome.bam',
'/tmp/RtmpNbUBPg/file75896140c440/sample2_align2genome.bam', and
'/tmp/RtmpNbUBPg/file75896140c440/sample3_align2genome.bam'
parsing /tmp/RtmpNbUBPg/file75896140c440/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 23.80gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 419396.85Read/s]
parsing /tmp/RtmpNbUBPg/file75896140c440/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 53.80gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1229856.91Read/s]
parsing /tmp/RtmpNbUBPg/file75896140c440/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 48.89gene_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, 1269462.47Read/s]
parsing /tmp/RtmpNbUBPg/file75896140c440/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 37.64gene_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, 794496.14Read/s]
-- Running step: isoform_identification @ Thu Feb 26 23:54:13 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Thu Feb 26 23:54:13 2026 -------------------
Checking for fastq file(s) /tmp/RtmpNbUBPg/file75896140c440/fastq, /tmp/RtmpNbUBPg/file75896140c440/fastq/sample1.fq.gz, /tmp/RtmpNbUBPg/file75896140c440/fastq/sample2.fq.gz, /tmp/RtmpNbUBPg/file75896140c440/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpNbUBPg/file75896140c440/sampleA_matched_reads.fastq.gz, /tmp/RtmpNbUBPg/file75896140c440/sample1_matched_reads.fastq.gz, /tmp/RtmpNbUBPg/file75896140c440/sample2_matched_reads.fastq.gz, /tmp/RtmpNbUBPg/file75896140c440/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpNbUBPg/file75896140c440/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpNbUBPg/file75896140c440/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpNbUBPg/file75896140c440/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpNbUBPg/file75896140c440/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpNbUBPg/file75896140c440/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpNbUBPg/file75896140c440/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/RtmpNbUBPg/file75896140c440/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpNbUBPg/file75896140c440/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/RtmpNbUBPg/file75896140c440/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpNbUBPg/file75896140c440/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/RtmpNbUBPg/file75896140c440/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpNbUBPg/file75896140c440/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 @ Thu Feb 26 23:54:14 2026 ----------
23:54:14 Thu Feb 26 2026 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpNbUBPg/file75896140c440/sampleA_realign2transcript.bam...
parsing /tmp/RtmpNbUBPg/file75896140c440/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpNbUBPg/file75896140c440/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpNbUBPg/file75896140c440/sample1_realign2transcript.bam...
parsing /tmp/RtmpNbUBPg/file75896140c440/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpNbUBPg/file75896140c440/sample1_realign2transcript.bamdone
parsing /tmp/RtmpNbUBPg/file75896140c440/sample2_realign2transcript.bam...
parsing /tmp/RtmpNbUBPg/file75896140c440/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpNbUBPg/file75896140c440/sample2_realign2transcript.bamdone
parsing /tmp/RtmpNbUBPg/file75896140c440/sample3_realign2transcript.bam...
parsing /tmp/RtmpNbUBPg/file75896140c440/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpNbUBPg/file75896140c440/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpNbUBPg/file758961652e10d/config_file_481430.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 @ Thu Feb 26 23:54:17 2026 ----------------
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/RtmpNbUBPg/file758961652e10d/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpNbUBPg/file758961652e10d/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpNbUBPg/file758961652e10d/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpNbUBPg/file758961652e10d/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/RtmpNbUBPg/file758961652e10d/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpNbUBPg/file758961652e10d/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/RtmpNbUBPg/file758961652e10d/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpNbUBPg/file758961652e10d/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/RtmpNbUBPg/file758961652e10d/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpNbUBPg/file758961652e10d/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 @ Thu Feb 26 23:54:18 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpNbUBPg/file758961652e10d/sampleA_matched_reads.fastq.gz ->/tmp/RtmpNbUBPg/file758961652e10d/sampleA_align2genome.bam
/tmp/RtmpNbUBPg/file758961652e10d/sample1_matched_reads.fastq.gz ->/tmp/RtmpNbUBPg/file758961652e10d/sample1_align2genome.bam
/tmp/RtmpNbUBPg/file758961652e10d/sample2_matched_reads.fastq.gz ->/tmp/RtmpNbUBPg/file758961652e10d/sample2_align2genome.bam
/tmp/RtmpNbUBPg/file758961652e10d/sample3_matched_reads.fastq.gz ->/tmp/RtmpNbUBPg/file758961652e10d/sample3_align2genome.bam
-- Running step: gene_quantification @ Thu Feb 26 23:54:37 2026 ----------------
23:54:37 Thu Feb 26 2026 quantify genes 
Using BAM(s): '/tmp/RtmpNbUBPg/file758961652e10d/sampleA_align2genome.bam',
'/tmp/RtmpNbUBPg/file758961652e10d/sample1_align2genome.bam',
'/tmp/RtmpNbUBPg/file758961652e10d/sample2_align2genome.bam', and
'/tmp/RtmpNbUBPg/file758961652e10d/sample3_align2genome.bam'
	Counter({'counted_reads': 358})
	Counter({'counted_reads': 91})
	Counter({'counted_reads': 95})
	Counter({'counted_reads': 176})
parsing /tmp/RtmpNbUBPg/file758961652e10d/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 14.31gene_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, 337825.32Read/s]
parsing /tmp/RtmpNbUBPg/file758961652e10d/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 53.21gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1385722.21Read/s]
parsing /tmp/RtmpNbUBPg/file758961652e10d/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 51.91gene_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, 1208872.49Read/s]
parsing /tmp/RtmpNbUBPg/file758961652e10d/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 36.80gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 705114.65Read/s]
-- Running step: isoform_identification @ Thu Feb 26 23:54:38 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Thu Feb 26 23:54:39 2026 -------------------
Checking for fastq file(s) /tmp/RtmpNbUBPg/file758961652e10d/fastq, /tmp/RtmpNbUBPg/file758961652e10d/fastq/sample1.fq.gz, /tmp/RtmpNbUBPg/file758961652e10d/fastq/sample2.fq.gz, /tmp/RtmpNbUBPg/file758961652e10d/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpNbUBPg/file758961652e10d/sampleA_matched_reads.fastq.gz, /tmp/RtmpNbUBPg/file758961652e10d/sample1_matched_reads.fastq.gz, /tmp/RtmpNbUBPg/file758961652e10d/sample2_matched_reads.fastq.gz, /tmp/RtmpNbUBPg/file758961652e10d/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpNbUBPg/file758961652e10d/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpNbUBPg/file758961652e10d/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpNbUBPg/file758961652e10d/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpNbUBPg/file758961652e10d/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpNbUBPg/file758961652e10d/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpNbUBPg/file758961652e10d/sampleA_realign2transcript.bam
/tmp/RtmpNbUBPg/file758961652e10d/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpNbUBPg/file758961652e10d/sample1_realign2transcript.bam
/tmp/RtmpNbUBPg/file758961652e10d/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpNbUBPg/file758961652e10d/sample2_realign2transcript.bam
/tmp/RtmpNbUBPg/file758961652e10d/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpNbUBPg/file758961652e10d/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Thu Feb 26 23:54:58 2026 ----------
23:54:58 Thu Feb 26 2026 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpNbUBPg/file758961652e10d/sampleA_realign2transcript.bam...
parsing /tmp/RtmpNbUBPg/file758961652e10d/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpNbUBPg/file758961652e10d/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpNbUBPg/file758961652e10d/sample1_realign2transcript.bam...
parsing /tmp/RtmpNbUBPg/file758961652e10d/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpNbUBPg/file758961652e10d/sample1_realign2transcript.bamdone
parsing /tmp/RtmpNbUBPg/file758961652e10d/sample2_realign2transcript.bam...
parsing /tmp/RtmpNbUBPg/file758961652e10d/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpNbUBPg/file758961652e10d/sample2_realign2transcript.bamdone
parsing /tmp/RtmpNbUBPg/file758961652e10d/sample3_realign2transcript.bam...
parsing /tmp/RtmpNbUBPg/file758961652e10d/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpNbUBPg/file758961652e10d/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
[ FAIL 0 | WARN 182 | SKIP 0 | PASS 59 ]

[ FAIL 0 | WARN 182 | 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 
723.177  41.414 753.457