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###
### Running command:
###
###   /home/biocbuild/bbs-3.23-bioc/R/bin/R CMD check --install=check:FLAMES.install-out.txt --library=/home/biocbuild/bbs-3.23-bioc/R/site-library --timings FLAMES_2.5.5.tar.gz
###
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* using log directory ‘/home/biocbuild/bbs-3.23-bioc/meat/FLAMES.Rcheck’
* using R version 4.6.0 RC (2026-04-17 r89917)
* using platform: x86_64-pc-linux-gnu
* R was compiled by
    gcc (Ubuntu 13.3.0-6ubuntu2~24.04.1) 13.3.0
    GNU Fortran (Ubuntu 13.3.0-6ubuntu2~24.04.1) 13.3.0
* running under: Ubuntu 24.04.4 LTS
* using session charset: UTF-8
* current time: 2026-04-22 04:02:06 UTC
* checking for file ‘FLAMES/DESCRIPTION’ ... OK
* this is package ‘FLAMES’ version ‘2.5.5’
* package encoding: UTF-8
* checking package namespace information ... OK
* checking package dependencies ... INFO
Imports includes 47 non-default packages.
Importing from so many packages makes the package vulnerable to any of
them becoming unavailable.  Move as many as possible to Suggests and
use conditionally.
* checking if this is a source package ... OK
* checking if there is a namespace ... OK
* checking for hidden files and directories ... OK
* 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-3.23-bioc/meat/FLAMES.Rcheck/00install.out’ for details.
* used C++ compiler: ‘g++ (Ubuntu 13.3.0-6ubuntu2~24.04.1) 13.3.0’
* checking C++ specification ... INFO
  specified C++17
* checking installed package size ... INFO
  installed size is  8.8Mb
  sub-directories of 1Mb or more:
    bin    4.6Mb
    data   1.9Mb
* 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
Namespace in Imports field not imported from: ‘scuttle’
  All declared Imports should be used.
There are ::: calls to the package's namespace in its code. A package
  almost never needs to use ::: for its own objects:
  ‘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’
barcode_group: no visible global function definition for ‘new’
barcode_segment: no visible global function definition for ‘new’
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_diversity: no visible global function definition for ‘as’
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 ‘CB’
plot_demultiplex_raw: no visible binding for global variable ‘UB’
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 ‘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_isoforms: no visible binding for global variable ‘xmin’
plot_isoforms: no visible binding for global variable ‘xmax’
plot_isoforms: no visible binding for global variable ‘ymin’
plot_isoforms: no visible binding for global variable ‘ymax’
plot_isoforms: no visible binding for global variable ‘x’
plot_isoforms: no visible binding for global variable ‘xend’
plot_isoforms: no visible binding for global variable ‘y’
plot_isoforms: no visible binding for global variable ‘yend’
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_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_files’
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:
  CB Count FSM_match FlankEditDist Freq Reads Sample Type UB UMI_count
  adj.p.value allele allele_count as bam_index barcode barcode_rank
  barcodes_file barcodes_files 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 xend xmax xmin y yend ymax ymin
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-3.23-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
find_variants                20.965  1.226  21.335
plot_isoform_reduced_dim     20.858  0.608  21.467
blaze                         5.107 14.601  13.242
bulk_long_pipeline            2.413 13.336   2.556
sc_long_multisample_pipeline  8.095  4.577   7.510
MultiSampleSCPipeline        10.326  0.712  11.283
sc_plot_genotype             10.536  0.176   9.540
create_sce_from_dir           6.000  2.837   6.786
sc_DTU_analysis               6.596  1.737   6.475
create_se_from_dir            5.267  0.155   5.406
plot_durations                5.081  0.130   5.199
experiment                    4.901  0.101   4.987
* 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: 4 NOTEs
See
  ‘/home/biocbuild/bbs-3.23-bioc/meat/FLAMES.Rcheck/00check.log’
for details.

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###
### Running command:
###
###   /home/biocbuild/bbs-3.23-bioc/R/bin/R CMD INSTALL FLAMES
###
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* installing to library ‘/home/biocbuild/bbs-3.23-bioc/R/site-library’
* installing *source* package ‘FLAMES’ ...
** this is package ‘FLAMES’ version ‘2.5.5’
** using non-staged installation via StagedInstall field
** libs
specified C++17
using C++ compiler: ‘g++ (Ubuntu 13.3.0-6ubuntu2~24.04.1) 13.3.0’
using C++17
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.23-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.23-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-3.23-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.23-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-3.23-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.23-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-3.23-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.23-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-3.23-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.23-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-3.23-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.23-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-3.23-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.23-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-3.23-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.23-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-3.23-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.23-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:142:56: warning: comparison of integer expressions of different signedness: ‘__gnu_cxx::__alloc_traits<std::allocator<unsigned int>, unsigned int>::value_type’ {aka ‘unsigned int’} and ‘int’ [-Wsign-compare]
  142 |       if (j == (len2 - 1) && dist_holder[i * len2 + j] < best) {
main-functions/flexiplex.cpp: In function ‘void refine_matched_segments(const std::string&, const std::vector<Segment>&, const std::unordered_map<std::__cxx11::basic_string<char>, std::unordered_set<std::__cxx11::basic_string<char> > >*, const std::vector<int>&, Barcode&, std::vector<int>&, std::vector<int>&)’:
main-functions/flexiplex.cpp:348:70: warning: comparison of integer expressions of different signedness: ‘unsigned int’ and ‘const int’ [-Wsign-compare]
  348 |         } else if (editDistance < best_edit_distance && editDistance <= s.max_edit_distance) {
      |                                                         ~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:356:30: warning: comparison of integer expressions of different signedness: ‘unsigned int’ and ‘const int’ [-Wsign-compare]
  356 |       if (best_edit_distance <= s.max_edit_distance && current_segment_unambiguous) {
      |           ~~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘void print_read(const std::string&, const std::string&, const std::string&, const std::vector<Barcode>&, BGZF*, bool, bool, bool, const std::vector<Segment>&, const std::unordered_map<std::__cxx11::basic_string<char>, BarcodeGroup>&)’:
main-functions/flexiplex.cpp:790:21: warning: comparison of integer expressions of different signedness: ‘int’ and ‘const size_t’ {aka ‘const long unsigned int’} [-Wsign-compare]
  790 |   for (int b = 0; b < vec_size; b++) {
      |                   ~~^~~~~~~~~~
main-functions/flexiplex.cpp:803:23: warning: comparison of integer expressions of different signedness: ‘int’ and ‘const size_t’ {aka ‘const long unsigned int’} [-Wsign-compare]
  803 |     for (int k = 0; k < vec_size; k++) {
      |                     ~~^~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.23-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.23-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-3.23-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.23-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-3.23-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.23-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-3.23-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.23-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-3.23-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.23-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-3.23-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.23-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-3.23-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.23-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-3.23-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.23-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-3.23-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-3.23-bioc/R/site-library/Rhtslib/usrlib/libhts.a -lcurl -lbz2 -llzma -lz -L/home/biocbuild/bbs-3.23-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-3.23-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-3.23-bioc/meat/FLAMES/src/submodule/minimap2'
echo "Installing binary to /home/biocbuild/bbs-3.23-bioc/meat/FLAMES/src/../inst/bin"
Installing binary to /home/biocbuild/bbs-3.23-bioc/meat/FLAMES/src/../inst/bin
mkdir -p ../inst/bin
cp submodule/minimap2/minimap2 ../inst/bin/
Building oarfish with cargo
mkdir -p ../inst/bin
mkdir cargo_temp
(cargo install oarfish --root cargo_temp --bin oarfish --vers 0.8.0)
    Updating crates.io index
  Installing oarfish v0.8.0
    Updating crates.io index
     Locking 279 packages to latest compatible versions
      Adding generic-array v0.14.7 (available: v0.14.9)
      Adding indicatif v0.17.11 (available: v0.18.4)
      Adding needletail v0.6.3 (available: v0.7.3)
      Adding noodles-bam v0.78.0 (available: v0.88.0)
      Adding noodles-bgzf v0.38.0 (available: v0.46.0)
      Adding noodles-sam v0.74.0 (available: v0.84.0)
      Adding rand v0.9.4 (available: v0.10.1)
      Adding tabled v0.18.0 (available: v0.20.0)
      Adding typed-builder v0.21.2 (available: v0.23.2)
   Compiling libc v0.2.185
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   Compiling proc-macro-error-attr2 v2.0.0
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   Compiling lexical-core v1.0.6
   Compiling block-buffer v0.10.4
   Compiling crypto-common v0.1.7
   Compiling jobserver v0.1.34
   Compiling digest v0.10.7
   Compiling getrandom v0.2.17
   Compiling num_cpus v1.17.0
   Compiling console v0.15.11
   Compiling cc v1.2.60
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   Compiling num-integer v0.1.46
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   Compiling approx v0.5.1
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   Compiling noisy_float v0.2.1
   Compiling chrono v0.4.44
   Compiling flate2 v1.1.9
   Compiling num-bigint v0.4.6
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   Compiling alga v0.9.3
   Compiling noodles-bgzf v0.38.0
   Compiling zstd-sys v2.0.16+zstd.1.5.7
   Compiling bzip2-sys v0.1.13+1.0.8
   Compiling liblzma-sys v0.3.13
   Compiling libz-sys v1.1.28
   Compiling sha2-asm v0.6.4
   Compiling minimap2-sys v0.1.30+minimap2.2.30
   Compiling lz4-sys v1.11.1+lz4-1.10.0
   Compiling ndarray v0.16.1
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   Compiling csv v1.4.0
   Compiling sha2 v0.10.9
   Compiling num-rational v0.4.2
   Compiling liblzma v0.3.6
   Compiling num v0.4.3
   Compiling proc-macro-error2 v2.0.1
   Compiling ppv-lite86 v0.2.21
   Compiling hashbrown v0.14.5
   Compiling rand_chacha v0.3.1
   Compiling rand_chacha v0.9.0
   Compiling bytemuck_derive v1.10.2
   Compiling futures-macro v0.3.32
   Compiling serde_derive v1.0.228
   Compiling async-trait v0.1.89
   Compiling tracing-attributes v0.1.31
   Compiling async-stream-impl v0.3.6
   Compiling thiserror-impl v1.0.69
   Compiling strum_macros v0.26.4
   Compiling derive-new v0.6.0
   Compiling clap_derive v4.6.1
   Compiling typed-builder-macro v0.21.2
   Compiling tabled_derive v0.10.0
   Compiling rand v0.8.6
   Compiling rand v0.9.4
   Compiling async-stream v0.3.6
   Compiling futures-util v0.3.32
   Compiling bytemuck v1.25.0
   Compiling tabled v0.18.0
   Compiling tracing v0.1.44
   Compiling safe_arch v0.7.4
   Compiling rand_distr v0.4.3
   Compiling typed-builder v0.21.2
   Compiling bstr v1.12.1
   Compiling matchers v0.2.0
   Compiling regex v1.12.3
   Compiling tracing-subscriber v0.3.23
   Compiling wide v0.7.33
   Compiling clap v4.6.1
   Compiling bzip2 v0.4.4
   Compiling noodles-core v0.17.0
   Compiling noodles-csi v0.46.0
   Compiling noodles-sam v0.74.0
   Compiling simba v0.9.1
   Compiling ndarray-stats v0.6.0
   Compiling bincode v1.3.3
   Compiling arrow-format v0.8.1
   Compiling bio-types v1.0.4
   Compiling kders v0.1.1
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   Compiling noodles-bam v0.78.0
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   Compiling seqcol_rs v0.4.1
   Compiling minimap2 v0.1.31+minimap2.2.30
   Compiling statrs v0.18.0
   Compiling oarfish v0.8.0
    Finished `release` profile [optimized] target(s) in 48.30s
  Installing /home/biocbuild/bbs-3.23-bioc/meat/FLAMES/src/cargo_temp/bin/oarfish
   Installed package `oarfish v0.8.0` (executable `oarfish`)
warning: be sure to add `/home/biocbuild/bbs-3.23-bioc/meat/FLAMES/src/cargo_temp/bin` to your PATH to be able to run the installed binaries
cp cargo_temp/bin/oarfish ../inst/bin/
cargo uninstall oarfish --root cargo_temp
    Removing cargo_temp/bin/oarfish
rm -rf cargo_temp
installing to /home/biocbuild/bbs-3.23-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 4.6.0 RC (2026-04-17 r89917) -- "Because it was There"
Copyright (C) 2026 The R Foundation for Statistical Computing
Platform: x86_64-pc-linux-gnu

R is free software and comes with ABSOLUTELY NO WARRANTY.
You are welcome to redistribute it under certain conditions.
Type 'license()' or 'licence()' for distribution details.

R is a collaborative project with many contributors.
Type 'contributors()' for more information and
'citation()' on how to cite R or R packages in publications.

Type 'demo()' for some demos, 'help()' for on-line help, or
'help.start()' for an HTML browser interface to help.
Type 'q()' to quit R.

> # This file is part of the standard setup for testthat.
> # It is recommended that you do not modify it.
> #
> # Where should you do additional test configuration?
> # Learn more about the roles of various files in:
> # * https://r-pkgs.org/tests.html
> # * https://testthat.r-lib.org/reference/test_package.html#special-files
> 
> library(testthat)
> library(FLAMES)
> 
> test_check("FLAMES")
i Writing configuration to: /tmp/RtmpYfu9rt/file25b9ed3176891b/config_file_2472429.json
i Writing configuration to: /tmp/RtmpYfu9rt/file25b9ed3176891b/config_file_2472429.json
i Writing configuration to: /tmp/RtmpYfu9rt/file25b9ed3176891b/config_file_2472429.json
i Writing configuration to: /tmp/RtmpYfu9rt/file25b9ed33fdc794/config_file_2472429.json
Converting legacy `pattern` argument to `segments`...
Loading known barcodes from /tmp/RtmpYfu9rt/file25b9ed63b05bd0/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
Processing file: /home/biocbuild/bbs-3.23-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 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...
Converting legacy `pattern` argument to `segments`...
Loading known barcodes from /tmp/RtmpYfu9rt/file25b9ed79311f90/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
Processing file: /home/biocbuild/bbs-3.23-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 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...
Converting legacy `pattern` argument to `segments`...
Loading known barcodes from /tmp/RtmpYfu9rt/file25b9ed79311f90/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
Processing file: /tmp/RtmpYfu9rt/file25b9ed551499da/musc_rps24_1.fastq
Searching for barcodes...
Processing file: /tmp/RtmpYfu9rt/file25b9ed551499da/musc_rps24_2.fastq
Searching for barcodes...
Processing file: /tmp/RtmpYfu9rt/file25b9ed551499da/musc_rps24_3.fastq
Searching for barcodes...
Processing file: /tmp/RtmpYfu9rt/file25b9ed551499da/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 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...
Converting legacy `pattern` argument to `segments`...
Loading known barcodes from /tmp/RtmpYfu9rt/file25b9ed11a28e39/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
Processing file: /home/biocbuild/bbs-3.23-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 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...
2 transcripts found in the BAM file.
1(50%) transcripts failed the filter.
Failed transcripts account for 100 reads, out of 200(50%) reads in total.
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
i Writing configuration to: /tmp/RtmpYfu9rt/file25b9ed33796628/config_file_2472429.json
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Wed Apr 22 00:11:25 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpYfu9rt/file25b9ed33796628/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpYfu9rt/file25b9ed33796628/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpYfu9rt/file25b9ed33796628/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Wed Apr 22 00:11:27 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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Detected 6 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Wed Apr 22 00:11:53 2026 -------------------
Realigning sample sample1 -> /tmp/RtmpYfu9rt/file25b9ed33796628/sample1_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample2 -> /tmp/RtmpYfu9rt/file25b9ed33796628/sample2_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample3 -> /tmp/RtmpYfu9rt/file25b9ed33796628/sample3_realign2transcript.bam
Skipped sorting BAM files.

-- Running step: transcript_quantification @ Wed Apr 22 00:11:54 2026 ----------
2026-04-22T04:11:54.807062Z  INFO oarfish: setting user-provided filter parameters.
2026-04-22T04:11:54.807555Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpYfu9rt/file25b9ed33796628/sample1_realign2transcript.bam, contains 5 reference sequences.
2026-04-22T04:11:54.807580Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-22T04:11:54.807588Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-22T04:11:54.807673Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-22T04:11:54.807693Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-04-22T04:11:54.811692Z  INFO oarfish::alignment_parser: the alignment file contained 1 unmapped read records.
2026-04-22T04:11:54.811947Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 686   │
│ aligned fraction too low        │ 16    │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 4     │
│ reads with valid best alignment │ 283   │
╰─────────────────────────────────┴───────╯

2026-04-22T04:11:54.811999Z  INFO oarfish::bulk: Total number of alignment records : 364
2026-04-22T04:11:54.812007Z  INFO oarfish::bulk: number of aligned reads : 283
2026-04-22T04:11:54.812018Z  INFO oarfish::bulk: number of unique alignments : 235
2026-04-22T04:11:54.812982Z  INFO oarfish: oarfish completed successfully.
2026-04-22T04:11:54.820744Z  INFO oarfish: setting user-provided filter parameters.
2026-04-22T04:11:54.821149Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpYfu9rt/file25b9ed33796628/sample2_realign2transcript.bam, contains 5 reference sequences.
2026-04-22T04:11:54.821195Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-22T04:11:54.821203Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-22T04:11:54.821261Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-22T04:11:54.821273Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-04-22T04:11:54.825465Z  INFO oarfish::alignment_parser: the alignment file contained 1 unmapped read records.
2026-04-22T04:11:54.825700Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 695   │
│ aligned fraction too low        │ 14    │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 7     │
│ reads with valid best alignment │ 285   │
╰─────────────────────────────────┴───────╯

2026-04-22T04:11:54.825761Z  INFO oarfish::bulk: Total number of alignment records : 369
2026-04-22T04:11:54.825769Z  INFO oarfish::bulk: number of aligned reads : 285
2026-04-22T04:11:54.825775Z  INFO oarfish::bulk: number of unique alignments : 235
2026-04-22T04:11:54.826759Z  INFO oarfish: oarfish completed successfully.
2026-04-22T04:11:54.834630Z  INFO oarfish: setting user-provided filter parameters.
2026-04-22T04:11:54.835065Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpYfu9rt/file25b9ed33796628/sample3_realign2transcript.bam, contains 5 reference sequences.
2026-04-22T04:11:54.835088Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-22T04:11:54.835121Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-22T04:11:54.835177Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-22T04:11:54.835189Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-04-22T04:11:54.839250Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2026-04-22T04:11:54.839501Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 696   │
│ aligned fraction too low        │ 14    │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 5     │
│ reads with valid best alignment │ 284   │
╰─────────────────────────────────┴───────╯

2026-04-22T04:11:54.839556Z  INFO oarfish::bulk: Total number of alignment records : 362
2026-04-22T04:11:54.839564Z  INFO oarfish::bulk: number of aligned reads : 284
2026-04-22T04:11:54.839571Z  INFO oarfish::bulk: number of unique alignments : 237
2026-04-22T04:11:54.840511Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
i Writing configuration to: /tmp/RtmpYfu9rt/file25b9ed3d59a01a/config_file_2472429.json
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Wed Apr 22 00:11:55 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpYfu9rt/file25b9ed3d59a01a/sample1_align2genome.bam
sample2 ->/tmp/RtmpYfu9rt/file25b9ed3d59a01a/sample2_align2genome.bam
sample3 ->/tmp/RtmpYfu9rt/file25b9ed3d59a01a/sample3_align2genome.bam
-- Running step: isoform_identification @ Wed Apr 22 00:12:18 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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Detected 6 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Wed Apr 22 00:12:44 2026 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpYfu9rt/file25b9ed3d59a01a/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpYfu9rt/file25b9ed3d59a01a/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpYfu9rt/file25b9ed3d59a01a/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Wed Apr 22 00:13:05 2026 ----------
2026-04-22T04:13:05.736286Z  INFO oarfish: setting user-provided filter parameters.
2026-04-22T04:13:05.736888Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpYfu9rt/file25b9ed3d59a01a/sample1_realign2transcript.bam, contains 5 reference sequences.
2026-04-22T04:13:05.736916Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-22T04:13:05.737139Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-22T04:13:05.737198Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-22T04:13:05.737210Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-04-22T04:13:05.741527Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2026-04-22T04:13:05.741769Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 702   │
│ aligned fraction too low        │ 13    │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 5     │
│ reads with valid best alignment │ 285   │
╰─────────────────────────────────┴───────╯

2026-04-22T04:13:05.741811Z  INFO oarfish::bulk: Total number of alignment records : 364
2026-04-22T04:13:05.741823Z  INFO oarfish::bulk: number of aligned reads : 285
2026-04-22T04:13:05.741830Z  INFO oarfish::bulk: number of unique alignments : 238
2026-04-22T04:13:05.742635Z  INFO oarfish: oarfish completed successfully.
2026-04-22T04:13:05.753755Z  INFO oarfish: setting user-provided filter parameters.
2026-04-22T04:13:05.754143Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpYfu9rt/file25b9ed3d59a01a/sample2_realign2transcript.bam, contains 5 reference sequences.
2026-04-22T04:13:05.754164Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-22T04:13:05.754171Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-22T04:13:05.754237Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-22T04:13:05.754248Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-04-22T04:13:05.758174Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2026-04-22T04:13:05.758371Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 687   │
│ aligned fraction too low        │ 16    │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 6     │
│ reads with valid best alignment │ 282   │
╰─────────────────────────────────┴───────╯

2026-04-22T04:13:05.758423Z  INFO oarfish::bulk: Total number of alignment records : 355
2026-04-22T04:13:05.758431Z  INFO oarfish::bulk: number of aligned reads : 282
2026-04-22T04:13:05.758443Z  INFO oarfish::bulk: number of unique alignments : 240
2026-04-22T04:13:05.759238Z  INFO oarfish: oarfish completed successfully.
2026-04-22T04:13:05.770445Z  INFO oarfish: setting user-provided filter parameters.
2026-04-22T04:13:05.770945Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpYfu9rt/file25b9ed3d59a01a/sample3_realign2transcript.bam, contains 5 reference sequences.
2026-04-22T04:13:05.770995Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-22T04:13:05.771003Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-22T04:13:05.771059Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-22T04:13:05.771071Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-04-22T04:13:05.775519Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2026-04-22T04:13:05.775729Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 688   │
│ aligned fraction too low        │ 15    │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 7     │
│ reads with valid best alignment │ 283   │
╰─────────────────────────────────┴───────╯

2026-04-22T04:13:05.775781Z  INFO oarfish::bulk: Total number of alignment records : 354
2026-04-22T04:13:05.775789Z  INFO oarfish::bulk: number of aligned reads : 283
2026-04-22T04:13:05.775796Z  INFO oarfish::bulk: number of unique alignments : 240
2026-04-22T04:13:05.776893Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
i Writing configuration to: /tmp/RtmpYfu9rt/file25b9ed3080cfd9/config_file_2472429.json
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Wed Apr 22 00:13:06 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpYfu9rt/file25b9ed3080cfd9/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpYfu9rt/file25b9ed3080cfd9/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpYfu9rt/file25b9ed3080cfd9/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Wed Apr 22 00:13:07 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 6 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Wed Apr 22 00:13:27 2026 -------------------
Realigning sample sample1 -> /tmp/RtmpYfu9rt/file25b9ed3080cfd9/sample1_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample2 -> /tmp/RtmpYfu9rt/file25b9ed3080cfd9/sample2_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample3 -> /tmp/RtmpYfu9rt/file25b9ed3080cfd9/sample3_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Wed Apr 22 00:13:28 2026 ----------
00:13:28 Wed Apr 22 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)
i Writing configuration to: /tmp/RtmpYfu9rt/file25b9ed7bef44bc/config_file_2472429.json
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Wed Apr 22 00:13:30 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpYfu9rt/file25b9ed7bef44bc/sample1_align2genome.bam
sample2 ->/tmp/RtmpYfu9rt/file25b9ed7bef44bc/sample2_align2genome.bam
sample3 ->/tmp/RtmpYfu9rt/file25b9ed7bef44bc/sample3_align2genome.bam
-- Running step: isoform_identification @ Wed Apr 22 00:13:52 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 5 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Wed Apr 22 00:14:12 2026 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpYfu9rt/file25b9ed7bef44bc/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpYfu9rt/file25b9ed7bef44bc/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpYfu9rt/file25b9ed7bef44bc/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Wed Apr 22 00:14:32 2026 ----------
00:14:32 Wed Apr 22 2026 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Inputs:  ['/tmp/RtmpYfu9rt/file25b9ed3080cfd9/sample1_realign2transcript.bam', '/tmp/RtmpYfu9rt/file25b9ed3080cfd9/sample2_realign2transcript.bam', '/tmp/RtmpYfu9rt/file25b9ed3080cfd9/sample3_realign2transcript.bam'] /tmp/RtmpYfu9rt/file25b9ed3080cfd9/transcript_assembly.fa.fai 5 0.4 0.4
	Counter({'counted_reads': 866, 'not_enough_coverage': 30, 'unmapped': 4})
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
i Writing configuration to: /tmp/RtmpYfu9rt/file25b9ed40a024df/config_file_2472429.json
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Wed Apr 22 00:14:33 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpYfu9rt/file25b9ed40a024df/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpYfu9rt/file25b9ed40a024df/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpYfu9rt/file25b9ed40a024df/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Wed Apr 22 00:14:35 2026 -------------
Inputs:  ['/tmp/RtmpYfu9rt/file25b9ed7bef44bc/sample1_realign2transcript.bam', '/tmp/RtmpYfu9rt/file25b9ed7bef44bc/sample2_realign2transcript.bam', '/tmp/RtmpYfu9rt/file25b9ed7bef44bc/sample3_realign2transcript.bam'] /tmp/RtmpYfu9rt/file25b9ed7bef44bc/transcript_assembly.fa.fai 5 0.4 0.4
	Counter({'counted_reads': 863, 'not_enough_coverage': 32, 'unmapped': 5})
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Wed Apr 22 00:14:36 2026 -------------------
Realigning sample sample1 -> /tmp/RtmpYfu9rt/file25b9ed40a024df/sample1_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample2 -> /tmp/RtmpYfu9rt/file25b9ed40a024df/sample2_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample3 -> /tmp/RtmpYfu9rt/file25b9ed40a024df/sample3_realign2transcript.bam
Skipped sorting BAM files.

-- Running step: transcript_quantification @ Wed Apr 22 00:14:38 2026 ----------
2026-04-22T04:14:38.917930Z  INFO oarfish: setting user-provided filter parameters.
2026-04-22T04:14:38.918381Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpYfu9rt/file25b9ed40a024df/sample1_realign2transcript.bam, contains 17 reference sequences.
2026-04-22T04:14:38.918403Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-22T04:14:38.918442Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-22T04:14:38.918534Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-22T04:14:38.918549Z  INFO oarfish: parsed reference information for 17 transcripts.
2026-04-22T04:14:38.928985Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-04-22T04:14:38.929251Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 3436  │
│ aligned fraction too low        │ 7     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 293   │
╰─────────────────────────────────┴───────╯

2026-04-22T04:14:38.929304Z  INFO oarfish::bulk: Total number of alignment records : 513
2026-04-22T04:14:38.929317Z  INFO oarfish::bulk: number of aligned reads : 293
2026-04-22T04:14:38.929323Z  INFO oarfish::bulk: number of unique alignments : 198
2026-04-22T04:14:38.930218Z  INFO oarfish: oarfish completed successfully.
2026-04-22T04:14:38.939035Z  INFO oarfish: setting user-provided filter parameters.
2026-04-22T04:14:38.939428Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpYfu9rt/file25b9ed40a024df/sample2_realign2transcript.bam, contains 17 reference sequences.
2026-04-22T04:14:38.939476Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-22T04:14:38.939486Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-22T04:14:38.939576Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-22T04:14:38.939600Z  INFO oarfish: parsed reference information for 17 transcripts.
2026-04-22T04:14:38.951370Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-04-22T04:14:38.951624Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 3402  │
│ aligned fraction too low        │ 8     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 292   │
╰─────────────────────────────────┴───────╯

2026-04-22T04:14:38.951706Z  INFO oarfish::bulk: Total number of alignment records : 525
2026-04-22T04:14:38.951713Z  INFO oarfish::bulk: number of aligned reads : 292
2026-04-22T04:14:38.951727Z  INFO oarfish::bulk: number of unique alignments : 190
2026-04-22T04:14:38.952731Z  INFO oarfish: oarfish completed successfully.
2026-04-22T04:14:38.961274Z  INFO oarfish: setting user-provided filter parameters.
2026-04-22T04:14:38.961622Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpYfu9rt/file25b9ed40a024df/sample3_realign2transcript.bam, contains 17 reference sequences.
2026-04-22T04:14:38.961691Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-22T04:14:38.961703Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-22T04:14:38.961792Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-22T04:14:38.961808Z  INFO oarfish: parsed reference information for 17 transcripts.
2026-04-22T04:14:38.972046Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-04-22T04:14:38.972286Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 3342  │
│ aligned fraction too low        │ 10    │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 290   │
╰─────────────────────────────────┴───────╯

2026-04-22T04:14:38.972351Z  INFO oarfish::bulk: Total number of alignment records : 517
2026-04-22T04:14:38.972359Z  INFO oarfish::bulk: number of aligned reads : 290
2026-04-22T04:14:38.972365Z  INFO oarfish::bulk: number of unique alignments : 192
2026-04-22T04:14:38.973336Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
i Writing configuration to: /tmp/RtmpYfu9rt/file25b9ed1cebab62/config_file_2472429.json
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Wed Apr 22 00:14:39 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpYfu9rt/file25b9ed1cebab62/sample1_align2genome.bam
sample2 ->/tmp/RtmpYfu9rt/file25b9ed1cebab62/sample2_align2genome.bam
sample3 ->/tmp/RtmpYfu9rt/file25b9ed1cebab62/sample3_align2genome.bam
-- Running step: isoform_identification @ Wed Apr 22 00:15:00 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Wed Apr 22 00:15:00 2026 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpYfu9rt/file25b9ed1cebab62/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpYfu9rt/file25b9ed1cebab62/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpYfu9rt/file25b9ed1cebab62/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Wed Apr 22 00:15:22 2026 ----------
2026-04-22T04:15:22.411242Z  INFO oarfish: setting user-provided filter parameters.
2026-04-22T04:15:22.411704Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpYfu9rt/file25b9ed1cebab62/sample1_realign2transcript.bam, contains 15 reference sequences.
2026-04-22T04:15:22.411726Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-22T04:15:22.411734Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-22T04:15:22.411815Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-22T04:15:22.411846Z  INFO oarfish: parsed reference information for 15 transcripts.
2026-04-22T04:15:22.421254Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-04-22T04:15:22.421477Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 3045  │
│ aligned fraction too low        │ 7     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 293   │
╰─────────────────────────────────┴───────╯

2026-04-22T04:15:22.421538Z  INFO oarfish::bulk: Total number of alignment records : 495
2026-04-22T04:15:22.421546Z  INFO oarfish::bulk: number of aligned reads : 293
2026-04-22T04:15:22.421559Z  INFO oarfish::bulk: number of unique alignments : 208
2026-04-22T04:15:22.422449Z  INFO oarfish: oarfish completed successfully.
2026-04-22T04:15:22.429822Z  INFO oarfish: setting user-provided filter parameters.
2026-04-22T04:15:22.430177Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpYfu9rt/file25b9ed1cebab62/sample2_realign2transcript.bam, contains 15 reference sequences.
2026-04-22T04:15:22.430224Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-22T04:15:22.430233Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-22T04:15:22.430311Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-22T04:15:22.430326Z  INFO oarfish: parsed reference information for 15 transcripts.
2026-04-22T04:15:22.439603Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-04-22T04:15:22.439845Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 2896  │
│ aligned fraction too low        │ 10    │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 290   │
╰─────────────────────────────────┴───────╯

2026-04-22T04:15:22.439914Z  INFO oarfish::bulk: Total number of alignment records : 511
2026-04-22T04:15:22.439923Z  INFO oarfish::bulk: number of aligned reads : 290
2026-04-22T04:15:22.439929Z  INFO oarfish::bulk: number of unique alignments : 198
2026-04-22T04:15:22.440785Z  INFO oarfish: oarfish completed successfully.
2026-04-22T04:15:22.448080Z  INFO oarfish: setting user-provided filter parameters.
2026-04-22T04:15:22.448440Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpYfu9rt/file25b9ed1cebab62/sample3_realign2transcript.bam, contains 15 reference sequences.
2026-04-22T04:15:22.448461Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-22T04:15:22.448493Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-22T04:15:22.448573Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-22T04:15:22.448588Z  INFO oarfish: parsed reference information for 15 transcripts.
2026-04-22T04:15:22.458187Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-04-22T04:15:22.458453Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 2880  │
│ aligned fraction too low        │ 9     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 291   │
╰─────────────────────────────────┴───────╯

2026-04-22T04:15:22.458505Z  INFO oarfish::bulk: Total number of alignment records : 496
2026-04-22T04:15:22.458513Z  INFO oarfish::bulk: number of aligned reads : 291
2026-04-22T04:15:22.458520Z  INFO oarfish::bulk: number of unique alignments : 206
2026-04-22T04:15:22.459359Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
i Writing configuration to: /tmp/RtmpYfu9rt/file25b9ed1131f90f/config_file_2472429.json
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Wed Apr 22 00:15:22 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpYfu9rt/file25b9ed1131f90f/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpYfu9rt/file25b9ed1131f90f/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpYfu9rt/file25b9ed1131f90f/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Wed Apr 22 00:15:24 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Wed Apr 22 00:15:25 2026 -------------------
Realigning sample sample1 -> /tmp/RtmpYfu9rt/file25b9ed1131f90f/sample1_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample2 -> /tmp/RtmpYfu9rt/file25b9ed1131f90f/sample2_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample3 -> /tmp/RtmpYfu9rt/file25b9ed1131f90f/sample3_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Wed Apr 22 00:15:26 2026 ----------
00:15:26 Wed Apr 22 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)
i Writing configuration to: /tmp/RtmpYfu9rt/file25b9ed4c464218/config_file_2472429.json
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Wed Apr 22 00:15:27 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpYfu9rt/file25b9ed4c464218/sample1_align2genome.bam
sample2 ->/tmp/RtmpYfu9rt/file25b9ed4c464218/sample2_align2genome.bam
sample3 ->/tmp/RtmpYfu9rt/file25b9ed4c464218/sample3_align2genome.bam
-- Running step: isoform_identification @ Wed Apr 22 00:15:48 2026 -------------
Inputs:  ['/tmp/RtmpYfu9rt/file25b9ed1131f90f/sample1_realign2transcript.bam', '/tmp/RtmpYfu9rt/file25b9ed1131f90f/sample2_realign2transcript.bam', '/tmp/RtmpYfu9rt/file25b9ed1131f90f/sample3_realign2transcript.bam'] /tmp/RtmpYfu9rt/file25b9ed1131f90f/transcript_assembly.fa.fai 5 0.4 0.4
	Counter({'counted_reads': 895, 'not_enough_coverage': 5})
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Wed Apr 22 00:15:48 2026 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpYfu9rt/file25b9ed4c464218/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpYfu9rt/file25b9ed4c464218/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpYfu9rt/file25b9ed4c464218/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Wed Apr 22 00:16:08 2026 ----------
00:16:08 Wed Apr 22 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)
i Writing configuration to: /tmp/RtmpYfu9rt/file25b9ed4c3656aa/config_file_2472429.json
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Wed Apr 22 00:16:09 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpYfu9rt/file25b9ed4c3656aa/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 8
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
Processing file: /home/biocbuild/bbs-3.23-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 chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Wed Apr 22 00:16:10 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpYfu9rt/file25b9ed4c3656aa/matched_reads.fastq.gz -> /tmp/RtmpYfu9rt/file25b9ed4c3656aa/align2genome.bam
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: isoform_identification @ Wed Apr 22 00:16:10 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 @ Wed Apr 22 00:16:19 2026 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.23-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpYfu9rt/file25b9ed4c3656aa/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpYfu9rt/file25b9ed4c3656aa/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/RtmpYfu9rt/file25b9ed4c3656aa/matched_reads.fastq.gz -> /tmp/RtmpYfu9rt/file25b9ed4c3656aa/realign2transcript.bam
Sorting BAM files by 8 with CB threads...

[bam_sort_core] merging from 0 files and 8 in-memory blocks...
-- Running step: transcript_quantification @ Wed Apr 22 00:16:20 2026 ----------
2026-04-22T04:16:20.126932Z  INFO oarfish: setting user-provided filter parameters.
2026-04-22T04:16:20.127478Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpYfu9rt/file25b9ed4c3656aa/realign2transcript.bam, contains 5 reference sequences.
2026-04-22T04:16:20.127534Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-22T04:16:20.127543Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-22T04:16:20.127599Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-22T04:16:20.127610Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-04-22T04:16:20.133960Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
i Writing configuration to: /tmp/RtmpYfu9rt/file25b9ed5035e57a/config_file_2472429.json
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Wed Apr 22 00:16:20 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpYfu9rt/file25b9ed5035e57a/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 8
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
Processing file: /home/biocbuild/bbs-3.23-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 chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Wed Apr 22 00:16:20 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpYfu9rt/file25b9ed5035e57a/matched_reads.fastq.gz ->/tmp/RtmpYfu9rt/file25b9ed5035e57a/align2genome.bam
-- Running step: isoform_identification @ Wed Apr 22 00:16:40 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 @ Wed Apr 22 00:16:50 2026 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.23-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpYfu9rt/file25b9ed5035e57a/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpYfu9rt/file25b9ed5035e57a/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/RtmpYfu9rt/file25b9ed5035e57a/matched_reads.fastq.gz ->/tmp/RtmpYfu9rt/file25b9ed5035e57a/realign2transcript.bam
-- Running step: transcript_quantification @ Wed Apr 22 00:17:09 2026 ----------
2026-04-22T04:17:09.436701Z  INFO oarfish: setting user-provided filter parameters.
2026-04-22T04:17:09.437097Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpYfu9rt/file25b9ed5035e57a/realign2transcript.bam, contains 5 reference sequences.
2026-04-22T04:17:09.437118Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-22T04:17:09.437163Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-22T04:17:09.437214Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-22T04:17:09.437224Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-04-22T04:17:09.443232Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
i Writing configuration to: /tmp/RtmpYfu9rt/file25b9ed65673258/config_file_2472429.json
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Wed Apr 22 00:17:09 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpYfu9rt/file25b9ed65673258/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 8
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
Processing file: /home/biocbuild/bbs-3.23-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 chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Wed Apr 22 00:17:10 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpYfu9rt/file25b9ed65673258/matched_reads.fastq.gz -> /tmp/RtmpYfu9rt/file25b9ed65673258/align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: isoform_identification @ Wed Apr 22 00:17:10 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 @ Wed Apr 22 00:17:19 2026 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.23-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpYfu9rt/file25b9ed65673258/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpYfu9rt/file25b9ed65673258/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/RtmpYfu9rt/file25b9ed65673258/matched_reads.fastq.gz -> /tmp/RtmpYfu9rt/file25b9ed65673258/realign2transcript.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 8 threads...

[bam_sort_core] merging from 0 files and 8 in-memory blocks...
Indexing bam files
-- Running step: transcript_quantification @ Wed Apr 22 00:17:19 2026 ----------
00:17:19 Wed Apr 22 2026 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Inputs:  ['/tmp/RtmpYfu9rt/file25b9ed4c464218/sample1_realign2transcript.bam', '/tmp/RtmpYfu9rt/file25b9ed4c464218/sample2_realign2transcript.bam', '/tmp/RtmpYfu9rt/file25b9ed4c464218/sample3_realign2transcript.bam'] /tmp/RtmpYfu9rt/file25b9ed4c464218/transcript_assembly.fa.fai 5 0.4 0.4
	Counter({'counted_reads': 895, 'not_enough_coverage': 5})
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
i Writing configuration to: /tmp/RtmpYfu9rt/file25b9ed43e3d5c3/config_file_2472429.json
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Wed Apr 22 00:17:20 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpYfu9rt/file25b9ed43e3d5c3/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 8
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
Processing file: /home/biocbuild/bbs-3.23-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 chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Wed Apr 22 00:17:20 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpYfu9rt/file25b9ed43e3d5c3/matched_reads.fastq.gz ->/tmp/RtmpYfu9rt/file25b9ed43e3d5c3/align2genome.bam
-- Running step: isoform_identification @ Wed Apr 22 00:17:44 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 @ Wed Apr 22 00:17:53 2026 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.23-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpYfu9rt/file25b9ed43e3d5c3/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpYfu9rt/file25b9ed43e3d5c3/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/RtmpYfu9rt/file25b9ed43e3d5c3/matched_reads.fastq.gz ->/tmp/RtmpYfu9rt/file25b9ed43e3d5c3/realign2transcript.bam
-- Running step: transcript_quantification @ Wed Apr 22 00:18:13 2026 ----------
00:18:13 Wed Apr 22 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)
i Writing configuration to: /tmp/RtmpYfu9rt/file25b9ed3e6e9dc4/config_file_2472429.json
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Wed Apr 22 00:18:14 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpYfu9rt/file25b9ed3e6e9dc4/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 8
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
Processing file: /home/biocbuild/bbs-3.23-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 chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Wed Apr 22 00:18:14 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpYfu9rt/file25b9ed3e6e9dc4/matched_reads.fastq.gz -> /tmp/RtmpYfu9rt/file25b9ed3e6e9dc4/align2genome.bam
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: isoform_identification @ Wed Apr 22 00:18:14 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 @ Wed Apr 22 00:18:15 2026 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.23-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpYfu9rt/file25b9ed3e6e9dc4/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpYfu9rt/file25b9ed3e6e9dc4/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/RtmpYfu9rt/file25b9ed3e6e9dc4/matched_reads.fastq.gz -> /tmp/RtmpYfu9rt/file25b9ed3e6e9dc4/realign2transcript.bam
Sorting BAM files by 8 with CB threads...

[bam_sort_core] merging from 0 files and 8 in-memory blocks...
-- Running step: transcript_quantification @ Wed Apr 22 00:18:15 2026 ----------
2026-04-22T04:18:15.372125Z  INFO oarfish: setting user-provided filter parameters.
2026-04-22T04:18:15.372690Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpYfu9rt/file25b9ed3e6e9dc4/realign2transcript.bam, contains 10 reference sequences.
2026-04-22T04:18:15.372756Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-22T04:18:15.372765Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-22T04:18:15.372836Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-22T04:18:15.372859Z  INFO oarfish: parsed reference information for 10 transcripts.
2026-04-22T04:18:15.382805Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
i Writing configuration to: /tmp/RtmpYfu9rt/file25b9ed21d7d0fc/config_file_2472429.json
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Wed Apr 22 00:18:16 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpYfu9rt/file25b9ed21d7d0fc/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 8
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
Processing file: /home/biocbuild/bbs-3.23-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 chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Wed Apr 22 00:18:16 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpYfu9rt/file25b9ed21d7d0fc/matched_reads.fastq.gz ->/tmp/RtmpYfu9rt/file25b9ed21d7d0fc/align2genome.bam
-- Running step: isoform_identification @ Wed Apr 22 00:18:35 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Wed Apr 22 00:18:36 2026 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.23-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpYfu9rt/file25b9ed21d7d0fc/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpYfu9rt/file25b9ed21d7d0fc/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/RtmpYfu9rt/file25b9ed21d7d0fc/matched_reads.fastq.gz ->/tmp/RtmpYfu9rt/file25b9ed21d7d0fc/realign2transcript.bam
-- Running step: transcript_quantification @ Wed Apr 22 00:18:55 2026 ----------
2026-04-22T04:18:55.855819Z  INFO oarfish: setting user-provided filter parameters.
2026-04-22T04:18:55.856307Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpYfu9rt/file25b9ed21d7d0fc/realign2transcript.bam, contains 10 reference sequences.
2026-04-22T04:18:55.856329Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-22T04:18:55.856337Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-22T04:18:55.856426Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-22T04:18:55.856439Z  INFO oarfish: parsed reference information for 10 transcripts.
2026-04-22T04:18:55.865985Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
i Writing configuration to: /tmp/RtmpYfu9rt/file25b9ed6b4202bf/config_file_2472429.json
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Wed Apr 22 00:18:56 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpYfu9rt/file25b9ed6b4202bf/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 8
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
Processing file: /home/biocbuild/bbs-3.23-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 chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Wed Apr 22 00:18:56 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpYfu9rt/file25b9ed6b4202bf/matched_reads.fastq.gz -> /tmp/RtmpYfu9rt/file25b9ed6b4202bf/align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: isoform_identification @ Wed Apr 22 00:18:57 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Wed Apr 22 00:18:57 2026 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.23-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpYfu9rt/file25b9ed6b4202bf/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpYfu9rt/file25b9ed6b4202bf/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/RtmpYfu9rt/file25b9ed6b4202bf/matched_reads.fastq.gz -> /tmp/RtmpYfu9rt/file25b9ed6b4202bf/realign2transcript.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 8 threads...

[bam_sort_core] merging from 0 files and 8 in-memory blocks...
Indexing bam files
-- Running step: transcript_quantification @ Wed Apr 22 00:18:57 2026 ----------
00:18:57 Wed Apr 22 2026 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
i Writing configuration to: /tmp/RtmpYfu9rt/file25b9ed64bebf92/config_file_2472429.json
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Wed Apr 22 00:18:58 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpYfu9rt/file25b9ed64bebf92/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 8
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
Processing file: /home/biocbuild/bbs-3.23-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 chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Wed Apr 22 00:18:59 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpYfu9rt/file25b9ed64bebf92/matched_reads.fastq.gz ->/tmp/RtmpYfu9rt/file25b9ed64bebf92/align2genome.bam
-- Running step: isoform_identification @ Wed Apr 22 00:19:17 2026 -------------
	Counter({'counted_reads': 368, 'unmapped': 4})
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Wed Apr 22 00:19:18 2026 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.23-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpYfu9rt/file25b9ed64bebf92/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpYfu9rt/file25b9ed64bebf92/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/RtmpYfu9rt/file25b9ed64bebf92/matched_reads.fastq.gz ->/tmp/RtmpYfu9rt/file25b9ed64bebf92/realign2transcript.bam
-- Running step: transcript_quantification @ Wed Apr 22 00:19:36 2026 ----------
00:19:36 Wed Apr 22 2026 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
i Writing configuration to: /tmp/RtmpYfu9rt/file25b9ed624df4dc/config_file_2472429.json
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Wed Apr 22 00:19:38 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpYfu9rt/file25b9ed624df4dc/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
Processing file: /tmp/RtmpYfu9rt/file25b9ed624df4dc/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpYfu9rt/file25b9ed624df4dc/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpYfu9rt/file25b9ed624df4dc/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 993
Number of reads where at least one barcode was found: 927
Number of chimera reads: 3
All done!
Reads	Barcodes
26	1
23	3
22	1
21	3
20	1
19	1
18	1
16	2
15	1
14	2
13	3
12	5
11	2
10	6
9	9
8	3
7	6
6	11
5	8
4	8
3	39
2	19
1	2
Loading known barcodes from /tmp/RtmpYfu9rt/file25b9ed624df4dc/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
Processing file: /tmp/RtmpYfu9rt/file25b9ed624df4dc/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 300
Number of reads where at least one barcode was found: 280
Number of chimera reads: 1
All done!
Reads	Barcodes
9	1
8	1
7	5
6	2
5	7
4	5
3	19
2	27
1	53
Loading known barcodes from /tmp/RtmpYfu9rt/file25b9ed624df4dc/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
Processing file: /tmp/RtmpYfu9rt/file25b9ed624df4dc/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 300
Number of reads where at least one barcode was found: 279
Number of chimera reads: 1
All done!
Reads	Barcodes
9	1
7	5
6	4
5	1
4	15
3	12
2	23
1	65
Loading known barcodes from /tmp/RtmpYfu9rt/file25b9ed624df4dc/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
Processing file: /tmp/RtmpYfu9rt/file25b9ed624df4dc/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 chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Wed Apr 22 00:19:39 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpYfu9rt/file25b9ed624df4dc/sampleA_matched_reads.fastq.gz -> /tmp/RtmpYfu9rt/file25b9ed624df4dc/sampleA_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpYfu9rt/file25b9ed624df4dc/sample1_matched_reads.fastq.gz -> /tmp/RtmpYfu9rt/file25b9ed624df4dc/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpYfu9rt/file25b9ed624df4dc/sample2_matched_reads.fastq.gz -> /tmp/RtmpYfu9rt/file25b9ed624df4dc/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpYfu9rt/file25b9ed624df4dc/sample3_matched_reads.fastq.gz -> /tmp/RtmpYfu9rt/file25b9ed624df4dc/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Wed Apr 22 00:19:42 2026 ----------------
00:19:42 Wed Apr 22 2026 quantify genes 
Using BAM(s): '/tmp/RtmpYfu9rt/file25b9ed624df4dc/sampleA_align2genome.bam',
'/tmp/RtmpYfu9rt/file25b9ed624df4dc/sample1_align2genome.bam',
'/tmp/RtmpYfu9rt/file25b9ed624df4dc/sample2_align2genome.bam', and
'/tmp/RtmpYfu9rt/file25b9ed624df4dc/sample3_align2genome.bam'
	Counter({'counted_reads': 368, 'unmapped': 4})
parsing /tmp/RtmpYfu9rt/file25b9ed624df4dc/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 13.70gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 177229.11Read/s]
parsing /tmp/RtmpYfu9rt/file25b9ed624df4dc/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 26.51gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 544856.33Read/s]
parsing /tmp/RtmpYfu9rt/file25b9ed624df4dc/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 28.53gene_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, 551533.77Read/s]
parsing /tmp/RtmpYfu9rt/file25b9ed624df4dc/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 23.71gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 432830.84Read/s]
-- Running step: isoform_identification @ Wed Apr 22 00:19:44 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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  |                                                                            
  |==================                                                    |  25%
  |                                                                            
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Detected 7 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)
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Wed Apr 22 00:20:08 2026 -------------------
Checking for fastq file(s) /tmp/RtmpYfu9rt/file25b9ed624df4dc/fastq, /tmp/RtmpYfu9rt/file25b9ed624df4dc/fastq/sample1.fq.gz, /tmp/RtmpYfu9rt/file25b9ed624df4dc/fastq/sample2.fq.gz, /tmp/RtmpYfu9rt/file25b9ed624df4dc/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpYfu9rt/file25b9ed624df4dc/sampleA_matched_reads.fastq.gz, /tmp/RtmpYfu9rt/file25b9ed624df4dc/sample1_matched_reads.fastq.gz, /tmp/RtmpYfu9rt/file25b9ed624df4dc/sample2_matched_reads.fastq.gz, /tmp/RtmpYfu9rt/file25b9ed624df4dc/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpYfu9rt/file25b9ed624df4dc/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpYfu9rt/file25b9ed624df4dc/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpYfu9rt/file25b9ed624df4dc/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpYfu9rt/file25b9ed624df4dc/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpYfu9rt/file25b9ed624df4dc/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpYfu9rt/file25b9ed624df4dc/sampleA_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpYfu9rt/file25b9ed624df4dc/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpYfu9rt/file25b9ed624df4dc/sample1_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpYfu9rt/file25b9ed624df4dc/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpYfu9rt/file25b9ed624df4dc/sample2_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpYfu9rt/file25b9ed624df4dc/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpYfu9rt/file25b9ed624df4dc/sample3_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

-- Running step: transcript_quantification @ Wed Apr 22 00:20:10 2026 ----------
2026-04-22T04:20:10.847772Z  INFO oarfish: setting user-provided filter parameters.
2026-04-22T04:20:10.848246Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpYfu9rt/file25b9ed624df4dc/sampleA_realign2transcript.bam, contains 6 reference sequences.
2026-04-22T04:20:10.848327Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-22T04:20:10.848336Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-22T04:20:10.848396Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-22T04:20:10.848421Z  INFO oarfish: parsed reference information for 6 transcripts.
2026-04-22T04:20:10.859333Z  INFO oarfish::single_cell: Processed 100 cells.
2026-04-22T04:20:11.175014Z  INFO oarfish: setting user-provided filter parameters.
2026-04-22T04:20:11.175386Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpYfu9rt/file25b9ed624df4dc/sample1_realign2transcript.bam, contains 6 reference sequences.
2026-04-22T04:20:11.175482Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-22T04:20:11.175491Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-22T04:20:11.175548Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-22T04:20:11.175573Z  INFO oarfish: parsed reference information for 6 transcripts.
2026-04-22T04:20:11.180790Z  INFO oarfish::single_cell: Processed 100 cells.
2026-04-22T04:20:11.531165Z  INFO oarfish: setting user-provided filter parameters.
2026-04-22T04:20:11.531539Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpYfu9rt/file25b9ed624df4dc/sample2_realign2transcript.bam, contains 6 reference sequences.
2026-04-22T04:20:11.531626Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-22T04:20:11.531634Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-22T04:20:11.531693Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-22T04:20:11.531705Z  INFO oarfish: parsed reference information for 6 transcripts.
2026-04-22T04:20:11.537187Z  INFO oarfish::single_cell: Processed 100 cells.
2026-04-22T04:20:11.843431Z  INFO oarfish: setting user-provided filter parameters.
2026-04-22T04:20:11.843947Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpYfu9rt/file25b9ed624df4dc/sample3_realign2transcript.bam, contains 6 reference sequences.
2026-04-22T04:20:11.844013Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-22T04:20:11.844022Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-22T04:20:11.844077Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-22T04:20:11.844087Z  INFO oarfish: parsed reference information for 6 transcripts.
2026-04-22T04:20:11.849760Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
i Writing configuration to: /tmp/RtmpYfu9rt/file25b9ed2a255c86/config_file_2472429.json
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Wed Apr 22 00:20:12 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpYfu9rt/file25b9ed2a255c86/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
Processing file: /tmp/RtmpYfu9rt/file25b9ed2a255c86/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpYfu9rt/file25b9ed2a255c86/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpYfu9rt/file25b9ed2a255c86/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 993
Number of reads where at least one barcode was found: 930
Number of chimera reads: 3
All done!
Reads	Barcodes
28	1
25	1
23	3
22	1
21	1
19	2
18	1
17	1
16	2
15	2
14	2
13	4
12	2
11	5
10	4
9	7
8	7
7	2
6	11
5	12
4	7
3	37
2	20
1	2
Loading known barcodes from /tmp/RtmpYfu9rt/file25b9ed2a255c86/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
Processing file: /tmp/RtmpYfu9rt/file25b9ed2a255c86/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 300
Number of reads where at least one barcode was found: 284
Number of chimera reads: 1
All done!
Reads	Barcodes
10	1
9	1
8	3
7	2
6	4
5	7
4	6
3	15
2	21
1	60
Loading known barcodes from /tmp/RtmpYfu9rt/file25b9ed2a255c86/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
Processing file: /tmp/RtmpYfu9rt/file25b9ed2a255c86/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 300
Number of reads where at least one barcode was found: 278
Number of chimera reads: 1
All done!
Reads	Barcodes
8	2
7	3
6	1
5	6
4	9
3	19
2	29
1	56
Loading known barcodes from /tmp/RtmpYfu9rt/file25b9ed2a255c86/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
Processing file: /tmp/RtmpYfu9rt/file25b9ed2a255c86/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 chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Wed Apr 22 00:20:13 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpYfu9rt/file25b9ed2a255c86/sampleA_matched_reads.fastq.gz ->/tmp/RtmpYfu9rt/file25b9ed2a255c86/sampleA_align2genome.bam
/tmp/RtmpYfu9rt/file25b9ed2a255c86/sample1_matched_reads.fastq.gz ->/tmp/RtmpYfu9rt/file25b9ed2a255c86/sample1_align2genome.bam
/tmp/RtmpYfu9rt/file25b9ed2a255c86/sample2_matched_reads.fastq.gz ->/tmp/RtmpYfu9rt/file25b9ed2a255c86/sample2_align2genome.bam
/tmp/RtmpYfu9rt/file25b9ed2a255c86/sample3_matched_reads.fastq.gz ->/tmp/RtmpYfu9rt/file25b9ed2a255c86/sample3_align2genome.bam
-- Running step: gene_quantification @ Wed Apr 22 00:20:36 2026 ----------------
00:20:36 Wed Apr 22 2026 quantify genes 
Using BAM(s): '/tmp/RtmpYfu9rt/file25b9ed2a255c86/sampleA_align2genome.bam',
'/tmp/RtmpYfu9rt/file25b9ed2a255c86/sample1_align2genome.bam',
'/tmp/RtmpYfu9rt/file25b9ed2a255c86/sample2_align2genome.bam', and
'/tmp/RtmpYfu9rt/file25b9ed2a255c86/sample3_align2genome.bam'
parsing /tmp/RtmpYfu9rt/file25b9ed2a255c86/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 13.88gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 151491.11Read/s]
parsing /tmp/RtmpYfu9rt/file25b9ed2a255c86/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 18.07gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 553309.06Read/s]
parsing /tmp/RtmpYfu9rt/file25b9ed2a255c86/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 27.67gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 496979.00Read/s]
parsing /tmp/RtmpYfu9rt/file25b9ed2a255c86/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 22.87gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 416630.64Read/s]
-- Running step: isoform_identification @ Wed Apr 22 00:20:37 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

  |                                                                            
  |                                                                      |   0%
  |                                                                            
  |==================                                                    |  25%
  |                                                                            
  |===================================                                   |  50%
  |                                                                            
  |====================================================                  |  75%
  |                                                                            
  |======================================================================| 100%

Detected 8 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)
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Wed Apr 22 00:21:02 2026 -------------------
Checking for fastq file(s) /tmp/RtmpYfu9rt/file25b9ed2a255c86/fastq, /tmp/RtmpYfu9rt/file25b9ed2a255c86/fastq/sample1.fq.gz, /tmp/RtmpYfu9rt/file25b9ed2a255c86/fastq/sample2.fq.gz, /tmp/RtmpYfu9rt/file25b9ed2a255c86/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpYfu9rt/file25b9ed2a255c86/sampleA_matched_reads.fastq.gz, /tmp/RtmpYfu9rt/file25b9ed2a255c86/sample1_matched_reads.fastq.gz, /tmp/RtmpYfu9rt/file25b9ed2a255c86/sample2_matched_reads.fastq.gz, /tmp/RtmpYfu9rt/file25b9ed2a255c86/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpYfu9rt/file25b9ed2a255c86/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpYfu9rt/file25b9ed2a255c86/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpYfu9rt/file25b9ed2a255c86/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpYfu9rt/file25b9ed2a255c86/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpYfu9rt/file25b9ed2a255c86/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpYfu9rt/file25b9ed2a255c86/sampleA_realign2transcript.bam
/tmp/RtmpYfu9rt/file25b9ed2a255c86/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpYfu9rt/file25b9ed2a255c86/sample1_realign2transcript.bam
/tmp/RtmpYfu9rt/file25b9ed2a255c86/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpYfu9rt/file25b9ed2a255c86/sample2_realign2transcript.bam
/tmp/RtmpYfu9rt/file25b9ed2a255c86/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpYfu9rt/file25b9ed2a255c86/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Wed Apr 22 00:21:22 2026 ----------
2026-04-22T04:21:22.921219Z  INFO oarfish: setting user-provided filter parameters.
2026-04-22T04:21:22.921582Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpYfu9rt/file25b9ed2a255c86/sampleA_realign2transcript.bam, contains 6 reference sequences.
2026-04-22T04:21:22.921659Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-22T04:21:22.921667Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-22T04:21:22.921729Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-22T04:21:22.921755Z  INFO oarfish: parsed reference information for 6 transcripts.
2026-04-22T04:21:22.932776Z  INFO oarfish::single_cell: Processed 100 cells.
2026-04-22T04:21:23.269316Z  INFO oarfish: setting user-provided filter parameters.
2026-04-22T04:21:23.269851Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpYfu9rt/file25b9ed2a255c86/sample1_realign2transcript.bam, contains 6 reference sequences.
2026-04-22T04:21:23.269921Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-22T04:21:23.269930Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-22T04:21:23.269986Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-22T04:21:23.270011Z  INFO oarfish: parsed reference information for 6 transcripts.
2026-04-22T04:21:23.275330Z  INFO oarfish::single_cell: Processed 100 cells.
2026-04-22T04:21:23.583580Z  INFO oarfish: setting user-provided filter parameters.
2026-04-22T04:21:23.584071Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpYfu9rt/file25b9ed2a255c86/sample2_realign2transcript.bam, contains 6 reference sequences.
2026-04-22T04:21:23.584148Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-22T04:21:23.584156Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-22T04:21:23.584210Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-22T04:21:23.584220Z  INFO oarfish: parsed reference information for 6 transcripts.
2026-04-22T04:21:23.589165Z  INFO oarfish::single_cell: Processed 100 cells.
2026-04-22T04:21:23.912102Z  INFO oarfish: setting user-provided filter parameters.
2026-04-22T04:21:23.912599Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpYfu9rt/file25b9ed2a255c86/sample3_realign2transcript.bam, contains 6 reference sequences.
2026-04-22T04:21:23.912667Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-22T04:21:23.912683Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-22T04:21:23.912740Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-22T04:21:23.912753Z  INFO oarfish: parsed reference information for 6 transcripts.
2026-04-22T04:21:23.918885Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
i Writing configuration to: /tmp/RtmpYfu9rt/file25b9ed78f8afcf/config_file_2472429.json
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Wed Apr 22 00:21:24 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpYfu9rt/file25b9ed78f8afcf/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
Processing file: /tmp/RtmpYfu9rt/file25b9ed78f8afcf/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpYfu9rt/file25b9ed78f8afcf/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpYfu9rt/file25b9ed78f8afcf/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 993
Number of reads where at least one barcode was found: 929
Number of chimera reads: 2
All done!
Reads	Barcodes
27	1
26	1
24	1
22	2
21	1
20	3
19	1
18	1
17	2
16	2
15	2
14	1
12	1
11	7
10	5
9	5
8	9
7	5
6	13
5	11
4	5
3	33
2	22
1	3
Loading known barcodes from /tmp/RtmpYfu9rt/file25b9ed78f8afcf/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
Processing file: /tmp/RtmpYfu9rt/file25b9ed78f8afcf/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 300
Number of reads where at least one barcode was found: 281
Number of chimera reads: 1
All done!
Reads	Barcodes
9	1
8	3
7	2
6	5
5	3
4	9
3	13
2	31
1	55
Loading known barcodes from /tmp/RtmpYfu9rt/file25b9ed78f8afcf/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
Processing file: /tmp/RtmpYfu9rt/file25b9ed78f8afcf/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 300
Number of reads where at least one barcode was found: 280
Number of chimera reads: 0
All done!
Reads	Barcodes
9	1
8	3
7	1
6	5
5	5
4	6
3	18
2	30
1	50
Loading known barcodes from /tmp/RtmpYfu9rt/file25b9ed78f8afcf/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
Processing file: /tmp/RtmpYfu9rt/file25b9ed78f8afcf/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 chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Wed Apr 22 00:21:25 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpYfu9rt/file25b9ed78f8afcf/sampleA_matched_reads.fastq.gz -> /tmp/RtmpYfu9rt/file25b9ed78f8afcf/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/RtmpYfu9rt/file25b9ed78f8afcf/sample1_matched_reads.fastq.gz -> /tmp/RtmpYfu9rt/file25b9ed78f8afcf/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/RtmpYfu9rt/file25b9ed78f8afcf/sample2_matched_reads.fastq.gz -> /tmp/RtmpYfu9rt/file25b9ed78f8afcf/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/RtmpYfu9rt/file25b9ed78f8afcf/sample3_matched_reads.fastq.gz -> /tmp/RtmpYfu9rt/file25b9ed78f8afcf/sample3_align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Wed Apr 22 00:21:29 2026 ----------------
00:21:29 Wed Apr 22 2026 quantify genes 
Using BAM(s): '/tmp/RtmpYfu9rt/file25b9ed78f8afcf/sampleA_align2genome.bam',
'/tmp/RtmpYfu9rt/file25b9ed78f8afcf/sample1_align2genome.bam',
'/tmp/RtmpYfu9rt/file25b9ed78f8afcf/sample2_align2genome.bam', and
'/tmp/RtmpYfu9rt/file25b9ed78f8afcf/sample3_align2genome.bam'
parsing /tmp/RtmpYfu9rt/file25b9ed78f8afcf/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 14.63gene_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, 147861.70Read/s]
parsing /tmp/RtmpYfu9rt/file25b9ed78f8afcf/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 22.20gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 489988.79Read/s]
parsing /tmp/RtmpYfu9rt/file25b9ed78f8afcf/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 29.94gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 496720.04Read/s]
parsing /tmp/RtmpYfu9rt/file25b9ed78f8afcf/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 25.33gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 403873.20Read/s]
-- Running step: isoform_identification @ Wed Apr 22 00:21:30 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 8 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)
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Wed Apr 22 00:21:53 2026 -------------------
Checking for fastq file(s) /tmp/RtmpYfu9rt/file25b9ed78f8afcf/fastq, /tmp/RtmpYfu9rt/file25b9ed78f8afcf/fastq/sample1.fq.gz, /tmp/RtmpYfu9rt/file25b9ed78f8afcf/fastq/sample2.fq.gz, /tmp/RtmpYfu9rt/file25b9ed78f8afcf/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpYfu9rt/file25b9ed78f8afcf/sampleA_matched_reads.fastq.gz, /tmp/RtmpYfu9rt/file25b9ed78f8afcf/sample1_matched_reads.fastq.gz, /tmp/RtmpYfu9rt/file25b9ed78f8afcf/sample2_matched_reads.fastq.gz, /tmp/RtmpYfu9rt/file25b9ed78f8afcf/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpYfu9rt/file25b9ed78f8afcf/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpYfu9rt/file25b9ed78f8afcf/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpYfu9rt/file25b9ed78f8afcf/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpYfu9rt/file25b9ed78f8afcf/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpYfu9rt/file25b9ed78f8afcf/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpYfu9rt/file25b9ed78f8afcf/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/RtmpYfu9rt/file25b9ed78f8afcf/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpYfu9rt/file25b9ed78f8afcf/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/RtmpYfu9rt/file25b9ed78f8afcf/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpYfu9rt/file25b9ed78f8afcf/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/RtmpYfu9rt/file25b9ed78f8afcf/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpYfu9rt/file25b9ed78f8afcf/sample3_realign2transcript.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Wed Apr 22 00:21:54 2026 ----------
00:21:54 Wed Apr 22 2026 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpYfu9rt/file25b9ed78f8afcf/sampleA_realign2transcript.bam...
parsing /tmp/RtmpYfu9rt/file25b9ed78f8afcf/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpYfu9rt/file25b9ed78f8afcf/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpYfu9rt/file25b9ed78f8afcf/sample1_realign2transcript.bam...
parsing /tmp/RtmpYfu9rt/file25b9ed78f8afcf/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpYfu9rt/file25b9ed78f8afcf/sample1_realign2transcript.bamdone
parsing /tmp/RtmpYfu9rt/file25b9ed78f8afcf/sample2_realign2transcript.bam...
parsing /tmp/RtmpYfu9rt/file25b9ed78f8afcf/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpYfu9rt/file25b9ed78f8afcf/sample2_realign2transcript.bamdone
parsing /tmp/RtmpYfu9rt/file25b9ed78f8afcf/sample3_realign2transcript.bam...
parsing /tmp/RtmpYfu9rt/file25b9ed78f8afcf/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpYfu9rt/file25b9ed78f8afcf/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
i Writing configuration to: /tmp/RtmpYfu9rt/file25b9ed4854b194/config_file_2472429.json
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Wed Apr 22 00:21:57 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpYfu9rt/file25b9ed4854b194/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
Processing file: /tmp/RtmpYfu9rt/file25b9ed4854b194/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpYfu9rt/file25b9ed4854b194/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpYfu9rt/file25b9ed4854b194/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 993
Number of reads where at least one barcode was found: 924
Number of chimera reads: 2
All done!
Reads	Barcodes
26	1
25	1
22	1
21	3
20	2
19	2
18	1
17	4
16	1
15	1
14	1
13	1
12	1
11	7
10	5
9	3
8	11
7	3
6	5
5	19
4	7
3	35
2	19
1	3
Loading known barcodes from /tmp/RtmpYfu9rt/file25b9ed4854b194/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
Processing file: /tmp/RtmpYfu9rt/file25b9ed4854b194/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 300
Number of reads where at least one barcode was found: 279
Number of chimera reads: 1
All done!
Reads	Barcodes
9	1
8	1
7	3
6	5
5	8
4	7
3	11
2	23
1	66
Loading known barcodes from /tmp/RtmpYfu9rt/file25b9ed4854b194/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
Processing file: /tmp/RtmpYfu9rt/file25b9ed4854b194/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 300
Number of reads where at least one barcode was found: 277
Number of chimera reads: 0
All done!
Reads	Barcodes
8	1
7	3
6	8
5	5
4	4
3	19
2	22
1	60
Loading known barcodes from /tmp/RtmpYfu9rt/file25b9ed4854b194/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
Processing file: /tmp/RtmpYfu9rt/file25b9ed4854b194/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 chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Wed Apr 22 00:21:58 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpYfu9rt/file25b9ed4854b194/sampleA_matched_reads.fastq.gz ->/tmp/RtmpYfu9rt/file25b9ed4854b194/sampleA_align2genome.bam
/tmp/RtmpYfu9rt/file25b9ed4854b194/sample1_matched_reads.fastq.gz ->/tmp/RtmpYfu9rt/file25b9ed4854b194/sample1_align2genome.bam
/tmp/RtmpYfu9rt/file25b9ed4854b194/sample2_matched_reads.fastq.gz ->/tmp/RtmpYfu9rt/file25b9ed4854b194/sample2_align2genome.bam
/tmp/RtmpYfu9rt/file25b9ed4854b194/sample3_matched_reads.fastq.gz ->/tmp/RtmpYfu9rt/file25b9ed4854b194/sample3_align2genome.bam
-- Running step: gene_quantification @ Wed Apr 22 00:22:20 2026 ----------------
00:22:20 Wed Apr 22 2026 quantify genes 
Using BAM(s): '/tmp/RtmpYfu9rt/file25b9ed4854b194/sampleA_align2genome.bam',
'/tmp/RtmpYfu9rt/file25b9ed4854b194/sample1_align2genome.bam',
'/tmp/RtmpYfu9rt/file25b9ed4854b194/sample2_align2genome.bam', and
'/tmp/RtmpYfu9rt/file25b9ed4854b194/sample3_align2genome.bam'
	Counter({'counted_reads': 347, 'not_enough_coverage': 9, 'unmapped': 5})
	Counter({'counted_reads': 262, 'not_enough_coverage': 8, 'unmapped': 2})
	Counter({'counted_reads': 264, 'not_enough_coverage': 8, 'unmapped': 1})
	Counter({'counted_reads': 347, 'not_enough_coverage': 9, 'unmapped': 2})
parsing /tmp/RtmpYfu9rt/file25b9ed4854b194/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 10.04gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 178620.88Read/s]
parsing /tmp/RtmpYfu9rt/file25b9ed4854b194/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 22.42gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 541005.06Read/s]
parsing /tmp/RtmpYfu9rt/file25b9ed4854b194/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 25.48gene_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, 490906.37Read/s]
parsing /tmp/RtmpYfu9rt/file25b9ed4854b194/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 23.42gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 426059.89Read/s]
-- Running step: isoform_identification @ Wed Apr 22 00:22:21 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

  |                                                                            
  |                                                                      |   0%
  |                                                                            
  |==================                                                    |  25%
  |                                                                            
  |===================================                                   |  50%
  |                                                                            
  |====================================================                  |  75%
  |                                                                            
  |======================================================================| 100%

Detected 8 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Wed Apr 22 00:22:49 2026 -------------------
Checking for fastq file(s) /tmp/RtmpYfu9rt/file25b9ed4854b194/fastq, /tmp/RtmpYfu9rt/file25b9ed4854b194/fastq/sample1.fq.gz, /tmp/RtmpYfu9rt/file25b9ed4854b194/fastq/sample2.fq.gz, /tmp/RtmpYfu9rt/file25b9ed4854b194/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpYfu9rt/file25b9ed4854b194/sampleA_matched_reads.fastq.gz, /tmp/RtmpYfu9rt/file25b9ed4854b194/sample1_matched_reads.fastq.gz, /tmp/RtmpYfu9rt/file25b9ed4854b194/sample2_matched_reads.fastq.gz, /tmp/RtmpYfu9rt/file25b9ed4854b194/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpYfu9rt/file25b9ed4854b194/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpYfu9rt/file25b9ed4854b194/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpYfu9rt/file25b9ed4854b194/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpYfu9rt/file25b9ed4854b194/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpYfu9rt/file25b9ed4854b194/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpYfu9rt/file25b9ed4854b194/sampleA_realign2transcript.bam
/tmp/RtmpYfu9rt/file25b9ed4854b194/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpYfu9rt/file25b9ed4854b194/sample1_realign2transcript.bam
/tmp/RtmpYfu9rt/file25b9ed4854b194/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpYfu9rt/file25b9ed4854b194/sample2_realign2transcript.bam
/tmp/RtmpYfu9rt/file25b9ed4854b194/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpYfu9rt/file25b9ed4854b194/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Wed Apr 22 00:23:09 2026 ----------
00:23:09 Wed Apr 22 2026 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpYfu9rt/file25b9ed4854b194/sampleA_realign2transcript.bam...
parsing /tmp/RtmpYfu9rt/file25b9ed4854b194/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpYfu9rt/file25b9ed4854b194/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpYfu9rt/file25b9ed4854b194/sample1_realign2transcript.bam...
parsing /tmp/RtmpYfu9rt/file25b9ed4854b194/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpYfu9rt/file25b9ed4854b194/sample1_realign2transcript.bamdone
parsing /tmp/RtmpYfu9rt/file25b9ed4854b194/sample2_realign2transcript.bam...
parsing /tmp/RtmpYfu9rt/file25b9ed4854b194/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpYfu9rt/file25b9ed4854b194/sample2_realign2transcript.bamdone
parsing /tmp/RtmpYfu9rt/file25b9ed4854b194/sample3_realign2transcript.bam...
parsing /tmp/RtmpYfu9rt/file25b9ed4854b194/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpYfu9rt/file25b9ed4854b194/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
i Writing configuration to: /tmp/RtmpYfu9rt/file25b9ed508fd5f1/config_file_2472429.json
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Wed Apr 22 00:23:12 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpYfu9rt/file25b9ed508fd5f1/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
Processing file: /tmp/RtmpYfu9rt/file25b9ed508fd5f1/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpYfu9rt/file25b9ed508fd5f1/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpYfu9rt/file25b9ed508fd5f1/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 993
Number of reads where at least one barcode was found: 928
Number of chimera reads: 2
All done!
Reads	Barcodes
28	1
26	1
21	2
20	3
19	4
17	2
14	4
13	2
12	5
11	7
10	1
9	6
8	4
7	7
6	12
5	15
4	4
3	35
2	19
1	3
Loading known barcodes from /tmp/RtmpYfu9rt/file25b9ed508fd5f1/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
Processing file: /tmp/RtmpYfu9rt/file25b9ed508fd5f1/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 300
Number of reads where at least one barcode was found: 278
Number of chimera reads: 1
All done!
Reads	Barcodes
9	1
8	1
7	4
6	3
5	5
4	8
3	12
2	36
1	54
Loading known barcodes from /tmp/RtmpYfu9rt/file25b9ed508fd5f1/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
Processing file: /tmp/RtmpYfu9rt/file25b9ed508fd5f1/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 300
Number of reads where at least one barcode was found: 282
Number of chimera reads: 0
All done!
Reads	Barcodes
9	2
7	1
6	4
5	8
4	14
3	9
2	27
1	59
Loading known barcodes from /tmp/RtmpYfu9rt/file25b9ed508fd5f1/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
Processing file: /tmp/RtmpYfu9rt/file25b9ed508fd5f1/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 chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Wed Apr 22 00:23:13 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpYfu9rt/file25b9ed508fd5f1/sampleA_matched_reads.fastq.gz -> /tmp/RtmpYfu9rt/file25b9ed508fd5f1/sampleA_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpYfu9rt/file25b9ed508fd5f1/sample1_matched_reads.fastq.gz -> /tmp/RtmpYfu9rt/file25b9ed508fd5f1/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpYfu9rt/file25b9ed508fd5f1/sample2_matched_reads.fastq.gz -> /tmp/RtmpYfu9rt/file25b9ed508fd5f1/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpYfu9rt/file25b9ed508fd5f1/sample3_matched_reads.fastq.gz -> /tmp/RtmpYfu9rt/file25b9ed508fd5f1/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Wed Apr 22 00:23:16 2026 ----------------
00:23:16 Wed Apr 22 2026 quantify genes 
Using BAM(s): '/tmp/RtmpYfu9rt/file25b9ed508fd5f1/sampleA_align2genome.bam',
'/tmp/RtmpYfu9rt/file25b9ed508fd5f1/sample1_align2genome.bam',
'/tmp/RtmpYfu9rt/file25b9ed508fd5f1/sample2_align2genome.bam', and
'/tmp/RtmpYfu9rt/file25b9ed508fd5f1/sample3_align2genome.bam'
	Counter({'counted_reads': 347, 'not_enough_coverage': 9, 'unmapped': 5})
	Counter({'counted_reads': 265, 'not_enough_coverage': 8, 'unmapped': 2})
	Counter({'counted_reads': 261, 'not_enough_coverage': 9, 'unmapped': 1})
	Counter({'counted_reads': 347, 'not_enough_coverage': 9, 'unmapped': 2})
parsing /tmp/RtmpYfu9rt/file25b9ed508fd5f1/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 14.52gene_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, 185670.83Read/s]
parsing /tmp/RtmpYfu9rt/file25b9ed508fd5f1/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 29.73gene_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, 493122.65Read/s]
parsing /tmp/RtmpYfu9rt/file25b9ed508fd5f1/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 28.31gene_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, 520824.52Read/s]
parsing /tmp/RtmpYfu9rt/file25b9ed508fd5f1/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 23.81gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 371967.36Read/s]
-- Running step: isoform_identification @ Wed Apr 22 00:23:17 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Wed Apr 22 00:23:18 2026 -------------------
Checking for fastq file(s) /tmp/RtmpYfu9rt/file25b9ed508fd5f1/fastq, /tmp/RtmpYfu9rt/file25b9ed508fd5f1/fastq/sample1.fq.gz, /tmp/RtmpYfu9rt/file25b9ed508fd5f1/fastq/sample2.fq.gz, /tmp/RtmpYfu9rt/file25b9ed508fd5f1/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpYfu9rt/file25b9ed508fd5f1/sampleA_matched_reads.fastq.gz, /tmp/RtmpYfu9rt/file25b9ed508fd5f1/sample1_matched_reads.fastq.gz, /tmp/RtmpYfu9rt/file25b9ed508fd5f1/sample2_matched_reads.fastq.gz, /tmp/RtmpYfu9rt/file25b9ed508fd5f1/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpYfu9rt/file25b9ed508fd5f1/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpYfu9rt/file25b9ed508fd5f1/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpYfu9rt/file25b9ed508fd5f1/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpYfu9rt/file25b9ed508fd5f1/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpYfu9rt/file25b9ed508fd5f1/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpYfu9rt/file25b9ed508fd5f1/sampleA_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpYfu9rt/file25b9ed508fd5f1/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpYfu9rt/file25b9ed508fd5f1/sample1_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpYfu9rt/file25b9ed508fd5f1/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpYfu9rt/file25b9ed508fd5f1/sample2_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpYfu9rt/file25b9ed508fd5f1/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpYfu9rt/file25b9ed508fd5f1/sample3_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

-- Running step: transcript_quantification @ Wed Apr 22 00:23:26 2026 ----------
2026-04-22T04:23:26.145655Z  INFO oarfish: setting user-provided filter parameters.
2026-04-22T04:23:26.146082Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpYfu9rt/file25b9ed508fd5f1/sampleA_realign2transcript.bam, contains 27 reference sequences.
2026-04-22T04:23:26.146111Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-22T04:23:26.146121Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-22T04:23:26.146510Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-22T04:23:26.146537Z  INFO oarfish: parsed reference information for 27 transcripts.
2026-04-22T04:23:26.189223Z  INFO oarfish::single_cell: Processed 100 cells.
2026-04-22T04:23:26.763212Z  INFO oarfish: setting user-provided filter parameters.
2026-04-22T04:23:26.763633Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpYfu9rt/file25b9ed508fd5f1/sample1_realign2transcript.bam, contains 27 reference sequences.
2026-04-22T04:23:26.763660Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-22T04:23:26.763669Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-22T04:23:26.763795Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-22T04:23:26.763816Z  INFO oarfish: parsed reference information for 27 transcripts.
2026-04-22T04:23:26.779758Z  INFO oarfish::single_cell: Processed 100 cells.
2026-04-22T04:23:27.336560Z  INFO oarfish: setting user-provided filter parameters.
2026-04-22T04:23:27.337113Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpYfu9rt/file25b9ed508fd5f1/sample2_realign2transcript.bam, contains 27 reference sequences.
2026-04-22T04:23:27.337138Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-22T04:23:27.337147Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-22T04:23:27.337260Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-22T04:23:27.337280Z  INFO oarfish: parsed reference information for 27 transcripts.
2026-04-22T04:23:27.353331Z  INFO oarfish::single_cell: Processed 100 cells.
2026-04-22T04:23:27.880935Z  INFO oarfish: setting user-provided filter parameters.
2026-04-22T04:23:27.881498Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpYfu9rt/file25b9ed508fd5f1/sample3_realign2transcript.bam, contains 27 reference sequences.
2026-04-22T04:23:27.881521Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-22T04:23:27.881531Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-22T04:23:27.881646Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-22T04:23:27.881665Z  INFO oarfish: parsed reference information for 27 transcripts.
2026-04-22T04:23:27.899907Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
i Writing configuration to: /tmp/RtmpYfu9rt/file25b9ed2932c277/config_file_2472429.json
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Wed Apr 22 00:23:28 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpYfu9rt/file25b9ed2932c277/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
Processing file: /tmp/RtmpYfu9rt/file25b9ed2932c277/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpYfu9rt/file25b9ed2932c277/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpYfu9rt/file25b9ed2932c277/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 993
Number of reads where at least one barcode was found: 932
Number of chimera reads: 3
All done!
Reads	Barcodes
26	1
25	1
24	1
22	2
21	1
20	4
19	1
18	1
15	3
14	1
13	1
12	3
11	9
10	5
9	5
8	6
7	3
6	15
5	10
4	6
3	37
2	20
1	1
Loading known barcodes from /tmp/RtmpYfu9rt/file25b9ed2932c277/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
Processing file: /tmp/RtmpYfu9rt/file25b9ed2932c277/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 300
Number of reads where at least one barcode was found: 280
Number of chimera reads: 1
All done!
Reads	Barcodes
8	2
7	4
6	5
5	2
4	9
3	18
2	27
1	56
Loading known barcodes from /tmp/RtmpYfu9rt/file25b9ed2932c277/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
Processing file: /tmp/RtmpYfu9rt/file25b9ed2932c277/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 300
Number of reads where at least one barcode was found: 284
Number of chimera reads: 1
All done!
Reads	Barcodes
8	3
7	4
6	4
5	3
4	9
3	17
2	26
1	58
Loading known barcodes from /tmp/RtmpYfu9rt/file25b9ed2932c277/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
Processing file: /tmp/RtmpYfu9rt/file25b9ed2932c277/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 chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Wed Apr 22 00:23:29 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpYfu9rt/file25b9ed2932c277/sampleA_matched_reads.fastq.gz ->/tmp/RtmpYfu9rt/file25b9ed2932c277/sampleA_align2genome.bam
/tmp/RtmpYfu9rt/file25b9ed2932c277/sample1_matched_reads.fastq.gz ->/tmp/RtmpYfu9rt/file25b9ed2932c277/sample1_align2genome.bam
/tmp/RtmpYfu9rt/file25b9ed2932c277/sample2_matched_reads.fastq.gz ->/tmp/RtmpYfu9rt/file25b9ed2932c277/sample2_align2genome.bam
/tmp/RtmpYfu9rt/file25b9ed2932c277/sample3_matched_reads.fastq.gz ->/tmp/RtmpYfu9rt/file25b9ed2932c277/sample3_align2genome.bam
-- Running step: gene_quantification @ Wed Apr 22 00:23:51 2026 ----------------
00:23:51 Wed Apr 22 2026 quantify genes 
Using BAM(s): '/tmp/RtmpYfu9rt/file25b9ed2932c277/sampleA_align2genome.bam',
'/tmp/RtmpYfu9rt/file25b9ed2932c277/sample1_align2genome.bam',
'/tmp/RtmpYfu9rt/file25b9ed2932c277/sample2_align2genome.bam', and
'/tmp/RtmpYfu9rt/file25b9ed2932c277/sample3_align2genome.bam'
parsing /tmp/RtmpYfu9rt/file25b9ed2932c277/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 14.23gene_group/s]
/home/biocbuild/bbs-3.23-bioc/R/site-library/FLAMES/python/count_gene.py:702: 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, 137167.38Read/s]
parsing /tmp/RtmpYfu9rt/file25b9ed2932c277/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 20.45gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 509784.63Read/s]
parsing /tmp/RtmpYfu9rt/file25b9ed2932c277/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 27.73gene_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, 459901.75Read/s]
parsing /tmp/RtmpYfu9rt/file25b9ed2932c277/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 24.04gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 398667.78Read/s]
-- Running step: isoform_identification @ Wed Apr 22 00:23:53 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Wed Apr 22 00:23:53 2026 -------------------
Checking for fastq file(s) /tmp/RtmpYfu9rt/file25b9ed2932c277/fastq, /tmp/RtmpYfu9rt/file25b9ed2932c277/fastq/sample1.fq.gz, /tmp/RtmpYfu9rt/file25b9ed2932c277/fastq/sample2.fq.gz, /tmp/RtmpYfu9rt/file25b9ed2932c277/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpYfu9rt/file25b9ed2932c277/sampleA_matched_reads.fastq.gz, /tmp/RtmpYfu9rt/file25b9ed2932c277/sample1_matched_reads.fastq.gz, /tmp/RtmpYfu9rt/file25b9ed2932c277/sample2_matched_reads.fastq.gz, /tmp/RtmpYfu9rt/file25b9ed2932c277/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpYfu9rt/file25b9ed2932c277/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpYfu9rt/file25b9ed2932c277/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpYfu9rt/file25b9ed2932c277/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpYfu9rt/file25b9ed2932c277/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpYfu9rt/file25b9ed2932c277/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpYfu9rt/file25b9ed2932c277/sampleA_realign2transcript.bam
/tmp/RtmpYfu9rt/file25b9ed2932c277/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpYfu9rt/file25b9ed2932c277/sample1_realign2transcript.bam
/tmp/RtmpYfu9rt/file25b9ed2932c277/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpYfu9rt/file25b9ed2932c277/sample2_realign2transcript.bam
/tmp/RtmpYfu9rt/file25b9ed2932c277/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpYfu9rt/file25b9ed2932c277/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Wed Apr 22 00:24:20 2026 ----------
2026-04-22T04:24:20.156155Z  INFO oarfish: setting user-provided filter parameters.
2026-04-22T04:24:20.156620Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpYfu9rt/file25b9ed2932c277/sampleA_realign2transcript.bam, contains 28 reference sequences.
2026-04-22T04:24:20.156645Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-22T04:24:20.156655Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-22T04:24:20.156780Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-22T04:24:20.156800Z  INFO oarfish: parsed reference information for 28 transcripts.
2026-04-22T04:24:20.197893Z  INFO oarfish::single_cell: Processed 100 cells.
2026-04-22T04:24:20.864471Z  INFO oarfish: setting user-provided filter parameters.
2026-04-22T04:24:20.865014Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpYfu9rt/file25b9ed2932c277/sample1_realign2transcript.bam, contains 28 reference sequences.
2026-04-22T04:24:20.865040Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-22T04:24:20.865049Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-22T04:24:20.865166Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-22T04:24:20.865186Z  INFO oarfish: parsed reference information for 28 transcripts.
2026-04-22T04:24:20.881324Z  INFO oarfish::single_cell: Processed 100 cells.
2026-04-22T04:24:21.484254Z  INFO oarfish: setting user-provided filter parameters.
2026-04-22T04:24:21.484776Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpYfu9rt/file25b9ed2932c277/sample2_realign2transcript.bam, contains 28 reference sequences.
2026-04-22T04:24:21.484802Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-22T04:24:21.484812Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-22T04:24:21.484932Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-22T04:24:21.484952Z  INFO oarfish: parsed reference information for 28 transcripts.
2026-04-22T04:24:21.501273Z  INFO oarfish::single_cell: Processed 100 cells.
2026-04-22T04:24:22.138786Z  INFO oarfish: setting user-provided filter parameters.
2026-04-22T04:24:22.139219Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpYfu9rt/file25b9ed2932c277/sample3_realign2transcript.bam, contains 28 reference sequences.
2026-04-22T04:24:22.139247Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-22T04:24:22.139256Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-22T04:24:22.139377Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-22T04:24:22.139397Z  INFO oarfish: parsed reference information for 28 transcripts.
2026-04-22T04:24:22.157853Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
i Writing configuration to: /tmp/RtmpYfu9rt/file25b9ed4c72e8f7/config_file_2472429.json
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Wed Apr 22 00:24:23 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpYfu9rt/file25b9ed4c72e8f7/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
Processing file: /tmp/RtmpYfu9rt/file25b9ed4c72e8f7/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpYfu9rt/file25b9ed4c72e8f7/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpYfu9rt/file25b9ed4c72e8f7/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 993
Number of reads where at least one barcode was found: 928
Number of chimera reads: 2
All done!
Reads	Barcodes
26	1
23	2
22	1
21	1
20	2
19	2
18	4
16	2
15	1
14	1
13	1
12	5
11	6
10	5
9	4
8	7
7	3
6	15
5	11
4	2
3	35
2	23
1	3
Loading known barcodes from /tmp/RtmpYfu9rt/file25b9ed4c72e8f7/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
Processing file: /tmp/RtmpYfu9rt/file25b9ed4c72e8f7/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 300
Number of reads where at least one barcode was found: 278
Number of chimera reads: 0
All done!
Reads	Barcodes
7	4
6	3
5	9
4	11
3	13
2	23
1	59
Loading known barcodes from /tmp/RtmpYfu9rt/file25b9ed4c72e8f7/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
Processing file: /tmp/RtmpYfu9rt/file25b9ed4c72e8f7/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 300
Number of reads where at least one barcode was found: 282
Number of chimera reads: 1
All done!
Reads	Barcodes
9	1
8	1
7	5
6	2
5	8
4	7
3	14
2	32
1	46
Loading known barcodes from /tmp/RtmpYfu9rt/file25b9ed4c72e8f7/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
Processing file: /tmp/RtmpYfu9rt/file25b9ed4c72e8f7/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 chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Wed Apr 22 00:24:24 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpYfu9rt/file25b9ed4c72e8f7/sampleA_matched_reads.fastq.gz -> /tmp/RtmpYfu9rt/file25b9ed4c72e8f7/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/RtmpYfu9rt/file25b9ed4c72e8f7/sample1_matched_reads.fastq.gz -> /tmp/RtmpYfu9rt/file25b9ed4c72e8f7/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/RtmpYfu9rt/file25b9ed4c72e8f7/sample2_matched_reads.fastq.gz -> /tmp/RtmpYfu9rt/file25b9ed4c72e8f7/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/RtmpYfu9rt/file25b9ed4c72e8f7/sample3_matched_reads.fastq.gz -> /tmp/RtmpYfu9rt/file25b9ed4c72e8f7/sample3_align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Wed Apr 22 00:24:27 2026 ----------------
00:24:27 Wed Apr 22 2026 quantify genes 
Using BAM(s): '/tmp/RtmpYfu9rt/file25b9ed4c72e8f7/sampleA_align2genome.bam',
'/tmp/RtmpYfu9rt/file25b9ed4c72e8f7/sample1_align2genome.bam',
'/tmp/RtmpYfu9rt/file25b9ed4c72e8f7/sample2_align2genome.bam', and
'/tmp/RtmpYfu9rt/file25b9ed4c72e8f7/sample3_align2genome.bam'
parsing /tmp/RtmpYfu9rt/file25b9ed4c72e8f7/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 14.40gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 173095.18Read/s]
parsing /tmp/RtmpYfu9rt/file25b9ed4c72e8f7/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 23.24gene_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, 554157.07Read/s]
parsing /tmp/RtmpYfu9rt/file25b9ed4c72e8f7/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 28.61gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 530736.45Read/s]
parsing /tmp/RtmpYfu9rt/file25b9ed4c72e8f7/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 23.55gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 409072.68Read/s]
-- Running step: isoform_identification @ Wed Apr 22 00:24:29 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Wed Apr 22 00:24:29 2026 -------------------
Checking for fastq file(s) /tmp/RtmpYfu9rt/file25b9ed4c72e8f7/fastq, /tmp/RtmpYfu9rt/file25b9ed4c72e8f7/fastq/sample1.fq.gz, /tmp/RtmpYfu9rt/file25b9ed4c72e8f7/fastq/sample2.fq.gz, /tmp/RtmpYfu9rt/file25b9ed4c72e8f7/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpYfu9rt/file25b9ed4c72e8f7/sampleA_matched_reads.fastq.gz, /tmp/RtmpYfu9rt/file25b9ed4c72e8f7/sample1_matched_reads.fastq.gz, /tmp/RtmpYfu9rt/file25b9ed4c72e8f7/sample2_matched_reads.fastq.gz, /tmp/RtmpYfu9rt/file25b9ed4c72e8f7/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpYfu9rt/file25b9ed4c72e8f7/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpYfu9rt/file25b9ed4c72e8f7/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpYfu9rt/file25b9ed4c72e8f7/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpYfu9rt/file25b9ed4c72e8f7/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpYfu9rt/file25b9ed4c72e8f7/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpYfu9rt/file25b9ed4c72e8f7/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/RtmpYfu9rt/file25b9ed4c72e8f7/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpYfu9rt/file25b9ed4c72e8f7/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/RtmpYfu9rt/file25b9ed4c72e8f7/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpYfu9rt/file25b9ed4c72e8f7/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/RtmpYfu9rt/file25b9ed4c72e8f7/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpYfu9rt/file25b9ed4c72e8f7/sample3_realign2transcript.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Wed Apr 22 00:24:32 2026 ----------
00:24:32 Wed Apr 22 2026 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpYfu9rt/file25b9ed4c72e8f7/sampleA_realign2transcript.bam...
parsing /tmp/RtmpYfu9rt/file25b9ed4c72e8f7/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpYfu9rt/file25b9ed4c72e8f7/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpYfu9rt/file25b9ed4c72e8f7/sample1_realign2transcript.bam...
parsing /tmp/RtmpYfu9rt/file25b9ed4c72e8f7/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpYfu9rt/file25b9ed4c72e8f7/sample1_realign2transcript.bamdone
parsing /tmp/RtmpYfu9rt/file25b9ed4c72e8f7/sample2_realign2transcript.bam...
parsing /tmp/RtmpYfu9rt/file25b9ed4c72e8f7/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpYfu9rt/file25b9ed4c72e8f7/sample2_realign2transcript.bamdone
parsing /tmp/RtmpYfu9rt/file25b9ed4c72e8f7/sample3_realign2transcript.bam...
parsing /tmp/RtmpYfu9rt/file25b9ed4c72e8f7/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpYfu9rt/file25b9ed4c72e8f7/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
	Counter({'counted_reads': 357, 'not_enough_coverage': 1})
	Counter({'counted_reads': 275})
	Counter({'counted_reads': 275, 'not_enough_coverage': 1})
	Counter({'counted_reads': 357, 'not_enough_coverage': 1})
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
i Writing configuration to: /tmp/RtmpYfu9rt/file25b9ed145ce688/config_file_2472429.json
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Wed Apr 22 00:24:35 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpYfu9rt/file25b9ed145ce688/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
Processing file: /tmp/RtmpYfu9rt/file25b9ed145ce688/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpYfu9rt/file25b9ed145ce688/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpYfu9rt/file25b9ed145ce688/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 993
Number of reads where at least one barcode was found: 929
Number of chimera reads: 2
All done!
Reads	Barcodes
29	1
24	1
22	1
21	2
20	3
19	1
18	2
16	4
15	1
14	1
13	5
11	4
10	7
9	5
8	9
7	3
6	13
5	14
4	2
3	32
2	20
1	6
Loading known barcodes from /tmp/RtmpYfu9rt/file25b9ed145ce688/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
Processing file: /tmp/RtmpYfu9rt/file25b9ed145ce688/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 300
Number of reads where at least one barcode was found: 281
Number of chimera reads: 1
All done!
Reads	Barcodes
9	1
8	3
7	1
6	4
5	5
4	8
3	16
2	33
1	50
Loading known barcodes from /tmp/RtmpYfu9rt/file25b9ed145ce688/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
Processing file: /tmp/RtmpYfu9rt/file25b9ed145ce688/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 300
Number of reads where at least one barcode was found: 280
Number of chimera reads: 0
All done!
Reads	Barcodes
10	1
9	1
7	1
6	4
5	10
4	7
3	16
2	28
1	51
Loading known barcodes from /tmp/RtmpYfu9rt/file25b9ed145ce688/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
Processing file: /tmp/RtmpYfu9rt/file25b9ed145ce688/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 chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Wed Apr 22 00:24:37 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpYfu9rt/file25b9ed145ce688/sampleA_matched_reads.fastq.gz ->/tmp/RtmpYfu9rt/file25b9ed145ce688/sampleA_align2genome.bam
/tmp/RtmpYfu9rt/file25b9ed145ce688/sample1_matched_reads.fastq.gz ->/tmp/RtmpYfu9rt/file25b9ed145ce688/sample1_align2genome.bam
/tmp/RtmpYfu9rt/file25b9ed145ce688/sample2_matched_reads.fastq.gz ->/tmp/RtmpYfu9rt/file25b9ed145ce688/sample2_align2genome.bam
/tmp/RtmpYfu9rt/file25b9ed145ce688/sample3_matched_reads.fastq.gz ->/tmp/RtmpYfu9rt/file25b9ed145ce688/sample3_align2genome.bam
-- Running step: gene_quantification @ Wed Apr 22 00:25:00 2026 ----------------
00:25:00 Wed Apr 22 2026 quantify genes 
Using BAM(s): '/tmp/RtmpYfu9rt/file25b9ed145ce688/sampleA_align2genome.bam',
'/tmp/RtmpYfu9rt/file25b9ed145ce688/sample1_align2genome.bam',
'/tmp/RtmpYfu9rt/file25b9ed145ce688/sample2_align2genome.bam', and
'/tmp/RtmpYfu9rt/file25b9ed145ce688/sample3_align2genome.bam'
parsing /tmp/RtmpYfu9rt/file25b9ed145ce688/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 13.92gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 152577.85Read/s]
parsing /tmp/RtmpYfu9rt/file25b9ed145ce688/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 21.97gene_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, 463581.94Read/s]
parsing /tmp/RtmpYfu9rt/file25b9ed145ce688/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 28.17gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 440689.25Read/s]
parsing /tmp/RtmpYfu9rt/file25b9ed145ce688/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 23.55gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 326547.29Read/s]
-- Running step: isoform_identification @ Wed Apr 22 00:25:01 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Wed Apr 22 00:25:01 2026 -------------------
Checking for fastq file(s) /tmp/RtmpYfu9rt/file25b9ed145ce688/fastq, /tmp/RtmpYfu9rt/file25b9ed145ce688/fastq/sample1.fq.gz, /tmp/RtmpYfu9rt/file25b9ed145ce688/fastq/sample2.fq.gz, /tmp/RtmpYfu9rt/file25b9ed145ce688/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpYfu9rt/file25b9ed145ce688/sampleA_matched_reads.fastq.gz, /tmp/RtmpYfu9rt/file25b9ed145ce688/sample1_matched_reads.fastq.gz, /tmp/RtmpYfu9rt/file25b9ed145ce688/sample2_matched_reads.fastq.gz, /tmp/RtmpYfu9rt/file25b9ed145ce688/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpYfu9rt/file25b9ed145ce688/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpYfu9rt/file25b9ed145ce688/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpYfu9rt/file25b9ed145ce688/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpYfu9rt/file25b9ed145ce688/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpYfu9rt/file25b9ed145ce688/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpYfu9rt/file25b9ed145ce688/sampleA_realign2transcript.bam
/tmp/RtmpYfu9rt/file25b9ed145ce688/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpYfu9rt/file25b9ed145ce688/sample1_realign2transcript.bam
/tmp/RtmpYfu9rt/file25b9ed145ce688/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpYfu9rt/file25b9ed145ce688/sample2_realign2transcript.bam
/tmp/RtmpYfu9rt/file25b9ed145ce688/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpYfu9rt/file25b9ed145ce688/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Wed Apr 22 00:25:24 2026 ----------
00:25:24 Wed Apr 22 2026 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpYfu9rt/file25b9ed145ce688/sampleA_realign2transcript.bam...
parsing /tmp/RtmpYfu9rt/file25b9ed145ce688/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpYfu9rt/file25b9ed145ce688/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpYfu9rt/file25b9ed145ce688/sample1_realign2transcript.bam...
parsing /tmp/RtmpYfu9rt/file25b9ed145ce688/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpYfu9rt/file25b9ed145ce688/sample1_realign2transcript.bamdone
parsing /tmp/RtmpYfu9rt/file25b9ed145ce688/sample2_realign2transcript.bam...
parsing /tmp/RtmpYfu9rt/file25b9ed145ce688/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpYfu9rt/file25b9ed145ce688/sample2_realign2transcript.bamdone
parsing /tmp/RtmpYfu9rt/file25b9ed145ce688/sample3_realign2transcript.bam...
parsing /tmp/RtmpYfu9rt/file25b9ed145ce688/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpYfu9rt/file25b9ed145ce688/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Filtering for genes with at least 2 detected isoforms ...
	6 isoform(s) left.

Aggregating counts by cluster labels ...

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

Filtering for genes with at least 2 isoforms expressing more than 1 counts ...
	3 gene(s), 6 transcript(s) left.

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

[ FAIL 0 | WARN 194 | SKIP 0 | PASS 98 ]

[ FAIL 0 | WARN 194 | SKIP 0 | PASS 98 ]
	Counter({'counted_reads': 358})
	Counter({'counted_reads': 275})
	Counter({'counted_reads': 272})
	Counter({'counted_reads': 358})
> 
> proc.time()
   user  system elapsed 
833.518  46.021 868.460