##############################################################################
##############################################################################
###
### Running command:
###
###   /home/biocbuild/bbs-3.24-bioc/R/bin/R CMD check --install=check:FLAMES.install-out.txt --library=/home/biocbuild/bbs-3.24-bioc/R/site-library --timings FLAMES_2.7.0.tar.gz
###
##############################################################################
##############################################################################


* using log directory ‘/home/biocbuild/bbs-3.24-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: 2026-05-08 03:47:25 UTC
* checking for file ‘FLAMES/DESCRIPTION’ ... OK
* this is package ‘FLAMES’ version ‘2.7.0’
* 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-3.24-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 13.3Mb
  sub-directories of 1Mb or more:
    bin    8.3Mb
    data   1.8Mb
    libs   1.5Mb
* checking package directory ... OK
* checking ‘build’ directory ... OK
* checking DESCRIPTION meta-information ... OK
* checking top-level files ... OK
* checking for left-over files ... OK
* checking index information ... OK
* checking package subdirectories ... OK
* checking code files for non-ASCII characters ... OK
* checking R files for syntax errors ... OK
* checking whether the package can be loaded ... OK
* checking whether the package can be loaded with stated dependencies ... OK
* checking whether the package can be unloaded cleanly ... OK
* checking whether the namespace can be loaded with stated dependencies ... OK
* checking whether the namespace can be unloaded cleanly ... OK
* checking loading without being on the library search path ... OK
* checking dependencies in R code ... NOTE
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-3.24-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                24.250  1.288  25.765
plot_isoform_reduced_dim     19.992  0.654  20.647
blaze                         4.761 15.192  12.779
sc_long_multisample_pipeline  8.368  5.212   7.921
bulk_long_pipeline            2.361 10.160   2.452
sc_plot_genotype             10.472  1.366  10.140
MultiSampleSCPipeline         9.897  0.666  10.640
create_sce_from_dir           5.919  3.053   6.809
sc_DTU_analysis               6.892  1.613   6.705
create_se_from_dir            5.226  0.142   5.355
plot_durations                5.058  0.146   5.192
experiment                    4.948  0.107   5.136
resume_FLAMES                 4.911  0.113   5.013
* 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-3.24-bioc/meat/FLAMES.Rcheck/00check.log’
for details.

##############################################################################
##############################################################################
###
### Running command:
###
###   /home/biocbuild/bbs-3.24-bioc/R/bin/R CMD INSTALL FLAMES
###
##############################################################################
##############################################################################


* installing to library ‘/home/biocbuild/bbs-3.24-bioc/R/site-library’
* installing *source* package ‘FLAMES’ ...
** this is package ‘FLAMES’ version ‘2.7.0’
** 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-3.24-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.24-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-3.24-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.24-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-3.24-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.24-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-3.24-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.24-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-3.24-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.24-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-3.24-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.24-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-3.24-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.24-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-3.24-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.24-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-3.24-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.24-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-3.24-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.24-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-3.24-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.24-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-3.24-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.24-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-3.24-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.24-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-3.24-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.24-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-3.24-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.24-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-3.24-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.24-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-3.24-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.24-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-3.24-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-3.24-bioc/R/site-library/Rhtslib/usrlib/libhts.a -lcurl -lbz2 -llzma -lz -L/home/biocbuild/bbs-3.24-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-3.24-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-3.24-bioc/meat/FLAMES/src/submodule/minimap2'
echo "Installing binary to /home/biocbuild/bbs-3.24-bioc/meat/FLAMES/src/../inst/bin"
Installing binary to /home/biocbuild/bbs-3.24-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: v0.89.0)
      Adding noodles-bgzf v0.38.0 (available: v0.47.0)
      Adding noodles-sam v0.74.0 (available: v0.85.0)
      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 v0.2.186
   Compiling proc-macro2 v1.0.106
   Compiling unicode-ident v1.0.24
   Compiling quote v1.0.45
   Compiling find-msvc-tools v0.1.9
   Compiling shlex v1.3.0
   Compiling cfg-if v1.0.4
   Compiling autocfg v1.5.0
   Compiling pkg-config v0.3.33
   Compiling libm v0.2.16
   Compiling memchr v2.8.0
   Compiling zerocopy v0.8.48
   Compiling crossbeam-utils v0.8.21
   Compiling version_check v0.9.5
   Compiling adler2 v2.0.1
   Compiling crc32fast v1.5.0
   Compiling once_cell v1.21.4
   Compiling simd-adler32 v0.3.9
   Compiling serde_core v1.0.228
   Compiling regex-syntax v0.8.10
   Compiling zlib-rs v0.6.3
   Compiling pin-project-lite v0.2.17
   Compiling rawpointer v0.2.1
   Compiling either v1.15.0
   Compiling getrandom v0.3.4
   Compiling equivalent v1.0.2
   Compiling hashbrown v0.17.0
   Compiling futures-core v0.3.32
   Compiling typenum v1.20.0
   Compiling futures-sink v0.3.32
   Compiling serde v1.0.228
   Compiling paste v1.0.15
   Compiling lexical-util v1.0.7
   Compiling vcpkg v0.2.15
   Compiling futures-io v0.3.32
   Compiling futures-task v0.3.32
   Compiling zstd-safe v7.2.4
   Compiling slab v0.4.12
   Compiling heck v0.5.0
   Compiling zstd-safe v6.0.6
   Compiling bitflags v2.11.1
   Compiling bytecount v0.6.9
   Compiling unicode-width v0.2.2
   Compiling utf8parse v0.2.2
   Compiling zmij v1.0.21
   Compiling bytes v1.11.1
   Compiling anyhow v1.0.102
   Compiling semver v1.0.28
   Compiling rayon-core v1.13.0
   Compiling byteorder v1.5.0
   Compiling rustversion v1.0.22
   Compiling itoa v1.0.18
   Compiling rustix v1.1.4
   Compiling lazy_static v1.5.0
   Compiling getrandom v0.4.2
   Compiling snap v1.1.1
   Compiling smallvec v1.15.1
   Compiling thiserror v1.0.69
   Compiling log v0.4.29
   Compiling array-init-cursor v0.2.1
   Compiling anstyle v1.0.14
   Compiling bit-vec v0.8.0
   Compiling anstyle-query v1.1.5
   Compiling static_assertions v1.1.0
   Compiling is_terminal_polyfill v1.70.2
   Compiling portable-atomic v1.13.1
   Compiling fallible-streaming-iterator v0.1.9
   Compiling linux-raw-sys v0.12.1
   Compiling serde_json v1.0.149
   Compiling colorchoice v1.0.5
   Compiling cpufeatures v0.2.17
   Compiling strsim v0.11.1
   Compiling anstyle-parse v1.0.0
   Compiling twox-hash v1.6.3
   Compiling thread_local v1.1.9
   Compiling streaming-decompression v0.1.2
   Compiling clap_lex v1.1.0
   Compiling fnv v1.0.7
   Compiling fastrand v2.4.1
   Compiling seq-macro v0.3.6
   Compiling miniz_oxide v0.8.9
   Compiling nu-ansi-term v0.50.3
   Compiling number_prefix v0.4.0
   Compiling tracing-core v0.1.36
   Compiling itertools v0.13.0
   Compiling itertools v0.14.0
   Compiling streaming-iterator v0.1.9
   Compiling ethnum v1.5.3
   Compiling foreign_vec v0.1.0
   Compiling ryu v1.0.23
   Compiling hash_hasher v2.0.4
   Compiling dyn-clone v1.0.20
   Compiling base64 v0.22.1
   Compiling generic-array v0.14.7
   Compiling ahash v0.8.12
   Compiling anstream v1.0.0
   Compiling futures-channel v0.3.32
   Compiling sharded-slab v0.1.7
   Compiling planus v0.3.1
   Compiling base64 v0.21.7
   Compiling simdutf8 v0.1.5
   Compiling hex v0.4.3
   Compiling rustc_version v0.4.1
   Compiling papergrid v0.14.0
   Compiling arrayvec v0.7.6
   Compiling num-traits v0.2.19
   Compiling matrixmultiply v0.3.10
   Compiling num-complex v0.2.4
   Compiling atomic_float v1.1.0
   Compiling parse-size v1.1.0
   Compiling aho-corasick v1.1.4
   Compiling buffer-redux v1.1.0
   Compiling lz4_flex v0.10.0
   Compiling csv-core v0.1.13
   Compiling rustc-hash v2.1.2
   Compiling path-tools v0.1.0
   Compiling lexical-parse-integer v1.0.6
   Compiling lexical-write-integer v1.0.6
   Compiling clap_builder v4.6.0
   Compiling indexmap v2.14.0
   Compiling arrow2 v0.18.0
   Compiling tracing-log v0.2.0
   Compiling num-format v0.4.4
   Compiling lexical-parse-float v1.0.6
   Compiling crossbeam-epoch v0.9.18
   Compiling crossbeam-channel v0.5.15
   Compiling crossbeam-queue v0.3.12
   Compiling lexical-write-float v1.0.6
   Compiling crossbeam-deque v0.8.6
   Compiling syn v2.0.117
   Compiling proc-macro-error-attr2 v2.0.0
   Compiling crypto-common v0.1.7
   Compiling block-buffer v0.10.4
   Compiling lexical-core v1.0.6
   Compiling digest v0.10.7
   Compiling crossbeam v0.8.4
   Compiling jobserver v0.1.34
   Compiling getrandom v0.2.17
   Compiling console v0.15.11
   Compiling num_cpus v1.17.0
   Compiling cc v1.2.61
   Compiling rand_core v0.9.5
   Compiling rand_core v0.6.4
   Compiling rayon v1.12.0
   Compiling indicatif v0.17.11
   Compiling num-integer v0.1.46
   Compiling num-complex v0.4.6
   Compiling approx v0.5.1
   Compiling noisy_float v0.2.1
   Compiling approx v0.3.2
   Compiling chrono v0.4.44
   Compiling regex-automata v0.4.14
   Compiling num-bigint v0.4.6
   Compiling num-iter v0.1.45
   Compiling tempfile v3.27.0
   Compiling zstd-sys v2.0.16+zstd.1.5.7
   Compiling liblzma-sys v0.3.13
   Compiling bzip2-sys v0.1.13+1.0.8
   Compiling libz-sys v1.1.28
   Compiling sha2-asm v0.6.4
   Compiling lz4-sys v1.11.1+lz4-1.10.0
   Compiling minimap2-sys v0.1.30+minimap2.2.30
   Compiling ndarray v0.16.1
   Compiling ndarray v0.15.6
   Compiling alga v0.9.3
   Compiling ndarray v0.17.2
   Compiling csv v1.4.0
   Compiling sha2 v0.10.9
   Compiling flate2 v1.1.9
   Compiling noodles-bgzf v0.38.0
   Compiling num-rational v0.4.2
   Compiling liblzma v0.3.6
   Compiling proc-macro-error2 v2.0.1
   Compiling num v0.4.3
   Compiling bytemuck_derive v1.10.2
   Compiling serde_derive v1.0.228
   Compiling futures-macro v0.3.32
   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 clap_derive v4.6.1
   Compiling derive-new v0.6.0
   Compiling tabled_derive v0.10.0
   Compiling typed-builder-macro v0.21.2
   Compiling async-stream v0.3.6
   Compiling ppv-lite86 v0.2.21
   Compiling futures-util v0.3.32
   Compiling hashbrown v0.14.5
   Compiling bzip2 v0.4.4
   Compiling tabled v0.18.0
   Compiling rand_chacha v0.3.1
   Compiling rand_chacha v0.9.0
   Compiling tracing v0.1.44
   Compiling bytemuck v1.25.0
   Compiling rand v0.9.4
   Compiling rand v0.8.6
   Compiling typed-builder v0.21.2
   Compiling safe_arch v0.7.4
   Compiling clap v4.6.1
   Compiling wide v0.7.33
   Compiling rand_distr v0.4.3
   Compiling bstr v1.12.1
   Compiling matchers v0.2.0
   Compiling regex v1.12.3
   Compiling tracing-subscriber v0.3.23
   Compiling ndarray-stats v0.6.0
   Compiling noodles-core v0.17.0
   Compiling noodles-csi v0.46.0
   Compiling kders v0.1.1
   Compiling noodles-sam v0.74.0
   Compiling bincode v1.3.3
   Compiling arrow-format v0.8.1
   Compiling bio-types v1.0.4
   Compiling simba v0.9.1
   Compiling futures-executor v0.3.32
   Compiling sendable-swapvec v0.4.3
   Compiling futures v0.3.32
   Compiling parquet-format-safe v0.2.4
   Compiling sprs v0.11.4
   Compiling noodles-bam v0.78.0
   Compiling lz4 v1.28.1
   Compiling nalgebra v0.33.3
   Compiling zstd v0.12.4
   Compiling zstd v0.13.3
   Compiling parquet2 v0.17.2
   Compiling needletail v0.6.3
   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 48.55s
  Installing /home/biocbuild/bbs-3.24-bioc/meat/FLAMES/src/cargo_temp/bin/oarfish
   Installed package `oarfish v0.8.0` (executable `oarfish`)
warning: be sure to add `/home/biocbuild/bbs-3.24-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-3.24-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/RtmpqKK2Mr/file3ffbc9106aa6cd/config_file_4193225.json
i Writing configuration to: /tmp/RtmpqKK2Mr/file3ffbc9106aa6cd/config_file_4193225.json
i Writing configuration to: /tmp/RtmpqKK2Mr/file3ffbc9106aa6cd/config_file_4193225.json
i Writing configuration to: /tmp/RtmpqKK2Mr/file3ffbc9799f0d61/config_file_4193225.json
Converting legacy `pattern` argument to `segments`...
Loading known barcodes from /tmp/RtmpqKK2Mr/file3ffbc9445654f3/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
CB:Z: tag field: CB
Processing file: /home/biocbuild/bbs-3.24-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/RtmpqKK2Mr/file3ffbc92d469229/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
CB:Z: tag field: CB
Processing file: /home/biocbuild/bbs-3.24-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/RtmpqKK2Mr/file3ffbc92d469229/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
CB:Z: tag field: CB
Processing file: /tmp/RtmpqKK2Mr/file3ffbc9dd04d47/musc_rps24_1.fastq
Searching for barcodes...
Processing file: /tmp/RtmpqKK2Mr/file3ffbc9dd04d47/musc_rps24_2.fastq
Searching for barcodes...
Processing file: /tmp/RtmpqKK2Mr/file3ffbc9dd04d47/musc_rps24_3.fastq
Searching for barcodes...
Processing file: /tmp/RtmpqKK2Mr/file3ffbc9dd04d47/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/RtmpqKK2Mr/file3ffbc9797a7692/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
CB:Z: tag field: CB
Processing file: /home/biocbuild/bbs-3.24-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/RtmpqKK2Mr/file3ffbc91d8b4b75/config_file_4193225.json
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Thu May  7 23:56:28 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpqKK2Mr/file3ffbc91d8b4b75/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpqKK2Mr/file3ffbc91d8b4b75/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpqKK2Mr/file3ffbc91d8b4b75/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Thu May  7 23:56:30 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 @ Thu May  7 23:56:54 2026 -------------------
Realigning sample sample1 -> /tmp/RtmpqKK2Mr/file3ffbc91d8b4b75/sample1_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample2 -> /tmp/RtmpqKK2Mr/file3ffbc91d8b4b75/sample2_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample3 -> /tmp/RtmpqKK2Mr/file3ffbc91d8b4b75/sample3_realign2transcript.bam
Skipped sorting BAM files.

-- Running step: transcript_quantification @ Thu May  7 23:56:55 2026 ----------
2026-05-08T03:56:55.724071Z  INFO oarfish: setting user-provided filter parameters.
2026-05-08T03:56:55.724482Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpqKK2Mr/file3ffbc91d8b4b75/sample1_realign2transcript.bam, contains 5 reference sequences.
2026-05-08T03:56:55.724496Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-08T03:56:55.724500Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-08T03:56:55.724569Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-08T03:56:55.724574Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-05-08T03:56:55.728711Z  INFO oarfish::alignment_parser: the alignment file contained 1 unmapped read records.
2026-05-08T03:56:55.728898Z  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   │
╰─────────────────────────────────┴───────╯

2026-05-08T03:56:55.728919Z  INFO oarfish::bulk: Total number of alignment records : 364
2026-05-08T03:56:55.728922Z  INFO oarfish::bulk: number of aligned reads : 283
2026-05-08T03:56:55.728924Z  INFO oarfish::bulk: number of unique alignments : 235
2026-05-08T03:56:55.729791Z  INFO oarfish: oarfish completed successfully.
2026-05-08T03:56:55.736706Z  INFO oarfish: setting user-provided filter parameters.
2026-05-08T03:56:55.737117Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpqKK2Mr/file3ffbc91d8b4b75/sample2_realign2transcript.bam, contains 5 reference sequences.
2026-05-08T03:56:55.737125Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-08T03:56:55.737128Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-08T03:56:55.737183Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-08T03:56:55.737188Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-05-08T03:56:55.741395Z  INFO oarfish::alignment_parser: the alignment file contained 1 unmapped read records.
2026-05-08T03:56:55.741555Z  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   │
╰─────────────────────────────────┴───────╯

2026-05-08T03:56:55.741584Z  INFO oarfish::bulk: Total number of alignment records : 369
2026-05-08T03:56:55.741586Z  INFO oarfish::bulk: number of aligned reads : 285
2026-05-08T03:56:55.741589Z  INFO oarfish::bulk: number of unique alignments : 235
2026-05-08T03:56:55.742405Z  INFO oarfish: oarfish completed successfully.
2026-05-08T03:56:55.748996Z  INFO oarfish: setting user-provided filter parameters.
2026-05-08T03:56:55.749339Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpqKK2Mr/file3ffbc91d8b4b75/sample3_realign2transcript.bam, contains 5 reference sequences.
2026-05-08T03:56:55.749348Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-08T03:56:55.749350Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-08T03:56:55.749408Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-08T03:56:55.749416Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-05-08T03:56:55.753508Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2026-05-08T03:56:55.753682Z  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   │
╰─────────────────────────────────┴───────╯

2026-05-08T03:56:55.753711Z  INFO oarfish::bulk: Total number of alignment records : 362
2026-05-08T03:56:55.753714Z  INFO oarfish::bulk: number of aligned reads : 284
2026-05-08T03:56:55.753716Z  INFO oarfish::bulk: number of unique alignments : 237
2026-05-08T03:56:55.754527Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
i Writing configuration to: /tmp/RtmpqKK2Mr/file3ffbc96433b953/config_file_4193225.json
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Thu May  7 23:56:55 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpqKK2Mr/file3ffbc96433b953/sample1_align2genome.bam
sample2 ->/tmp/RtmpqKK2Mr/file3ffbc96433b953/sample2_align2genome.bam
sample3 ->/tmp/RtmpqKK2Mr/file3ffbc96433b953/sample3_align2genome.bam
-- Running step: isoform_identification @ Thu May  7 23:57:17 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 @ Thu May  7 23:57:38 2026 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpqKK2Mr/file3ffbc96433b953/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpqKK2Mr/file3ffbc96433b953/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpqKK2Mr/file3ffbc96433b953/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Thu May  7 23:57:59 2026 ----------
2026-05-08T03:57:59.471059Z  INFO oarfish: setting user-provided filter parameters.
2026-05-08T03:57:59.471557Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpqKK2Mr/file3ffbc96433b953/sample1_realign2transcript.bam, contains 5 reference sequences.
2026-05-08T03:57:59.471570Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-08T03:57:59.471575Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-08T03:57:59.471638Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-08T03:57:59.471645Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-05-08T03:57:59.475689Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2026-05-08T03:57:59.475933Z  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   │
╰─────────────────────────────────┴───────╯

2026-05-08T03:57:59.475961Z  INFO oarfish::bulk: Total number of alignment records : 364
2026-05-08T03:57:59.475965Z  INFO oarfish::bulk: number of aligned reads : 285
2026-05-08T03:57:59.475968Z  INFO oarfish::bulk: number of unique alignments : 238
2026-05-08T03:57:59.476888Z  INFO oarfish: oarfish completed successfully.
2026-05-08T03:57:59.485294Z  INFO oarfish: setting user-provided filter parameters.
2026-05-08T03:57:59.485693Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpqKK2Mr/file3ffbc96433b953/sample2_realign2transcript.bam, contains 5 reference sequences.
2026-05-08T03:57:59.485705Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-08T03:57:59.485709Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-08T03:57:59.485782Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-08T03:57:59.485787Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-05-08T03:57:59.489792Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2026-05-08T03:57:59.490030Z  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   │
╰─────────────────────────────────┴───────╯

2026-05-08T03:57:59.490055Z  INFO oarfish::bulk: Total number of alignment records : 355
2026-05-08T03:57:59.490059Z  INFO oarfish::bulk: number of aligned reads : 282
2026-05-08T03:57:59.490061Z  INFO oarfish::bulk: number of unique alignments : 240
2026-05-08T03:57:59.490948Z  INFO oarfish: oarfish completed successfully.
2026-05-08T03:57:59.499247Z  INFO oarfish: setting user-provided filter parameters.
2026-05-08T03:57:59.499646Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpqKK2Mr/file3ffbc96433b953/sample3_realign2transcript.bam, contains 5 reference sequences.
2026-05-08T03:57:59.499659Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-08T03:57:59.499663Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-08T03:57:59.499730Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-08T03:57:59.499735Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-05-08T03:57:59.503827Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2026-05-08T03:57:59.504052Z  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   │
╰─────────────────────────────────┴───────╯

2026-05-08T03:57:59.504084Z  INFO oarfish::bulk: Total number of alignment records : 354
2026-05-08T03:57:59.504087Z  INFO oarfish::bulk: number of aligned reads : 283
2026-05-08T03:57:59.504090Z  INFO oarfish::bulk: number of unique alignments : 240
2026-05-08T03:57:59.505012Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
i Writing configuration to: /tmp/RtmpqKK2Mr/file3ffbc9644fa428/config_file_4193225.json
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Thu May  7 23:57:59 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpqKK2Mr/file3ffbc9644fa428/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpqKK2Mr/file3ffbc9644fa428/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpqKK2Mr/file3ffbc9644fa428/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Thu May  7 23:58:01 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 @ Thu May  7 23:58:21 2026 -------------------
Realigning sample sample1 -> /tmp/RtmpqKK2Mr/file3ffbc9644fa428/sample1_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample2 -> /tmp/RtmpqKK2Mr/file3ffbc9644fa428/sample2_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample3 -> /tmp/RtmpqKK2Mr/file3ffbc9644fa428/sample3_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Thu May  7 23:58:22 2026 ----------
23:58:22 Thu May 07 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/RtmpqKK2Mr/file3ffbc96bd18e6c/config_file_4193225.json
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Thu May  7 23:58:24 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpqKK2Mr/file3ffbc96bd18e6c/sample1_align2genome.bam
sample2 ->/tmp/RtmpqKK2Mr/file3ffbc96bd18e6c/sample2_align2genome.bam
sample3 ->/tmp/RtmpqKK2Mr/file3ffbc96bd18e6c/sample3_align2genome.bam
-- Running step: isoform_identification @ Thu May  7 23:58:46 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 @ Thu May  7 23:59:06 2026 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpqKK2Mr/file3ffbc96bd18e6c/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpqKK2Mr/file3ffbc96bd18e6c/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpqKK2Mr/file3ffbc96bd18e6c/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Thu May  7 23:59:26 2026 ----------
23:59:26 Thu May 07 2026 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Inputs:  ['/tmp/RtmpqKK2Mr/file3ffbc9644fa428/sample1_realign2transcript.bam', '/tmp/RtmpqKK2Mr/file3ffbc9644fa428/sample2_realign2transcript.bam', '/tmp/RtmpqKK2Mr/file3ffbc9644fa428/sample3_realign2transcript.bam'] /tmp/RtmpqKK2Mr/file3ffbc9644fa428/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/RtmpqKK2Mr/file3ffbc920d95f15/config_file_4193225.json
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Thu May  7 23:59:27 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpqKK2Mr/file3ffbc920d95f15/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpqKK2Mr/file3ffbc920d95f15/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpqKK2Mr/file3ffbc920d95f15/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Thu May  7 23:59:29 2026 -------------
Inputs:  ['/tmp/RtmpqKK2Mr/file3ffbc96bd18e6c/sample1_realign2transcript.bam', '/tmp/RtmpqKK2Mr/file3ffbc96bd18e6c/sample2_realign2transcript.bam', '/tmp/RtmpqKK2Mr/file3ffbc96bd18e6c/sample3_realign2transcript.bam'] /tmp/RtmpqKK2Mr/file3ffbc96bd18e6c/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 @ Thu May  7 23:59:29 2026 -------------------
Realigning sample sample1 -> /tmp/RtmpqKK2Mr/file3ffbc920d95f15/sample1_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample2 -> /tmp/RtmpqKK2Mr/file3ffbc920d95f15/sample2_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample3 -> /tmp/RtmpqKK2Mr/file3ffbc920d95f15/sample3_realign2transcript.bam
Skipped sorting BAM files.

-- Running step: transcript_quantification @ Thu May  7 23:59:32 2026 ----------
2026-05-08T03:59:32.477656Z  INFO oarfish: setting user-provided filter parameters.
2026-05-08T03:59:32.478191Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpqKK2Mr/file3ffbc920d95f15/sample1_realign2transcript.bam, contains 17 reference sequences.
2026-05-08T03:59:32.478201Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-08T03:59:32.478205Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-08T03:59:32.478309Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-08T03:59:32.478320Z  INFO oarfish: parsed reference information for 17 transcripts.
2026-05-08T03:59:32.488870Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-05-08T03:59:32.489139Z  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   │
╰─────────────────────────────────┴───────╯

2026-05-08T03:59:32.489173Z  INFO oarfish::bulk: Total number of alignment records : 513
2026-05-08T03:59:32.489176Z  INFO oarfish::bulk: number of aligned reads : 293
2026-05-08T03:59:32.489179Z  INFO oarfish::bulk: number of unique alignments : 198
2026-05-08T03:59:32.490090Z  INFO oarfish: oarfish completed successfully.
2026-05-08T03:59:32.498135Z  INFO oarfish: setting user-provided filter parameters.
2026-05-08T03:59:32.498559Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpqKK2Mr/file3ffbc920d95f15/sample2_realign2transcript.bam, contains 17 reference sequences.
2026-05-08T03:59:32.498573Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-08T03:59:32.498577Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-08T03:59:32.498681Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-08T03:59:32.498691Z  INFO oarfish: parsed reference information for 17 transcripts.
2026-05-08T03:59:32.509257Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-05-08T03:59:32.509557Z  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   │
╰─────────────────────────────────┴───────╯

2026-05-08T03:59:32.509607Z  INFO oarfish::bulk: Total number of alignment records : 525
2026-05-08T03:59:32.509609Z  INFO oarfish::bulk: number of aligned reads : 292
2026-05-08T03:59:32.509611Z  INFO oarfish::bulk: number of unique alignments : 190
2026-05-08T03:59:32.510632Z  INFO oarfish: oarfish completed successfully.
2026-05-08T03:59:32.518261Z  INFO oarfish: setting user-provided filter parameters.
2026-05-08T03:59:32.518747Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpqKK2Mr/file3ffbc920d95f15/sample3_realign2transcript.bam, contains 17 reference sequences.
2026-05-08T03:59:32.518760Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-08T03:59:32.518763Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-08T03:59:32.518861Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-08T03:59:32.518870Z  INFO oarfish: parsed reference information for 17 transcripts.
2026-05-08T03:59:32.529283Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-05-08T03:59:32.529557Z  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   │
╰─────────────────────────────────┴───────╯

2026-05-08T03:59:32.529593Z  INFO oarfish::bulk: Total number of alignment records : 517
2026-05-08T03:59:32.529596Z  INFO oarfish::bulk: number of aligned reads : 290
2026-05-08T03:59:32.529598Z  INFO oarfish::bulk: number of unique alignments : 192
2026-05-08T03:59:32.530524Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
i Writing configuration to: /tmp/RtmpqKK2Mr/file3ffbc91128dcab/config_file_4193225.json
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Thu May  7 23:59:32 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpqKK2Mr/file3ffbc91128dcab/sample1_align2genome.bam
sample2 ->/tmp/RtmpqKK2Mr/file3ffbc91128dcab/sample2_align2genome.bam
sample3 ->/tmp/RtmpqKK2Mr/file3ffbc91128dcab/sample3_align2genome.bam
-- Running step: isoform_identification @ Thu May  7 23:59:57 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Thu May  7 23:59:58 2026 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpqKK2Mr/file3ffbc91128dcab/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpqKK2Mr/file3ffbc91128dcab/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpqKK2Mr/file3ffbc91128dcab/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Fri May  8 00:00:19 2026 ----------
2026-05-08T04:00:19.760597Z  INFO oarfish: setting user-provided filter parameters.
2026-05-08T04:00:19.761100Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpqKK2Mr/file3ffbc91128dcab/sample1_realign2transcript.bam, contains 15 reference sequences.
2026-05-08T04:00:19.761112Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-08T04:00:19.761116Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-08T04:00:19.761201Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-08T04:00:19.761211Z  INFO oarfish: parsed reference information for 15 transcripts.
2026-05-08T04:00:19.770691Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-05-08T04:00:19.770895Z  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   │
╰─────────────────────────────────┴───────╯

2026-05-08T04:00:19.770926Z  INFO oarfish::bulk: Total number of alignment records : 495
2026-05-08T04:00:19.770929Z  INFO oarfish::bulk: number of aligned reads : 293
2026-05-08T04:00:19.770932Z  INFO oarfish::bulk: number of unique alignments : 208
2026-05-08T04:00:19.771804Z  INFO oarfish: oarfish completed successfully.
2026-05-08T04:00:19.778898Z  INFO oarfish: setting user-provided filter parameters.
2026-05-08T04:00:19.779229Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpqKK2Mr/file3ffbc91128dcab/sample2_realign2transcript.bam, contains 15 reference sequences.
2026-05-08T04:00:19.779240Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-08T04:00:19.779243Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-08T04:00:19.779335Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-08T04:00:19.779353Z  INFO oarfish: parsed reference information for 15 transcripts.
2026-05-08T04:00:19.788688Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-05-08T04:00:19.788889Z  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   │
╰─────────────────────────────────┴───────╯

2026-05-08T04:00:19.788920Z  INFO oarfish::bulk: Total number of alignment records : 511
2026-05-08T04:00:19.788922Z  INFO oarfish::bulk: number of aligned reads : 290
2026-05-08T04:00:19.788925Z  INFO oarfish::bulk: number of unique alignments : 198
2026-05-08T04:00:19.789824Z  INFO oarfish: oarfish completed successfully.
2026-05-08T04:00:19.797150Z  INFO oarfish: setting user-provided filter parameters.
2026-05-08T04:00:19.797607Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpqKK2Mr/file3ffbc91128dcab/sample3_realign2transcript.bam, contains 15 reference sequences.
2026-05-08T04:00:19.797618Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-08T04:00:19.797622Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-08T04:00:19.797704Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-08T04:00:19.797712Z  INFO oarfish: parsed reference information for 15 transcripts.
2026-05-08T04:00:19.806894Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-05-08T04:00:19.807100Z  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   │
╰─────────────────────────────────┴───────╯

2026-05-08T04:00:19.807129Z  INFO oarfish::bulk: Total number of alignment records : 496
2026-05-08T04:00:19.807131Z  INFO oarfish::bulk: number of aligned reads : 291
2026-05-08T04:00:19.807134Z  INFO oarfish::bulk: number of unique alignments : 206
2026-05-08T04:00:19.808035Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
i Writing configuration to: /tmp/RtmpqKK2Mr/file3ffbc975fe295d/config_file_4193225.json
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Fri May  8 00:00:20 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpqKK2Mr/file3ffbc975fe295d/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpqKK2Mr/file3ffbc975fe295d/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpqKK2Mr/file3ffbc975fe295d/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Fri May  8 00:00:21 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri May  8 00:00:22 2026 -------------------
Realigning sample sample1 -> /tmp/RtmpqKK2Mr/file3ffbc975fe295d/sample1_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample2 -> /tmp/RtmpqKK2Mr/file3ffbc975fe295d/sample2_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample3 -> /tmp/RtmpqKK2Mr/file3ffbc975fe295d/sample3_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Fri May  8 00:00:23 2026 ----------
00:00:23 Fri May 08 2026 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Inputs:  ['/tmp/RtmpqKK2Mr/file3ffbc975fe295d/sample1_realign2transcript.bam', '/tmp/RtmpqKK2Mr/file3ffbc975fe295d/sample2_realign2transcript.bam', '/tmp/RtmpqKK2Mr/file3ffbc975fe295d/sample3_realign2transcript.bam'] /tmp/RtmpqKK2Mr/file3ffbc975fe295d/transcript_assembly.fa.fai 5 0.4 0.4
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
i Writing configuration to: /tmp/RtmpqKK2Mr/file3ffbc93bdf9129/config_file_4193225.json
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Fri May  8 00:00:24 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpqKK2Mr/file3ffbc93bdf9129/sample1_align2genome.bam
sample2 ->/tmp/RtmpqKK2Mr/file3ffbc93bdf9129/sample2_align2genome.bam
sample3 ->/tmp/RtmpqKK2Mr/file3ffbc93bdf9129/sample3_align2genome.bam
-- Running step: isoform_identification @ Fri May  8 00:00:45 2026 -------------
	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 @ Fri May  8 00:00:45 2026 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpqKK2Mr/file3ffbc93bdf9129/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpqKK2Mr/file3ffbc93bdf9129/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpqKK2Mr/file3ffbc93bdf9129/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Fri May  8 00:01:05 2026 ----------
00:01:05 Fri May 08 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/RtmpqKK2Mr/file3ffbc961b8662/config_file_4193225.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 @ Fri May  8 00:01:06 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpqKK2Mr/file3ffbc961b8662/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
CB:Z: tag field: CB
Processing file: /home/biocbuild/bbs-3.24-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 @ Fri May  8 00:01:07 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpqKK2Mr/file3ffbc961b8662/matched_reads.fastq.gz -> /tmp/RtmpqKK2Mr/file3ffbc961b8662/align2genome.bam
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: gene_quantification @ Fri May  8 00:01:07 2026 ----------------
00:01:07 Fri May 08 2026 quantify genes 
Using BAM(s): '/tmp/RtmpqKK2Mr/file3ffbc961b8662/align2genome.bam'
Inputs:  ['/tmp/RtmpqKK2Mr/file3ffbc93bdf9129/sample1_realign2transcript.bam', '/tmp/RtmpqKK2Mr/file3ffbc93bdf9129/sample2_realign2transcript.bam', '/tmp/RtmpqKK2Mr/file3ffbc93bdf9129/sample3_realign2transcript.bam'] /tmp/RtmpqKK2Mr/file3ffbc93bdf9129/transcript_assembly.fa.fai 5 0.4 0.4
	Counter({'counted_reads': 895, 'not_enough_coverage': 5})
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.68gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.67gene_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, 434390.82Read/s]
-- Running step: isoform_identification @ Fri May  8 00:01:08 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 @ Fri May  8 00:01:17 2026 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.24-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpqKK2Mr/file3ffbc961b8662/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpqKK2Mr/file3ffbc961b8662/matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpqKK2Mr/file3ffbc961b8662/matched_reads_dedup.fastq.gz -> /tmp/RtmpqKK2Mr/file3ffbc961b8662/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 @ Fri May  8 00:01:18 2026 ----------
2026-05-08T04:01:18.032993Z  INFO oarfish: setting user-provided filter parameters.
2026-05-08T04:01:18.033498Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpqKK2Mr/file3ffbc961b8662/realign2transcript.bam, contains 5 reference sequences.
2026-05-08T04:01:18.033520Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-08T04:01:18.033523Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-08T04:01:18.033578Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-08T04:01:18.033583Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-05-08T04:01:18.039518Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
i Writing configuration to: /tmp/RtmpqKK2Mr/file3ffbc91a6673b5/config_file_4193225.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 @ Fri May  8 00:01:18 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpqKK2Mr/file3ffbc91a6673b5/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
CB:Z: tag field: CB
Processing file: /home/biocbuild/bbs-3.24-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 @ Fri May  8 00:01:18 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpqKK2Mr/file3ffbc91a6673b5/matched_reads.fastq.gz ->/tmp/RtmpqKK2Mr/file3ffbc91a6673b5/align2genome.bam
-- Running step: gene_quantification @ Fri May  8 00:01:37 2026 ----------------
00:01:37 Fri May 08 2026 quantify genes 
Using BAM(s): '/tmp/RtmpqKK2Mr/file3ffbc91a6673b5/align2genome.bam'
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.58gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.58gene_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, 446202.55Read/s]
-- Running step: isoform_identification @ Fri May  8 00:01:38 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 @ Fri May  8 00:01:48 2026 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.24-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpqKK2Mr/file3ffbc91a6673b5/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpqKK2Mr/file3ffbc91a6673b5/matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpqKK2Mr/file3ffbc91a6673b5/matched_reads_dedup.fastq.gz ->/tmp/RtmpqKK2Mr/file3ffbc91a6673b5/realign2transcript.bam
-- Running step: transcript_quantification @ Fri May  8 00:02:08 2026 ----------
2026-05-08T04:02:08.464850Z  INFO oarfish: setting user-provided filter parameters.
2026-05-08T04:02:08.465246Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpqKK2Mr/file3ffbc91a6673b5/realign2transcript.bam, contains 5 reference sequences.
2026-05-08T04:02:08.465254Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-08T04:02:08.465257Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-08T04:02:08.465315Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-08T04:02:08.465321Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-05-08T04:02:08.472023Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
i Writing configuration to: /tmp/RtmpqKK2Mr/file3ffbc9682d95d1/config_file_4193225.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 @ Fri May  8 00:02:08 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpqKK2Mr/file3ffbc9682d95d1/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
CB:Z: tag field: CB
Processing file: /home/biocbuild/bbs-3.24-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 @ Fri May  8 00:02:09 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpqKK2Mr/file3ffbc9682d95d1/matched_reads.fastq.gz -> /tmp/RtmpqKK2Mr/file3ffbc9682d95d1/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: gene_quantification @ Fri May  8 00:02:09 2026 ----------------
00:02:09 Fri May 08 2026 quantify genes 
Using BAM(s): '/tmp/RtmpqKK2Mr/file3ffbc9682d95d1/align2genome.bam'
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.78gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.77gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 373358.02Read/s]
-- Running step: isoform_identification @ Fri May  8 00:02:10 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 @ Fri May  8 00:02:20 2026 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.24-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpqKK2Mr/file3ffbc9682d95d1/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpqKK2Mr/file3ffbc9682d95d1/matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpqKK2Mr/file3ffbc9682d95d1/matched_reads_dedup.fastq.gz -> /tmp/RtmpqKK2Mr/file3ffbc9682d95d1/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 @ Fri May  8 00:02:20 2026 ----------
00:02:20 Fri May 08 2026 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
i Writing configuration to: /tmp/RtmpqKK2Mr/file3ffbc93922e74c/config_file_4193225.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 @ Fri May  8 00:02:21 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpqKK2Mr/file3ffbc93922e74c/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
CB:Z: tag field: CB
Processing file: /home/biocbuild/bbs-3.24-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 @ Fri May  8 00:02:21 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpqKK2Mr/file3ffbc93922e74c/matched_reads.fastq.gz ->/tmp/RtmpqKK2Mr/file3ffbc93922e74c/align2genome.bam
-- Running step: gene_quantification @ Fri May  8 00:02:39 2026 ----------------
00:02:39 Fri May 08 2026 quantify genes 
Using BAM(s): '/tmp/RtmpqKK2Mr/file3ffbc93922e74c/align2genome.bam'
	Counter({'counted_reads': 347, 'not_enough_coverage': 9, 'unmapped': 2})
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.37gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.37gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 439949.65Read/s]
-- Running step: isoform_identification @ Fri May  8 00:02:40 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 @ Fri May  8 00:02:50 2026 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.24-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpqKK2Mr/file3ffbc93922e74c/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpqKK2Mr/file3ffbc93922e74c/matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpqKK2Mr/file3ffbc93922e74c/matched_reads_dedup.fastq.gz ->/tmp/RtmpqKK2Mr/file3ffbc93922e74c/realign2transcript.bam
-- Running step: transcript_quantification @ Fri May  8 00:03:08 2026 ----------
00:03:08 Fri May 08 2026 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
i Writing configuration to: /tmp/RtmpqKK2Mr/file3ffbc98139d47/config_file_4193225.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 @ Fri May  8 00:03:09 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpqKK2Mr/file3ffbc98139d47/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
CB:Z: tag field: CB
Processing file: /home/biocbuild/bbs-3.24-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 @ Fri May  8 00:03:09 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpqKK2Mr/file3ffbc98139d47/matched_reads.fastq.gz -> /tmp/RtmpqKK2Mr/file3ffbc98139d47/align2genome.bam
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: gene_quantification @ Fri May  8 00:03:09 2026 ----------------
00:03:09 Fri May 08 2026 quantify genes 
Using BAM(s): '/tmp/RtmpqKK2Mr/file3ffbc98139d47/align2genome.bam'
	Counter({'counted_reads': 347, 'not_enough_coverage': 9, 'unmapped': 2})
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.59gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.58gene_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, 363206.10Read/s]
-- Running step: isoform_identification @ Fri May  8 00:03:10 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri May  8 00:03:10 2026 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.24-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpqKK2Mr/file3ffbc98139d47/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpqKK2Mr/file3ffbc98139d47/matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpqKK2Mr/file3ffbc98139d47/matched_reads_dedup.fastq.gz -> /tmp/RtmpqKK2Mr/file3ffbc98139d47/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 @ Fri May  8 00:03:11 2026 ----------
2026-05-08T04:03:11.107583Z  INFO oarfish: setting user-provided filter parameters.
2026-05-08T04:03:11.108002Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpqKK2Mr/file3ffbc98139d47/realign2transcript.bam, contains 10 reference sequences.
2026-05-08T04:03:11.108011Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-08T04:03:11.108014Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-08T04:03:11.108088Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-08T04:03:11.108096Z  INFO oarfish: parsed reference information for 10 transcripts.
2026-05-08T04:03:11.117594Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
i Writing configuration to: /tmp/RtmpqKK2Mr/file3ffbc9f54e399/config_file_4193225.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 @ Fri May  8 00:03:11 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpqKK2Mr/file3ffbc9f54e399/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
CB:Z: tag field: CB
Processing file: /home/biocbuild/bbs-3.24-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 @ Fri May  8 00:03:12 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpqKK2Mr/file3ffbc9f54e399/matched_reads.fastq.gz ->/tmp/RtmpqKK2Mr/file3ffbc9f54e399/align2genome.bam
-- Running step: gene_quantification @ Fri May  8 00:03:30 2026 ----------------
00:03:30 Fri May 08 2026 quantify genes 
Using BAM(s): '/tmp/RtmpqKK2Mr/file3ffbc9f54e399/align2genome.bam'
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.56gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.56gene_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, 382384.95Read/s]
-- Running step: isoform_identification @ Fri May  8 00:03:31 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri May  8 00:03:31 2026 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.24-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpqKK2Mr/file3ffbc9f54e399/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpqKK2Mr/file3ffbc9f54e399/matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpqKK2Mr/file3ffbc9f54e399/matched_reads_dedup.fastq.gz ->/tmp/RtmpqKK2Mr/file3ffbc9f54e399/realign2transcript.bam
-- Running step: transcript_quantification @ Fri May  8 00:03:49 2026 ----------
2026-05-08T04:03:50.001886Z  INFO oarfish: setting user-provided filter parameters.
2026-05-08T04:03:50.002276Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpqKK2Mr/file3ffbc9f54e399/realign2transcript.bam, contains 10 reference sequences.
2026-05-08T04:03:50.002294Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-08T04:03:50.002297Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-08T04:03:50.002363Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-08T04:03:50.002370Z  INFO oarfish: parsed reference information for 10 transcripts.
2026-05-08T04:03:50.012694Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
i Writing configuration to: /tmp/RtmpqKK2Mr/file3ffbc93f1c7f61/config_file_4193225.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 @ Fri May  8 00:03:50 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpqKK2Mr/file3ffbc93f1c7f61/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
CB:Z: tag field: CB
Processing file: /home/biocbuild/bbs-3.24-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 @ Fri May  8 00:03:51 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpqKK2Mr/file3ffbc93f1c7f61/matched_reads.fastq.gz -> /tmp/RtmpqKK2Mr/file3ffbc93f1c7f61/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: gene_quantification @ Fri May  8 00:03:51 2026 ----------------
00:03:51 Fri May 08 2026 quantify genes 
Using BAM(s): '/tmp/RtmpqKK2Mr/file3ffbc93f1c7f61/align2genome.bam'
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.74gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.74gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 371229.91Read/s]
-- Running step: isoform_identification @ Fri May  8 00:03:52 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri May  8 00:03:52 2026 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.24-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpqKK2Mr/file3ffbc93f1c7f61/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpqKK2Mr/file3ffbc93f1c7f61/matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpqKK2Mr/file3ffbc93f1c7f61/matched_reads_dedup.fastq.gz -> /tmp/RtmpqKK2Mr/file3ffbc93f1c7f61/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 @ Fri May  8 00:03:52 2026 ----------
00:03:52 Fri May 08 2026 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
i Writing configuration to: /tmp/RtmpqKK2Mr/file3ffbc914452ea5/config_file_4193225.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 @ Fri May  8 00:03:53 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpqKK2Mr/file3ffbc914452ea5/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
CB:Z: tag field: CB
Processing file: /home/biocbuild/bbs-3.24-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 @ Fri May  8 00:03:53 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpqKK2Mr/file3ffbc914452ea5/matched_reads.fastq.gz ->/tmp/RtmpqKK2Mr/file3ffbc914452ea5/align2genome.bam
-- Running step: gene_quantification @ Fri May  8 00:04:12 2026 ----------------
00:04:12 Fri May 08 2026 quantify genes 
Using BAM(s): '/tmp/RtmpqKK2Mr/file3ffbc914452ea5/align2genome.bam'
	Counter({'counted_reads': 358})
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.81gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.81gene_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, 370063.88Read/s]
-- Running step: isoform_identification @ Fri May  8 00:04:12 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri May  8 00:04:13 2026 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.24-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpqKK2Mr/file3ffbc914452ea5/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpqKK2Mr/file3ffbc914452ea5/matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpqKK2Mr/file3ffbc914452ea5/matched_reads_dedup.fastq.gz ->/tmp/RtmpqKK2Mr/file3ffbc914452ea5/realign2transcript.bam
-- Running step: transcript_quantification @ Fri May  8 00:04:30 2026 ----------
00:04:30 Fri May 08 2026 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
i Writing configuration to: /tmp/RtmpqKK2Mr/file3ffbc945c2eeb0/config_file_4193225.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 @ Fri May  8 00:04:32 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpqKK2Mr/file3ffbc945c2eeb0/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
CB:Z: tag field: CB
Processing file: /tmp/RtmpqKK2Mr/file3ffbc945c2eeb0/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpqKK2Mr/file3ffbc945c2eeb0/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpqKK2Mr/file3ffbc945c2eeb0/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/RtmpqKK2Mr/file3ffbc945c2eeb0/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
CB:Z: tag field: CB
Processing file: /tmp/RtmpqKK2Mr/file3ffbc945c2eeb0/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/RtmpqKK2Mr/file3ffbc945c2eeb0/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
CB:Z: tag field: CB
Processing file: /tmp/RtmpqKK2Mr/file3ffbc945c2eeb0/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/RtmpqKK2Mr/file3ffbc945c2eeb0/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
CB:Z: tag field: CB
Processing file: /tmp/RtmpqKK2Mr/file3ffbc945c2eeb0/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 @ Fri May  8 00:04:33 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpqKK2Mr/file3ffbc945c2eeb0/sampleA_matched_reads.fastq.gz -> /tmp/RtmpqKK2Mr/file3ffbc945c2eeb0/sampleA_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpqKK2Mr/file3ffbc945c2eeb0/sample1_matched_reads.fastq.gz -> /tmp/RtmpqKK2Mr/file3ffbc945c2eeb0/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpqKK2Mr/file3ffbc945c2eeb0/sample2_matched_reads.fastq.gz -> /tmp/RtmpqKK2Mr/file3ffbc945c2eeb0/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpqKK2Mr/file3ffbc945c2eeb0/sample3_matched_reads.fastq.gz -> /tmp/RtmpqKK2Mr/file3ffbc945c2eeb0/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Fri May  8 00:04:37 2026 ----------------
00:04:37 Fri May 08 2026 quantify genes 
Using BAM(s): '/tmp/RtmpqKK2Mr/file3ffbc945c2eeb0/sampleA_align2genome.bam',
'/tmp/RtmpqKK2Mr/file3ffbc945c2eeb0/sample1_align2genome.bam',
'/tmp/RtmpqKK2Mr/file3ffbc945c2eeb0/sample2_align2genome.bam', and
'/tmp/RtmpqKK2Mr/file3ffbc945c2eeb0/sample3_align2genome.bam'
	Counter({'counted_reads': 358})
parsing /tmp/RtmpqKK2Mr/file3ffbc945c2eeb0/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 12.36gene_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, 175582.05Read/s]
parsing /tmp/RtmpqKK2Mr/file3ffbc945c2eeb0/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 27.63gene_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, 570684.66Read/s]
parsing /tmp/RtmpqKK2Mr/file3ffbc945c2eeb0/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 18.20gene_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, 562782.31Read/s]
parsing /tmp/RtmpqKK2Mr/file3ffbc945c2eeb0/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  7.88gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  7.87gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 426649.31Read/s]
-- Running step: isoform_identification @ Fri May  8 00:04:38 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

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 @ Fri May  8 00:05:04 2026 -------------------
Checking for fastq file(s) /tmp/RtmpqKK2Mr/file3ffbc945c2eeb0/fastq, /tmp/RtmpqKK2Mr/file3ffbc945c2eeb0/fastq/sample1.fq.gz, /tmp/RtmpqKK2Mr/file3ffbc945c2eeb0/fastq/sample2.fq.gz, /tmp/RtmpqKK2Mr/file3ffbc945c2eeb0/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpqKK2Mr/file3ffbc945c2eeb0/sampleA_matched_reads.fastq.gz, /tmp/RtmpqKK2Mr/file3ffbc945c2eeb0/sample1_matched_reads.fastq.gz, /tmp/RtmpqKK2Mr/file3ffbc945c2eeb0/sample2_matched_reads.fastq.gz, /tmp/RtmpqKK2Mr/file3ffbc945c2eeb0/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpqKK2Mr/file3ffbc945c2eeb0/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpqKK2Mr/file3ffbc945c2eeb0/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpqKK2Mr/file3ffbc945c2eeb0/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpqKK2Mr/file3ffbc945c2eeb0/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpqKK2Mr/file3ffbc945c2eeb0/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpqKK2Mr/file3ffbc945c2eeb0/sampleA_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpqKK2Mr/file3ffbc945c2eeb0/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpqKK2Mr/file3ffbc945c2eeb0/sample1_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpqKK2Mr/file3ffbc945c2eeb0/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpqKK2Mr/file3ffbc945c2eeb0/sample2_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpqKK2Mr/file3ffbc945c2eeb0/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpqKK2Mr/file3ffbc945c2eeb0/sample3_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

-- Running step: transcript_quantification @ Fri May  8 00:05:06 2026 ----------
2026-05-08T04:05:06.589941Z  INFO oarfish: setting user-provided filter parameters.
2026-05-08T04:05:06.590265Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpqKK2Mr/file3ffbc945c2eeb0/sampleA_realign2transcript.bam, contains 6 reference sequences.
2026-05-08T04:05:06.590273Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-08T04:05:06.590276Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-08T04:05:06.590335Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-08T04:05:06.590340Z  INFO oarfish: parsed reference information for 6 transcripts.
2026-05-08T04:05:06.600685Z  INFO oarfish::single_cell: Processed 100 cells.
2026-05-08T04:05:06.898985Z  INFO oarfish: setting user-provided filter parameters.
2026-05-08T04:05:06.899329Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpqKK2Mr/file3ffbc945c2eeb0/sample1_realign2transcript.bam, contains 6 reference sequences.
2026-05-08T04:05:06.899337Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-08T04:05:06.899341Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-08T04:05:06.899407Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-08T04:05:06.899416Z  INFO oarfish: parsed reference information for 6 transcripts.
2026-05-08T04:05:06.904536Z  INFO oarfish::single_cell: Processed 100 cells.
2026-05-08T04:05:07.183311Z  INFO oarfish: setting user-provided filter parameters.
2026-05-08T04:05:07.183774Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpqKK2Mr/file3ffbc945c2eeb0/sample2_realign2transcript.bam, contains 6 reference sequences.
2026-05-08T04:05:07.183786Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-08T04:05:07.183790Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-08T04:05:07.183848Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-08T04:05:07.183854Z  INFO oarfish: parsed reference information for 6 transcripts.
2026-05-08T04:05:07.188886Z  INFO oarfish::single_cell: Processed 100 cells.
2026-05-08T04:05:07.471295Z  INFO oarfish: setting user-provided filter parameters.
2026-05-08T04:05:07.471718Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpqKK2Mr/file3ffbc945c2eeb0/sample3_realign2transcript.bam, contains 6 reference sequences.
2026-05-08T04:05:07.471729Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-08T04:05:07.471733Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-08T04:05:07.471796Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-08T04:05:07.471802Z  INFO oarfish: parsed reference information for 6 transcripts.
2026-05-08T04:05:07.477505Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
i Writing configuration to: /tmp/RtmpqKK2Mr/file3ffbc92a3aba0f/config_file_4193225.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 @ Fri May  8 00:05:08 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpqKK2Mr/file3ffbc92a3aba0f/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
CB:Z: tag field: CB
Processing file: /tmp/RtmpqKK2Mr/file3ffbc92a3aba0f/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpqKK2Mr/file3ffbc92a3aba0f/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpqKK2Mr/file3ffbc92a3aba0f/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/RtmpqKK2Mr/file3ffbc92a3aba0f/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
CB:Z: tag field: CB
Processing file: /tmp/RtmpqKK2Mr/file3ffbc92a3aba0f/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/RtmpqKK2Mr/file3ffbc92a3aba0f/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
CB:Z: tag field: CB
Processing file: /tmp/RtmpqKK2Mr/file3ffbc92a3aba0f/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/RtmpqKK2Mr/file3ffbc92a3aba0f/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
CB:Z: tag field: CB
Processing file: /tmp/RtmpqKK2Mr/file3ffbc92a3aba0f/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 @ Fri May  8 00:05:09 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpqKK2Mr/file3ffbc92a3aba0f/sampleA_matched_reads.fastq.gz ->/tmp/RtmpqKK2Mr/file3ffbc92a3aba0f/sampleA_align2genome.bam
/tmp/RtmpqKK2Mr/file3ffbc92a3aba0f/sample1_matched_reads.fastq.gz ->/tmp/RtmpqKK2Mr/file3ffbc92a3aba0f/sample1_align2genome.bam
/tmp/RtmpqKK2Mr/file3ffbc92a3aba0f/sample2_matched_reads.fastq.gz ->/tmp/RtmpqKK2Mr/file3ffbc92a3aba0f/sample2_align2genome.bam
/tmp/RtmpqKK2Mr/file3ffbc92a3aba0f/sample3_matched_reads.fastq.gz ->/tmp/RtmpqKK2Mr/file3ffbc92a3aba0f/sample3_align2genome.bam
-- Running step: gene_quantification @ Fri May  8 00:05:30 2026 ----------------
00:05:30 Fri May 08 2026 quantify genes 
Using BAM(s): '/tmp/RtmpqKK2Mr/file3ffbc92a3aba0f/sampleA_align2genome.bam',
'/tmp/RtmpqKK2Mr/file3ffbc92a3aba0f/sample1_align2genome.bam',
'/tmp/RtmpqKK2Mr/file3ffbc92a3aba0f/sample2_align2genome.bam', and
'/tmp/RtmpqKK2Mr/file3ffbc92a3aba0f/sample3_align2genome.bam'
parsing /tmp/RtmpqKK2Mr/file3ffbc92a3aba0f/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 13.93gene_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, 134087.29Read/s]
parsing /tmp/RtmpqKK2Mr/file3ffbc92a3aba0f/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 19.45gene_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, 528835.99Read/s]
parsing /tmp/RtmpqKK2Mr/file3ffbc92a3aba0f/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 23.41gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 469329.51Read/s]
parsing /tmp/RtmpqKK2Mr/file3ffbc92a3aba0f/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 24.00gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 352273.06Read/s]
-- Running step: isoform_identification @ Fri May  8 00:05:31 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 @ Fri May  8 00:05:55 2026 -------------------
Checking for fastq file(s) /tmp/RtmpqKK2Mr/file3ffbc92a3aba0f/fastq, /tmp/RtmpqKK2Mr/file3ffbc92a3aba0f/fastq/sample1.fq.gz, /tmp/RtmpqKK2Mr/file3ffbc92a3aba0f/fastq/sample2.fq.gz, /tmp/RtmpqKK2Mr/file3ffbc92a3aba0f/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpqKK2Mr/file3ffbc92a3aba0f/sampleA_matched_reads.fastq.gz, /tmp/RtmpqKK2Mr/file3ffbc92a3aba0f/sample1_matched_reads.fastq.gz, /tmp/RtmpqKK2Mr/file3ffbc92a3aba0f/sample2_matched_reads.fastq.gz, /tmp/RtmpqKK2Mr/file3ffbc92a3aba0f/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpqKK2Mr/file3ffbc92a3aba0f/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpqKK2Mr/file3ffbc92a3aba0f/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpqKK2Mr/file3ffbc92a3aba0f/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpqKK2Mr/file3ffbc92a3aba0f/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpqKK2Mr/file3ffbc92a3aba0f/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpqKK2Mr/file3ffbc92a3aba0f/sampleA_realign2transcript.bam
/tmp/RtmpqKK2Mr/file3ffbc92a3aba0f/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpqKK2Mr/file3ffbc92a3aba0f/sample1_realign2transcript.bam
/tmp/RtmpqKK2Mr/file3ffbc92a3aba0f/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpqKK2Mr/file3ffbc92a3aba0f/sample2_realign2transcript.bam
/tmp/RtmpqKK2Mr/file3ffbc92a3aba0f/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpqKK2Mr/file3ffbc92a3aba0f/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Fri May  8 00:06:15 2026 ----------
2026-05-08T04:06:15.865145Z  INFO oarfish: setting user-provided filter parameters.
2026-05-08T04:06:15.865642Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpqKK2Mr/file3ffbc92a3aba0f/sampleA_realign2transcript.bam, contains 6 reference sequences.
2026-05-08T04:06:15.865654Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-08T04:06:15.865658Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-08T04:06:15.865720Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-08T04:06:15.865727Z  INFO oarfish: parsed reference information for 6 transcripts.
2026-05-08T04:06:15.876999Z  INFO oarfish::single_cell: Processed 100 cells.
2026-05-08T04:06:16.235148Z  INFO oarfish: setting user-provided filter parameters.
2026-05-08T04:06:16.235755Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpqKK2Mr/file3ffbc92a3aba0f/sample1_realign2transcript.bam, contains 6 reference sequences.
2026-05-08T04:06:16.235767Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-08T04:06:16.235772Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-08T04:06:16.235832Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-08T04:06:16.235839Z  INFO oarfish: parsed reference information for 6 transcripts.
2026-05-08T04:06:16.241154Z  INFO oarfish::single_cell: Processed 100 cells.
2026-05-08T04:06:16.550013Z  INFO oarfish: setting user-provided filter parameters.
2026-05-08T04:06:16.550510Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpqKK2Mr/file3ffbc92a3aba0f/sample2_realign2transcript.bam, contains 6 reference sequences.
2026-05-08T04:06:16.550523Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-08T04:06:16.550527Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-08T04:06:16.550585Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-08T04:06:16.550592Z  INFO oarfish: parsed reference information for 6 transcripts.
2026-05-08T04:06:16.555687Z  INFO oarfish::single_cell: Processed 100 cells.
2026-05-08T04:06:16.886672Z  INFO oarfish: setting user-provided filter parameters.
2026-05-08T04:06:16.887174Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpqKK2Mr/file3ffbc92a3aba0f/sample3_realign2transcript.bam, contains 6 reference sequences.
2026-05-08T04:06:16.887182Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-08T04:06:16.887185Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-08T04:06:16.887248Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-08T04:06:16.887253Z  INFO oarfish: parsed reference information for 6 transcripts.
2026-05-08T04:06:16.892879Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
i Writing configuration to: /tmp/RtmpqKK2Mr/file3ffbc92cbea804/config_file_4193225.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 @ Fri May  8 00:06:17 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpqKK2Mr/file3ffbc92cbea804/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
CB:Z: tag field: CB
Processing file: /tmp/RtmpqKK2Mr/file3ffbc92cbea804/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpqKK2Mr/file3ffbc92cbea804/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpqKK2Mr/file3ffbc92cbea804/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/RtmpqKK2Mr/file3ffbc92cbea804/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
CB:Z: tag field: CB
Processing file: /tmp/RtmpqKK2Mr/file3ffbc92cbea804/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/RtmpqKK2Mr/file3ffbc92cbea804/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
CB:Z: tag field: CB
Processing file: /tmp/RtmpqKK2Mr/file3ffbc92cbea804/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/RtmpqKK2Mr/file3ffbc92cbea804/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
CB:Z: tag field: CB
Processing file: /tmp/RtmpqKK2Mr/file3ffbc92cbea804/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 @ Fri May  8 00:06:18 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpqKK2Mr/file3ffbc92cbea804/sampleA_matched_reads.fastq.gz -> /tmp/RtmpqKK2Mr/file3ffbc92cbea804/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/RtmpqKK2Mr/file3ffbc92cbea804/sample1_matched_reads.fastq.gz -> /tmp/RtmpqKK2Mr/file3ffbc92cbea804/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/RtmpqKK2Mr/file3ffbc92cbea804/sample2_matched_reads.fastq.gz -> /tmp/RtmpqKK2Mr/file3ffbc92cbea804/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/RtmpqKK2Mr/file3ffbc92cbea804/sample3_matched_reads.fastq.gz -> /tmp/RtmpqKK2Mr/file3ffbc92cbea804/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 @ Fri May  8 00:06:22 2026 ----------------
00:06:22 Fri May 08 2026 quantify genes 
Using BAM(s): '/tmp/RtmpqKK2Mr/file3ffbc92cbea804/sampleA_align2genome.bam',
'/tmp/RtmpqKK2Mr/file3ffbc92cbea804/sample1_align2genome.bam',
'/tmp/RtmpqKK2Mr/file3ffbc92cbea804/sample2_align2genome.bam', and
'/tmp/RtmpqKK2Mr/file3ffbc92cbea804/sample3_align2genome.bam'
parsing /tmp/RtmpqKK2Mr/file3ffbc92cbea804/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 14.03gene_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, 149927.22Read/s]
parsing /tmp/RtmpqKK2Mr/file3ffbc92cbea804/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 28.92gene_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, 487347.09Read/s]
parsing /tmp/RtmpqKK2Mr/file3ffbc92cbea804/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 28.93gene_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, 517789.74Read/s]
parsing /tmp/RtmpqKK2Mr/file3ffbc92cbea804/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 22.99gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 428147.48Read/s]
-- Running step: isoform_identification @ Fri May  8 00:06:23 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 @ Fri May  8 00:06:46 2026 -------------------
Checking for fastq file(s) /tmp/RtmpqKK2Mr/file3ffbc92cbea804/fastq, /tmp/RtmpqKK2Mr/file3ffbc92cbea804/fastq/sample1.fq.gz, /tmp/RtmpqKK2Mr/file3ffbc92cbea804/fastq/sample2.fq.gz, /tmp/RtmpqKK2Mr/file3ffbc92cbea804/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpqKK2Mr/file3ffbc92cbea804/sampleA_matched_reads.fastq.gz, /tmp/RtmpqKK2Mr/file3ffbc92cbea804/sample1_matched_reads.fastq.gz, /tmp/RtmpqKK2Mr/file3ffbc92cbea804/sample2_matched_reads.fastq.gz, /tmp/RtmpqKK2Mr/file3ffbc92cbea804/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpqKK2Mr/file3ffbc92cbea804/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpqKK2Mr/file3ffbc92cbea804/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpqKK2Mr/file3ffbc92cbea804/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpqKK2Mr/file3ffbc92cbea804/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpqKK2Mr/file3ffbc92cbea804/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpqKK2Mr/file3ffbc92cbea804/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/RtmpqKK2Mr/file3ffbc92cbea804/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpqKK2Mr/file3ffbc92cbea804/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/RtmpqKK2Mr/file3ffbc92cbea804/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpqKK2Mr/file3ffbc92cbea804/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/RtmpqKK2Mr/file3ffbc92cbea804/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpqKK2Mr/file3ffbc92cbea804/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 @ Fri May  8 00:06:48 2026 ----------
00:06:48 Fri May 08 2026 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpqKK2Mr/file3ffbc92cbea804/sampleA_realign2transcript.bam...
parsing /tmp/RtmpqKK2Mr/file3ffbc92cbea804/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpqKK2Mr/file3ffbc92cbea804/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpqKK2Mr/file3ffbc92cbea804/sample1_realign2transcript.bam...
parsing /tmp/RtmpqKK2Mr/file3ffbc92cbea804/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpqKK2Mr/file3ffbc92cbea804/sample1_realign2transcript.bamdone
parsing /tmp/RtmpqKK2Mr/file3ffbc92cbea804/sample2_realign2transcript.bam...
parsing /tmp/RtmpqKK2Mr/file3ffbc92cbea804/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpqKK2Mr/file3ffbc92cbea804/sample2_realign2transcript.bamdone
parsing /tmp/RtmpqKK2Mr/file3ffbc92cbea804/sample3_realign2transcript.bam...
parsing /tmp/RtmpqKK2Mr/file3ffbc92cbea804/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpqKK2Mr/file3ffbc92cbea804/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
i Writing configuration to: /tmp/RtmpqKK2Mr/file3ffbc95881f660/config_file_4193225.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 @ Fri May  8 00:06:50 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpqKK2Mr/file3ffbc95881f660/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
CB:Z: tag field: CB
Processing file: /tmp/RtmpqKK2Mr/file3ffbc95881f660/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpqKK2Mr/file3ffbc95881f660/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpqKK2Mr/file3ffbc95881f660/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/RtmpqKK2Mr/file3ffbc95881f660/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
CB:Z: tag field: CB
Processing file: /tmp/RtmpqKK2Mr/file3ffbc95881f660/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/RtmpqKK2Mr/file3ffbc95881f660/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
CB:Z: tag field: CB
Processing file: /tmp/RtmpqKK2Mr/file3ffbc95881f660/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/RtmpqKK2Mr/file3ffbc95881f660/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
CB:Z: tag field: CB
Processing file: /tmp/RtmpqKK2Mr/file3ffbc95881f660/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 @ Fri May  8 00:06:51 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpqKK2Mr/file3ffbc95881f660/sampleA_matched_reads.fastq.gz ->/tmp/RtmpqKK2Mr/file3ffbc95881f660/sampleA_align2genome.bam
/tmp/RtmpqKK2Mr/file3ffbc95881f660/sample1_matched_reads.fastq.gz ->/tmp/RtmpqKK2Mr/file3ffbc95881f660/sample1_align2genome.bam
/tmp/RtmpqKK2Mr/file3ffbc95881f660/sample2_matched_reads.fastq.gz ->/tmp/RtmpqKK2Mr/file3ffbc95881f660/sample2_align2genome.bam
/tmp/RtmpqKK2Mr/file3ffbc95881f660/sample3_matched_reads.fastq.gz ->/tmp/RtmpqKK2Mr/file3ffbc95881f660/sample3_align2genome.bam
-- Running step: gene_quantification @ Fri May  8 00:07:13 2026 ----------------
00:07:13 Fri May 08 2026 quantify genes 
Using BAM(s): '/tmp/RtmpqKK2Mr/file3ffbc95881f660/sampleA_align2genome.bam',
'/tmp/RtmpqKK2Mr/file3ffbc95881f660/sample1_align2genome.bam',
'/tmp/RtmpqKK2Mr/file3ffbc95881f660/sample2_align2genome.bam', and
'/tmp/RtmpqKK2Mr/file3ffbc95881f660/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/RtmpqKK2Mr/file3ffbc95881f660/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]
/home/biocbuild/bbs-3.24-bioc/R/site-library/FLAMES/python/count_gene.py:704: 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, 149237.99Read/s]
parsing /tmp/RtmpqKK2Mr/file3ffbc95881f660/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 28.84gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 497639.41Read/s]
parsing /tmp/RtmpqKK2Mr/file3ffbc95881f660/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 29.39gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 531112.80Read/s]
parsing /tmp/RtmpqKK2Mr/file3ffbc95881f660/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 24.52gene_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, 396467.03Read/s]
-- Running step: isoform_identification @ Fri May  8 00:07:14 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 @ Fri May  8 00:07:37 2026 -------------------
Checking for fastq file(s) /tmp/RtmpqKK2Mr/file3ffbc95881f660/fastq, /tmp/RtmpqKK2Mr/file3ffbc95881f660/fastq/sample1.fq.gz, /tmp/RtmpqKK2Mr/file3ffbc95881f660/fastq/sample2.fq.gz, /tmp/RtmpqKK2Mr/file3ffbc95881f660/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpqKK2Mr/file3ffbc95881f660/sampleA_matched_reads.fastq.gz, /tmp/RtmpqKK2Mr/file3ffbc95881f660/sample1_matched_reads.fastq.gz, /tmp/RtmpqKK2Mr/file3ffbc95881f660/sample2_matched_reads.fastq.gz, /tmp/RtmpqKK2Mr/file3ffbc95881f660/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpqKK2Mr/file3ffbc95881f660/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpqKK2Mr/file3ffbc95881f660/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpqKK2Mr/file3ffbc95881f660/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpqKK2Mr/file3ffbc95881f660/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpqKK2Mr/file3ffbc95881f660/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpqKK2Mr/file3ffbc95881f660/sampleA_realign2transcript.bam
/tmp/RtmpqKK2Mr/file3ffbc95881f660/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpqKK2Mr/file3ffbc95881f660/sample1_realign2transcript.bam
/tmp/RtmpqKK2Mr/file3ffbc95881f660/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpqKK2Mr/file3ffbc95881f660/sample2_realign2transcript.bam
/tmp/RtmpqKK2Mr/file3ffbc95881f660/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpqKK2Mr/file3ffbc95881f660/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Fri May  8 00:07:57 2026 ----------
00:07:57 Fri May 08 2026 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpqKK2Mr/file3ffbc95881f660/sampleA_realign2transcript.bam...
parsing /tmp/RtmpqKK2Mr/file3ffbc95881f660/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpqKK2Mr/file3ffbc95881f660/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpqKK2Mr/file3ffbc95881f660/sample1_realign2transcript.bam...
parsing /tmp/RtmpqKK2Mr/file3ffbc95881f660/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpqKK2Mr/file3ffbc95881f660/sample1_realign2transcript.bamdone
parsing /tmp/RtmpqKK2Mr/file3ffbc95881f660/sample2_realign2transcript.bam...
parsing /tmp/RtmpqKK2Mr/file3ffbc95881f660/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpqKK2Mr/file3ffbc95881f660/sample2_realign2transcript.bamdone
parsing /tmp/RtmpqKK2Mr/file3ffbc95881f660/sample3_realign2transcript.bam...
parsing /tmp/RtmpqKK2Mr/file3ffbc95881f660/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpqKK2Mr/file3ffbc95881f660/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
i Writing configuration to: /tmp/RtmpqKK2Mr/file3ffbc9736cbb92/config_file_4193225.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 @ Fri May  8 00:07:59 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpqKK2Mr/file3ffbc9736cbb92/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
CB:Z: tag field: CB
Processing file: /tmp/RtmpqKK2Mr/file3ffbc9736cbb92/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpqKK2Mr/file3ffbc9736cbb92/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpqKK2Mr/file3ffbc9736cbb92/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/RtmpqKK2Mr/file3ffbc9736cbb92/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
CB:Z: tag field: CB
Processing file: /tmp/RtmpqKK2Mr/file3ffbc9736cbb92/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/RtmpqKK2Mr/file3ffbc9736cbb92/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
CB:Z: tag field: CB
Processing file: /tmp/RtmpqKK2Mr/file3ffbc9736cbb92/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/RtmpqKK2Mr/file3ffbc9736cbb92/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
CB:Z: tag field: CB
Processing file: /tmp/RtmpqKK2Mr/file3ffbc9736cbb92/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 @ Fri May  8 00:08:01 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpqKK2Mr/file3ffbc9736cbb92/sampleA_matched_reads.fastq.gz -> /tmp/RtmpqKK2Mr/file3ffbc9736cbb92/sampleA_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpqKK2Mr/file3ffbc9736cbb92/sample1_matched_reads.fastq.gz -> /tmp/RtmpqKK2Mr/file3ffbc9736cbb92/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpqKK2Mr/file3ffbc9736cbb92/sample2_matched_reads.fastq.gz -> /tmp/RtmpqKK2Mr/file3ffbc9736cbb92/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpqKK2Mr/file3ffbc9736cbb92/sample3_matched_reads.fastq.gz -> /tmp/RtmpqKK2Mr/file3ffbc9736cbb92/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Fri May  8 00:08:04 2026 ----------------
00:08:04 Fri May 08 2026 quantify genes 
Using BAM(s): '/tmp/RtmpqKK2Mr/file3ffbc9736cbb92/sampleA_align2genome.bam',
'/tmp/RtmpqKK2Mr/file3ffbc9736cbb92/sample1_align2genome.bam',
'/tmp/RtmpqKK2Mr/file3ffbc9736cbb92/sample2_align2genome.bam', and
'/tmp/RtmpqKK2Mr/file3ffbc9736cbb92/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/RtmpqKK2Mr/file3ffbc9736cbb92/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 14.60gene_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, 170032.92Read/s]
parsing /tmp/RtmpqKK2Mr/file3ffbc9736cbb92/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 20.78gene_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, 534442.41Read/s]
parsing /tmp/RtmpqKK2Mr/file3ffbc9736cbb92/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 28.06gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 560555.97Read/s]
parsing /tmp/RtmpqKK2Mr/file3ffbc9736cbb92/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 24.40gene_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, 419867.06Read/s]
-- Running step: isoform_identification @ Fri May  8 00:08:05 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri May  8 00:08:06 2026 -------------------
Checking for fastq file(s) /tmp/RtmpqKK2Mr/file3ffbc9736cbb92/fastq, /tmp/RtmpqKK2Mr/file3ffbc9736cbb92/fastq/sample1.fq.gz, /tmp/RtmpqKK2Mr/file3ffbc9736cbb92/fastq/sample2.fq.gz, /tmp/RtmpqKK2Mr/file3ffbc9736cbb92/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpqKK2Mr/file3ffbc9736cbb92/sampleA_matched_reads.fastq.gz, /tmp/RtmpqKK2Mr/file3ffbc9736cbb92/sample1_matched_reads.fastq.gz, /tmp/RtmpqKK2Mr/file3ffbc9736cbb92/sample2_matched_reads.fastq.gz, /tmp/RtmpqKK2Mr/file3ffbc9736cbb92/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpqKK2Mr/file3ffbc9736cbb92/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpqKK2Mr/file3ffbc9736cbb92/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpqKK2Mr/file3ffbc9736cbb92/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpqKK2Mr/file3ffbc9736cbb92/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpqKK2Mr/file3ffbc9736cbb92/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpqKK2Mr/file3ffbc9736cbb92/sampleA_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpqKK2Mr/file3ffbc9736cbb92/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpqKK2Mr/file3ffbc9736cbb92/sample1_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpqKK2Mr/file3ffbc9736cbb92/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpqKK2Mr/file3ffbc9736cbb92/sample2_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpqKK2Mr/file3ffbc9736cbb92/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpqKK2Mr/file3ffbc9736cbb92/sample3_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

-- Running step: transcript_quantification @ Fri May  8 00:08:13 2026 ----------
2026-05-08T04:08:13.767005Z  INFO oarfish: setting user-provided filter parameters.
2026-05-08T04:08:13.767527Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpqKK2Mr/file3ffbc9736cbb92/sampleA_realign2transcript.bam, contains 27 reference sequences.
2026-05-08T04:08:13.767542Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-08T04:08:13.767545Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-08T04:08:13.767674Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-08T04:08:13.767687Z  INFO oarfish: parsed reference information for 27 transcripts.
2026-05-08T04:08:13.811182Z  INFO oarfish::single_cell: Processed 100 cells.
2026-05-08T04:08:14.359821Z  INFO oarfish: setting user-provided filter parameters.
2026-05-08T04:08:14.360479Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpqKK2Mr/file3ffbc9736cbb92/sample1_realign2transcript.bam, contains 27 reference sequences.
2026-05-08T04:08:14.360492Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-08T04:08:14.360496Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-08T04:08:14.360616Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-08T04:08:14.360628Z  INFO oarfish: parsed reference information for 27 transcripts.
2026-05-08T04:08:14.376833Z  INFO oarfish::single_cell: Processed 100 cells.
2026-05-08T04:08:14.957661Z  INFO oarfish: setting user-provided filter parameters.
2026-05-08T04:08:14.958083Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpqKK2Mr/file3ffbc9736cbb92/sample2_realign2transcript.bam, contains 27 reference sequences.
2026-05-08T04:08:14.958092Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-08T04:08:14.958095Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-08T04:08:14.958210Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-08T04:08:14.958223Z  INFO oarfish: parsed reference information for 27 transcripts.
2026-05-08T04:08:14.974471Z  INFO oarfish::single_cell: Processed 100 cells.
2026-05-08T04:08:15.487764Z  INFO oarfish: setting user-provided filter parameters.
2026-05-08T04:08:15.488364Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpqKK2Mr/file3ffbc9736cbb92/sample3_realign2transcript.bam, contains 27 reference sequences.
2026-05-08T04:08:15.488386Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-08T04:08:15.488392Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-08T04:08:15.488513Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-08T04:08:15.488527Z  INFO oarfish: parsed reference information for 27 transcripts.
2026-05-08T04:08:15.506803Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
i Writing configuration to: /tmp/RtmpqKK2Mr/file3ffbc928febfe4/config_file_4193225.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 @ Fri May  8 00:08:16 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpqKK2Mr/file3ffbc928febfe4/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
CB:Z: tag field: CB
Processing file: /tmp/RtmpqKK2Mr/file3ffbc928febfe4/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpqKK2Mr/file3ffbc928febfe4/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpqKK2Mr/file3ffbc928febfe4/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/RtmpqKK2Mr/file3ffbc928febfe4/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
CB:Z: tag field: CB
Processing file: /tmp/RtmpqKK2Mr/file3ffbc928febfe4/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/RtmpqKK2Mr/file3ffbc928febfe4/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
CB:Z: tag field: CB
Processing file: /tmp/RtmpqKK2Mr/file3ffbc928febfe4/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/RtmpqKK2Mr/file3ffbc928febfe4/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
CB:Z: tag field: CB
Processing file: /tmp/RtmpqKK2Mr/file3ffbc928febfe4/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 @ Fri May  8 00:08:17 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpqKK2Mr/file3ffbc928febfe4/sampleA_matched_reads.fastq.gz ->/tmp/RtmpqKK2Mr/file3ffbc928febfe4/sampleA_align2genome.bam
/tmp/RtmpqKK2Mr/file3ffbc928febfe4/sample1_matched_reads.fastq.gz ->/tmp/RtmpqKK2Mr/file3ffbc928febfe4/sample1_align2genome.bam
/tmp/RtmpqKK2Mr/file3ffbc928febfe4/sample2_matched_reads.fastq.gz ->/tmp/RtmpqKK2Mr/file3ffbc928febfe4/sample2_align2genome.bam
/tmp/RtmpqKK2Mr/file3ffbc928febfe4/sample3_matched_reads.fastq.gz ->/tmp/RtmpqKK2Mr/file3ffbc928febfe4/sample3_align2genome.bam
-- Running step: gene_quantification @ Fri May  8 00:08:42 2026 ----------------
00:08:42 Fri May 08 2026 quantify genes 
Using BAM(s): '/tmp/RtmpqKK2Mr/file3ffbc928febfe4/sampleA_align2genome.bam',
'/tmp/RtmpqKK2Mr/file3ffbc928febfe4/sample1_align2genome.bam',
'/tmp/RtmpqKK2Mr/file3ffbc928febfe4/sample2_align2genome.bam', and
'/tmp/RtmpqKK2Mr/file3ffbc928febfe4/sample3_align2genome.bam'
parsing /tmp/RtmpqKK2Mr/file3ffbc928febfe4/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 13.73gene_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, 134157.63Read/s]
parsing /tmp/RtmpqKK2Mr/file3ffbc928febfe4/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 19.76gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 527930.72Read/s]
parsing /tmp/RtmpqKK2Mr/file3ffbc928febfe4/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 27.36gene_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, 442979.17Read/s]
parsing /tmp/RtmpqKK2Mr/file3ffbc928febfe4/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 23.39gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 379272.98Read/s]
-- Running step: isoform_identification @ Fri May  8 00:08:43 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri May  8 00:08:44 2026 -------------------
Checking for fastq file(s) /tmp/RtmpqKK2Mr/file3ffbc928febfe4/fastq, /tmp/RtmpqKK2Mr/file3ffbc928febfe4/fastq/sample1.fq.gz, /tmp/RtmpqKK2Mr/file3ffbc928febfe4/fastq/sample2.fq.gz, /tmp/RtmpqKK2Mr/file3ffbc928febfe4/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpqKK2Mr/file3ffbc928febfe4/sampleA_matched_reads.fastq.gz, /tmp/RtmpqKK2Mr/file3ffbc928febfe4/sample1_matched_reads.fastq.gz, /tmp/RtmpqKK2Mr/file3ffbc928febfe4/sample2_matched_reads.fastq.gz, /tmp/RtmpqKK2Mr/file3ffbc928febfe4/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpqKK2Mr/file3ffbc928febfe4/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpqKK2Mr/file3ffbc928febfe4/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpqKK2Mr/file3ffbc928febfe4/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpqKK2Mr/file3ffbc928febfe4/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpqKK2Mr/file3ffbc928febfe4/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpqKK2Mr/file3ffbc928febfe4/sampleA_realign2transcript.bam
/tmp/RtmpqKK2Mr/file3ffbc928febfe4/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpqKK2Mr/file3ffbc928febfe4/sample1_realign2transcript.bam
/tmp/RtmpqKK2Mr/file3ffbc928febfe4/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpqKK2Mr/file3ffbc928febfe4/sample2_realign2transcript.bam
/tmp/RtmpqKK2Mr/file3ffbc928febfe4/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpqKK2Mr/file3ffbc928febfe4/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Fri May  8 00:09:09 2026 ----------
2026-05-08T04:09:09.943856Z  INFO oarfish: setting user-provided filter parameters.
2026-05-08T04:09:09.944345Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpqKK2Mr/file3ffbc928febfe4/sampleA_realign2transcript.bam, contains 28 reference sequences.
2026-05-08T04:09:09.944356Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-08T04:09:09.944369Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-08T04:09:09.944497Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-08T04:09:09.944510Z  INFO oarfish: parsed reference information for 28 transcripts.
2026-05-08T04:09:09.986505Z  INFO oarfish::single_cell: Processed 100 cells.
2026-05-08T04:09:10.663948Z  INFO oarfish: setting user-provided filter parameters.
2026-05-08T04:09:10.664341Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpqKK2Mr/file3ffbc928febfe4/sample1_realign2transcript.bam, contains 28 reference sequences.
2026-05-08T04:09:10.664350Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-08T04:09:10.664353Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-08T04:09:10.664472Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-08T04:09:10.664488Z  INFO oarfish: parsed reference information for 28 transcripts.
2026-05-08T04:09:10.679871Z  INFO oarfish::single_cell: Processed 100 cells.
2026-05-08T04:09:11.280082Z  INFO oarfish: setting user-provided filter parameters.
2026-05-08T04:09:11.280494Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpqKK2Mr/file3ffbc928febfe4/sample2_realign2transcript.bam, contains 28 reference sequences.
2026-05-08T04:09:11.280506Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-08T04:09:11.280510Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-08T04:09:11.280634Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-08T04:09:11.280647Z  INFO oarfish: parsed reference information for 28 transcripts.
2026-05-08T04:09:11.296654Z  INFO oarfish::single_cell: Processed 100 cells.
2026-05-08T04:09:11.841762Z  INFO oarfish: setting user-provided filter parameters.
2026-05-08T04:09:11.842250Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpqKK2Mr/file3ffbc928febfe4/sample3_realign2transcript.bam, contains 28 reference sequences.
2026-05-08T04:09:11.842259Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-08T04:09:11.842262Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-08T04:09:11.842386Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-08T04:09:11.842404Z  INFO oarfish: parsed reference information for 28 transcripts.
2026-05-08T04:09:11.862189Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
i Writing configuration to: /tmp/RtmpqKK2Mr/file3ffbc96e0aab4b/config_file_4193225.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 @ Fri May  8 00:09:12 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpqKK2Mr/file3ffbc96e0aab4b/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
CB:Z: tag field: CB
Processing file: /tmp/RtmpqKK2Mr/file3ffbc96e0aab4b/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpqKK2Mr/file3ffbc96e0aab4b/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpqKK2Mr/file3ffbc96e0aab4b/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/RtmpqKK2Mr/file3ffbc96e0aab4b/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
CB:Z: tag field: CB
Processing file: /tmp/RtmpqKK2Mr/file3ffbc96e0aab4b/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/RtmpqKK2Mr/file3ffbc96e0aab4b/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
CB:Z: tag field: CB
Processing file: /tmp/RtmpqKK2Mr/file3ffbc96e0aab4b/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/RtmpqKK2Mr/file3ffbc96e0aab4b/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
CB:Z: tag field: CB
Processing file: /tmp/RtmpqKK2Mr/file3ffbc96e0aab4b/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 @ Fri May  8 00:09:13 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpqKK2Mr/file3ffbc96e0aab4b/sampleA_matched_reads.fastq.gz -> /tmp/RtmpqKK2Mr/file3ffbc96e0aab4b/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/RtmpqKK2Mr/file3ffbc96e0aab4b/sample1_matched_reads.fastq.gz -> /tmp/RtmpqKK2Mr/file3ffbc96e0aab4b/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/RtmpqKK2Mr/file3ffbc96e0aab4b/sample2_matched_reads.fastq.gz -> /tmp/RtmpqKK2Mr/file3ffbc96e0aab4b/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/RtmpqKK2Mr/file3ffbc96e0aab4b/sample3_matched_reads.fastq.gz -> /tmp/RtmpqKK2Mr/file3ffbc96e0aab4b/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 @ Fri May  8 00:09:17 2026 ----------------
00:09:17 Fri May 08 2026 quantify genes 
Using BAM(s): '/tmp/RtmpqKK2Mr/file3ffbc96e0aab4b/sampleA_align2genome.bam',
'/tmp/RtmpqKK2Mr/file3ffbc96e0aab4b/sample1_align2genome.bam',
'/tmp/RtmpqKK2Mr/file3ffbc96e0aab4b/sample2_align2genome.bam', and
'/tmp/RtmpqKK2Mr/file3ffbc96e0aab4b/sample3_align2genome.bam'
parsing /tmp/RtmpqKK2Mr/file3ffbc96e0aab4b/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 14.91gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 156380.18Read/s]
parsing /tmp/RtmpqKK2Mr/file3ffbc96e0aab4b/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  6.21gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  6.20gene_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, 533925.35Read/s]
parsing /tmp/RtmpqKK2Mr/file3ffbc96e0aab4b/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 29.28gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 476495.50Read/s]
parsing /tmp/RtmpqKK2Mr/file3ffbc96e0aab4b/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 21.04gene_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, 378246.88Read/s]
-- Running step: isoform_identification @ Fri May  8 00:09:18 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri May  8 00:09:19 2026 -------------------
Checking for fastq file(s) /tmp/RtmpqKK2Mr/file3ffbc96e0aab4b/fastq, /tmp/RtmpqKK2Mr/file3ffbc96e0aab4b/fastq/sample1.fq.gz, /tmp/RtmpqKK2Mr/file3ffbc96e0aab4b/fastq/sample2.fq.gz, /tmp/RtmpqKK2Mr/file3ffbc96e0aab4b/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpqKK2Mr/file3ffbc96e0aab4b/sampleA_matched_reads.fastq.gz, /tmp/RtmpqKK2Mr/file3ffbc96e0aab4b/sample1_matched_reads.fastq.gz, /tmp/RtmpqKK2Mr/file3ffbc96e0aab4b/sample2_matched_reads.fastq.gz, /tmp/RtmpqKK2Mr/file3ffbc96e0aab4b/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpqKK2Mr/file3ffbc96e0aab4b/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpqKK2Mr/file3ffbc96e0aab4b/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpqKK2Mr/file3ffbc96e0aab4b/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpqKK2Mr/file3ffbc96e0aab4b/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpqKK2Mr/file3ffbc96e0aab4b/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpqKK2Mr/file3ffbc96e0aab4b/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/RtmpqKK2Mr/file3ffbc96e0aab4b/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpqKK2Mr/file3ffbc96e0aab4b/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/RtmpqKK2Mr/file3ffbc96e0aab4b/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpqKK2Mr/file3ffbc96e0aab4b/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/RtmpqKK2Mr/file3ffbc96e0aab4b/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpqKK2Mr/file3ffbc96e0aab4b/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 @ Fri May  8 00:09:22 2026 ----------
00:09:22 Fri May 08 2026 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpqKK2Mr/file3ffbc96e0aab4b/sampleA_realign2transcript.bam...
parsing /tmp/RtmpqKK2Mr/file3ffbc96e0aab4b/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpqKK2Mr/file3ffbc96e0aab4b/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpqKK2Mr/file3ffbc96e0aab4b/sample1_realign2transcript.bam...
parsing /tmp/RtmpqKK2Mr/file3ffbc96e0aab4b/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpqKK2Mr/file3ffbc96e0aab4b/sample1_realign2transcript.bamdone
parsing /tmp/RtmpqKK2Mr/file3ffbc96e0aab4b/sample2_realign2transcript.bam...
parsing /tmp/RtmpqKK2Mr/file3ffbc96e0aab4b/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpqKK2Mr/file3ffbc96e0aab4b/sample2_realign2transcript.bamdone
parsing /tmp/RtmpqKK2Mr/file3ffbc96e0aab4b/sample3_realign2transcript.bam...
parsing /tmp/RtmpqKK2Mr/file3ffbc96e0aab4b/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpqKK2Mr/file3ffbc96e0aab4b/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
i Writing configuration to: /tmp/RtmpqKK2Mr/file3ffbc94709d26a/config_file_4193225.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 @ Fri May  8 00:09:25 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpqKK2Mr/file3ffbc94709d26a/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
CB:Z: tag field: CB
Processing file: /tmp/RtmpqKK2Mr/file3ffbc94709d26a/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpqKK2Mr/file3ffbc94709d26a/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpqKK2Mr/file3ffbc94709d26a/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/RtmpqKK2Mr/file3ffbc94709d26a/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
CB:Z: tag field: CB
Processing file: /tmp/RtmpqKK2Mr/file3ffbc94709d26a/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/RtmpqKK2Mr/file3ffbc94709d26a/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
CB:Z: tag field: CB
Processing file: /tmp/RtmpqKK2Mr/file3ffbc94709d26a/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/RtmpqKK2Mr/file3ffbc94709d26a/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
CB:Z: tag field: CB
Processing file: /tmp/RtmpqKK2Mr/file3ffbc94709d26a/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 @ Fri May  8 00:09:26 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpqKK2Mr/file3ffbc94709d26a/sampleA_matched_reads.fastq.gz ->/tmp/RtmpqKK2Mr/file3ffbc94709d26a/sampleA_align2genome.bam
/tmp/RtmpqKK2Mr/file3ffbc94709d26a/sample1_matched_reads.fastq.gz ->/tmp/RtmpqKK2Mr/file3ffbc94709d26a/sample1_align2genome.bam
/tmp/RtmpqKK2Mr/file3ffbc94709d26a/sample2_matched_reads.fastq.gz ->/tmp/RtmpqKK2Mr/file3ffbc94709d26a/sample2_align2genome.bam
/tmp/RtmpqKK2Mr/file3ffbc94709d26a/sample3_matched_reads.fastq.gz ->/tmp/RtmpqKK2Mr/file3ffbc94709d26a/sample3_align2genome.bam
-- Running step: gene_quantification @ Fri May  8 00:09:51 2026 ----------------
00:09:51 Fri May 08 2026 quantify genes 
Using BAM(s): '/tmp/RtmpqKK2Mr/file3ffbc94709d26a/sampleA_align2genome.bam',
'/tmp/RtmpqKK2Mr/file3ffbc94709d26a/sample1_align2genome.bam',
'/tmp/RtmpqKK2Mr/file3ffbc94709d26a/sample2_align2genome.bam', and
'/tmp/RtmpqKK2Mr/file3ffbc94709d26a/sample3_align2genome.bam'
	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})
parsing /tmp/RtmpqKK2Mr/file3ffbc94709d26a/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 13.89gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 144790.94Read/s]
parsing /tmp/RtmpqKK2Mr/file3ffbc94709d26a/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 29.15gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 502480.35Read/s]
parsing /tmp/RtmpqKK2Mr/file3ffbc94709d26a/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 27.43gene_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, 524366.65Read/s]
parsing /tmp/RtmpqKK2Mr/file3ffbc94709d26a/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 21.18gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 413574.19Read/s]
-- Running step: isoform_identification @ Fri May  8 00:09:52 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri May  8 00:09:52 2026 -------------------
Checking for fastq file(s) /tmp/RtmpqKK2Mr/file3ffbc94709d26a/fastq, /tmp/RtmpqKK2Mr/file3ffbc94709d26a/fastq/sample1.fq.gz, /tmp/RtmpqKK2Mr/file3ffbc94709d26a/fastq/sample2.fq.gz, /tmp/RtmpqKK2Mr/file3ffbc94709d26a/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpqKK2Mr/file3ffbc94709d26a/sampleA_matched_reads.fastq.gz, /tmp/RtmpqKK2Mr/file3ffbc94709d26a/sample1_matched_reads.fastq.gz, /tmp/RtmpqKK2Mr/file3ffbc94709d26a/sample2_matched_reads.fastq.gz, /tmp/RtmpqKK2Mr/file3ffbc94709d26a/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpqKK2Mr/file3ffbc94709d26a/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpqKK2Mr/file3ffbc94709d26a/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpqKK2Mr/file3ffbc94709d26a/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpqKK2Mr/file3ffbc94709d26a/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpqKK2Mr/file3ffbc94709d26a/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpqKK2Mr/file3ffbc94709d26a/sampleA_realign2transcript.bam
/tmp/RtmpqKK2Mr/file3ffbc94709d26a/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpqKK2Mr/file3ffbc94709d26a/sample1_realign2transcript.bam
/tmp/RtmpqKK2Mr/file3ffbc94709d26a/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpqKK2Mr/file3ffbc94709d26a/sample2_realign2transcript.bam
/tmp/RtmpqKK2Mr/file3ffbc94709d26a/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpqKK2Mr/file3ffbc94709d26a/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Fri May  8 00:10:15 2026 ----------
00:10:15 Fri May 08 2026 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpqKK2Mr/file3ffbc94709d26a/sampleA_realign2transcript.bam...
parsing /tmp/RtmpqKK2Mr/file3ffbc94709d26a/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpqKK2Mr/file3ffbc94709d26a/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpqKK2Mr/file3ffbc94709d26a/sample1_realign2transcript.bam...
parsing /tmp/RtmpqKK2Mr/file3ffbc94709d26a/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpqKK2Mr/file3ffbc94709d26a/sample1_realign2transcript.bamdone
parsing /tmp/RtmpqKK2Mr/file3ffbc94709d26a/sample2_realign2transcript.bam...
parsing /tmp/RtmpqKK2Mr/file3ffbc94709d26a/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpqKK2Mr/file3ffbc94709d26a/sample2_realign2transcript.bamdone
parsing /tmp/RtmpqKK2Mr/file3ffbc94709d26a/sample3_realign2transcript.bam...
parsing /tmp/RtmpqKK2Mr/file3ffbc94709d26a/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpqKK2Mr/file3ffbc94709d26a/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 ...

  |                                                                            
  |                                                                      |   0%
  |                                                                            
  |=======================                                               |  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 182 | SKIP 0 | PASS 98 ]

[ FAIL 0 | WARN 182 | 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 
815.581  55.919 855.931