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###
### Running command:
###
###   /home/biocbuild/bbs-3.22-bioc/R/bin/R CMD check --install=check:FLAMES.install-out.txt --library=/home/biocbuild/bbs-3.22-bioc/R/site-library --timings FLAMES_2.3.5.tar.gz
###
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* using log directory ‘/home/biocbuild/bbs-3.22-bioc/meat/FLAMES.Rcheck’
* using R version 4.5.1 (2025-06-13)
* using platform: x86_64-pc-linux-gnu
* R was compiled by
    gcc (Ubuntu 13.3.0-6ubuntu2~24.04) 13.3.0
    GNU Fortran (Ubuntu 13.3.0-6ubuntu2~24.04) 13.3.0
* running under: Ubuntu 24.04.3 LTS
* using session charset: UTF-8
* checking for file ‘FLAMES/DESCRIPTION’ ... OK
* this is package ‘FLAMES’ version ‘2.3.5’
* package encoding: UTF-8
* checking package namespace information ... OK
* checking package dependencies ... INFO
Imports includes 47 non-default packages.
Importing from so many packages makes the package vulnerable to any of
them becoming unavailable.  Move as many as possible to Suggests and
use conditionally.
* checking if this is a source package ... OK
* checking if there is a namespace ... OK
* checking for hidden files and directories ... NOTE
Found the following hidden files and directories:
  .dockerignore
These were most likely included in error. See section ‘Package
structure’ in the ‘Writing R Extensions’ manual.
* checking for portable file names ... OK
* checking for sufficient/correct file permissions ... OK
* checking whether package ‘FLAMES’ can be installed ... NOTE
Found the following notes/warnings:
  Non-staged installation was used
See ‘/home/biocbuild/bbs-3.22-bioc/meat/FLAMES.Rcheck/00install.out’ for details.
* used C++ compiler: ‘g++ (Ubuntu 13.3.0-6ubuntu2~24.04) 13.3.0’
* checking C++ specification ... OK
* checking installed package size ... INFO
  installed size is  6.0Mb
  sub-directories of 1Mb or more:
    bin    1.1Mb
    data   1.8Mb
    libs   1.5Mb
* checking package directory ... OK
* checking ‘build’ directory ... OK
* checking DESCRIPTION meta-information ... OK
* checking top-level files ... OK
* checking for left-over files ... OK
* checking index information ... OK
* checking package subdirectories ... OK
* checking code files for non-ASCII characters ... OK
* checking R files for syntax errors ... OK
* checking whether the package can be loaded ... OK
* checking whether the package can be loaded with stated dependencies ... OK
* checking whether the package can be unloaded cleanly ... OK
* checking whether the namespace can be loaded with stated dependencies ... OK
* checking whether the namespace can be unloaded cleanly ... OK
* checking loading without being on the library search path ... OK
* checking dependencies in R code ... NOTE
There are ::: calls to the package's namespace in its code. A package
  almost never needs to use ::: for its own objects:
  'blaze' 'find_barcode' 'gene_quantification' 'isoform_identification'
  'minimap2_align' 'transcript_quantification'
* checking S3 generic/method consistency ... OK
* checking replacement functions ... OK
* checking foreign function calls ... OK
* checking R code for possible problems ... NOTE
BulkPipeline: no visible global function definition for ‘new’
BulkPipeline: no visible global function definition for ‘setNames’
MultiSampleSCPipeline: no visible global function definition for ‘new’
MultiSampleSCPipeline: no visible global function definition for
  ‘setNames’
SingleCellPipeline: no visible global function definition for ‘new’
SingleCellPipeline: no visible global function definition for
  ‘setNames’
addRowRanges: no visible global function definition for ‘head’
addRowRanges: no visible global function definition for ‘as’
add_gene_counts: no visible global function definition for ‘as’
cache_dir: no visible global function definition for ‘packageVersion’
chisq_test_by_gene: no visible global function definition for
  ‘chisq.test’
create_sce_from_dir: no visible global function definition for
  ‘setNames’
create_spe: no visible binding for global variable ‘barcode’
create_spe: no visible binding for global variable ‘in_tissue’
download_oarfish: no visible global function definition for
  ‘download.file’
download_oarfish: no visible global function definition for ‘unzip’
filter_coverage: no visible global function definition for
  ‘starts_with’
filter_coverage: no visible binding for global variable ‘filter_res’
find_variants: no visible global function definition for ‘setNames’
find_variants_grange: no visible binding for global variable
  ‘which_label’
find_variants_grange: no visible binding for global variable
  ‘nucleotide’
find_variants_grange: no visible binding for global variable ‘pos’
find_variants_grange: no visible binding for global variable ‘count’
find_variants_grange: no visible binding for global variable
  ‘counts_no_ins’
find_variants_grange: no visible binding for global variable ‘ref’
generate_sc_sce: no visible binding for global variable ‘FSM_match’
get_coverage: no visible binding for global variable ‘Freq’
homopolymer_pct : <anonymous>: no visible binding for global variable
  ‘Freq’
homopolymer_pct : <anonymous>: no visible binding for global variable
  ‘pct’
plot_coverage: no visible binding for global variable ‘tr_length’
plot_coverage: no visible binding for global variable ‘read_counts’
plot_coverage: no visible binding for global variable ‘total_counts’
plot_coverage: no visible binding for global variable ‘cumpct’
plot_coverage: no visible binding for global variable ‘length_bin’
plot_coverage: no visible binding for global variable ‘min_length’
plot_coverage: no visible binding for global variable ‘max_length’
plot_coverage: no visible global function definition for ‘head’
plot_coverage: no visible binding for global variable ‘transcript’
plot_demultiplex_raw: no visible binding for global variable ‘Sample’
plot_demultiplex_raw: no visible binding for global variable
  ‘CellBarcode’
plot_demultiplex_raw: no visible binding for global variable ‘UMI’
plot_demultiplex_raw: no visible binding for global variable
  ‘UMI_count’
plot_demultiplex_raw: no visible binding for global variable
  ‘barcode_rank’
plot_demultiplex_raw: no visible binding for global variable
  ‘FlankEditDist’
plot_demultiplex_raw: no visible binding for global variable ‘n_reads’
plot_demultiplex_raw: no visible binding for global variable
  ‘BarcodeEditDist’
plot_demultiplex_raw: no visible binding for global variable ‘total
  reads’
plot_demultiplex_raw: no visible binding for global variable
  ‘demultiplexed reads’
plot_demultiplex_raw: no visible binding for global variable ‘single
  match reads’
plot_demultiplex_raw: no visible binding for global variable
  ‘undemultiplexted reads’
plot_demultiplex_raw: no visible binding for global variable
  ‘multi-matching reads’
plot_demultiplex_raw: no visible binding for global variable ‘Type’
plot_demultiplex_raw: no visible binding for global variable ‘Reads’
plot_demultiplex_raw: no visible binding for global variable ‘input’
plot_demultiplex_raw: no visible binding for global variable ‘output’
plot_demultiplex_raw: no visible binding for global variable
  ‘read1_with_adapter’
plot_demultiplex_raw: no visible binding for global variable ‘Count’
plot_flagstat: no visible global function definition for ‘everything’
plot_flagstat: no visible binding for global variable ‘name’
plot_flagstat: no visible binding for global variable ‘value’
plot_isoform_reduced_dim: no visible binding for global variable ‘x’
plot_isoform_reduced_dim: no visible binding for global variable ‘y’
plot_isoform_reduced_dim: no visible binding for global variable ‘expr’
plot_spatial: no visible binding for global variable ‘imageX’
plot_spatial: no visible binding for global variable ‘imageY’
plot_spatial_feature: no visible binding for global variable ‘imageX’
plot_spatial_feature: no visible binding for global variable ‘imageY’
plot_spatial_feature: no visible binding for global variable ‘x’
plot_spatial_feature: no visible binding for global variable ‘y’
plot_spatial_feature: no visible global function definition for
  ‘scale_alpha_continuous’
plot_spatial_feature: no visible global function definition for
  ‘scale_colour_gradient’
plot_spatial_isoform: no visible global function definition for ‘head’
plot_spatial_pie: no visible global function definition for ‘setNames’
plot_spatial_pie: no visible global function definition for ‘head’
plot_spatial_pie: no visible binding for global variable ‘imageX’
plot_spatial_pie: no visible binding for global variable ‘imageY’
sc_gene_entropy: no visible global function definition for ‘as’
sc_genotype: no visible binding for global variable ‘allele’
sc_genotype: no visible binding for global variable ‘allele_count’
sc_genotype: no visible binding for global variable ‘barcode’
sc_genotype: no visible binding for global variable ‘pct’
sc_mutations: no visible binding for global variable ‘mutation_index’
sc_mutations: no visible binding for global variable ‘bam_index’
sc_plot_genotype: no visible global function definition for ‘setNames’
sc_plot_genotype: no visible binding for global variable ‘barcode’
sc_plot_genotype: no visible binding for global variable ‘genotype’
sc_plot_genotype: no visible binding for global variable ‘x’
sc_plot_genotype: no visible binding for global variable ‘y’
sc_transcript_usage_chisq: no visible global function definition for
  ‘as’
sc_transcript_usage_chisq: no visible binding for global variable
  ‘p.value’
sc_transcript_usage_chisq: no visible binding for global variable
  ‘adj.p.value’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘total’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘test’
sc_transcript_usage_permutation: no visible global function definition
  for ‘as’
sc_transcript_usage_permutation : <anonymous>: no visible global
  function definition for ‘as’
sc_transcript_usage_permutation : <anonymous> : <anonymous>: no visible
  global function definition for ‘na.omit’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘transcript’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘p.value’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘adj.p.value’
variant_count_tb: no visible binding for global variable ‘barcode’
variant_count_tb: no visible binding for global variable ‘allele_count’
variant_count_tb: no visible binding for global variable
  ‘cell_total_reads’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘expect_cell_number’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘fastq’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘demultiplexed_fastq’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘barcodes_file’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘outdir’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible global
  function definition for ‘setNames’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline : <anonymous>: no
  visible global function definition for ‘setNames’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘expect_cell_number’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘fastq’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘demultiplexed_fastq’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘barcodes_file’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘outdir’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible global
  function definition for ‘setNames’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘j’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘genome_bam’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘minimap2’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘samtools’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘threads’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘outdir’
plot_durations,FLAMES.Pipeline: no visible binding for global variable
  ‘step’
plot_durations,FLAMES.Pipeline: no visible binding for global variable
  ‘duration’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘j’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘transcriptome_assembly’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘transcriptome_bam’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘minimap2’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘samtools’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘outdir’
resume_FLAMES,FLAMES.Pipeline : <anonymous>: no visible global function
  definition for ‘capture.output’
run_FLAMES,FLAMES.Pipeline : <anonymous>: no visible global function
  definition for ‘capture.output’
Undefined global functions or variables:
  BarcodeEditDist CellBarcode Count FSM_match FlankEditDist Freq Reads
  Sample Type UMI UMI_count adj.p.value allele allele_count as
  bam_index barcode barcode_rank barcodes_file capture.output
  cell_total_reads chisq.test count counts_no_ins cumpct demultiplexed
  reads demultiplexed_fastq download.file duration everything
  expect_cell_number expr fastq filter_res genome_bam genotype head
  imageX imageY in_tissue input j length_bin max_length min_length
  minimap2 multi-matching reads mutation_index n_reads na.omit name new
  nucleotide outdir output p.value packageVersion pct pos
  read1_with_adapter read_counts ref samtools scale_alpha_continuous
  scale_colour_gradient setNames single match reads starts_with step
  test threads total total reads total_counts tr_length transcript
  transcriptome_assembly transcriptome_bam undemultiplexted reads unzip
  value which_label x y
Consider adding
  importFrom("base", "match", "single")
  importFrom("methods", "as", "new")
  importFrom("stats", "chisq.test", "na.omit", "setNames", "step")
  importFrom("utils", "capture.output", "download.file", "head",
             "packageVersion", "unzip")
to your NAMESPACE file (and ensure that your DESCRIPTION Imports field
contains 'methods').
* checking Rd files ... OK
* checking Rd metadata ... OK
* checking Rd cross-references ... OK
* checking for missing documentation entries ... OK
* checking for code/documentation mismatches ... OK
* checking Rd \usage sections ... OK
* checking Rd contents ... OK
* checking for unstated dependencies in examples ... OK
* checking contents of ‘data’ directory ... OK
* checking data for non-ASCII characters ... OK
* checking data for ASCII and uncompressed saves ... OK
* checking line endings in shell scripts ... OK
* checking line endings in C/C++/Fortran sources/headers ... OK
* checking line endings in Makefiles ... OK
* checking compilation flags in Makevars ... OK
* checking for GNU extensions in Makefiles ... INFO
GNU make is a SystemRequirements.
* checking for portable use of $(BLAS_LIBS) and $(LAPACK_LIBS) ... OK
* checking use of PKG_*FLAGS in Makefiles ... OK
* checking include directives in Makefiles ... OK
* checking compiled code ... NOTE
Note: information on .o files is not available
File ‘/home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/libs/FLAMES.so’:
  Found ‘abort’, possibly from ‘abort’ (C)
  Found ‘exit’, possibly from ‘exit’ (C)
  Found ‘stderr’, possibly from ‘stderr’ (C)
  Found ‘stdout’, possibly from ‘stdout’ (C)

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

See ‘Writing portable packages’ in the ‘Writing R Extensions’ manual.
* checking files in ‘vignettes’ ... OK
* checking examples ... OK
Examples with CPU (user + system) or elapsed time > 5s
                               user system elapsed
plot_isoform_reduced_dim     23.329  0.158  23.489
find_variants                21.854  0.246  21.503
blaze                         4.326 17.556  12.612
sc_long_multisample_pipeline  8.540  7.730   9.127
bulk_long_pipeline            2.347 12.957   2.566
sc_plot_genotype             10.540  0.492   9.882
MultiSampleSCPipeline        10.280  0.692  11.431
sc_DTU_analysis               7.272  2.262   7.200
plot_isoform_heatmap          7.058  0.105   7.163
create_sce_from_dir           3.602  2.704   3.928
* checking for unstated dependencies in ‘tests’ ... OK
* checking tests ...
  Running ‘testthat.R’
 OK
* checking for unstated dependencies in vignettes ... OK
* checking package vignettes ... OK
* checking re-building of vignette outputs ... OK
* checking PDF version of manual ... OK
* DONE

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

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###
### Running command:
###
###   /home/biocbuild/bbs-3.22-bioc/R/bin/R CMD INSTALL FLAMES
###
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* installing to library ‘/home/biocbuild/bbs-3.22-bioc/R/site-library’
* installing *source* package ‘FLAMES’ ...
** this is package ‘FLAMES’ version ‘2.3.5’
** using non-staged installation via StagedInstall field
** libs
using C++ compiler: ‘g++ (Ubuntu 13.3.0-6ubuntu2~24.04) 13.3.0’
using C++17
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c RcppExports.cpp -o RcppExports.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c RcppFunctions.cpp -o RcppFunctions.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c classes/BamRecord.cpp -o classes/BamRecord.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c classes/GFFRecord.cpp -o classes/GFFRecord.o
In file included from classes/GFFRecord.cpp:8:
classes/../utility/utility.h: In function ‘std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > parseSpace(const std::string&)’:
classes/../utility/utility.h:255:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  255 |         for (int i = 0; i < s.size(); i++) {
      |                         ~~^~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c classes/GeneAnnotationParser.cpp -o classes/GeneAnnotationParser.o
In file included from classes/GeneAnnotationParser.cpp:15:
classes/../utility/utility.h: In function ‘std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > parseSpace(const std::string&)’:
classes/../utility/utility.h:255:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  255 |         for (int i = 0; i < s.size(); i++) {
      |                         ~~^~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c classes/Isoforms.cpp -o classes/Isoforms.o
In file included from classes/Isoforms.cpp:16:
classes/../utility/utility.h: In function ‘std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > parseSpace(const std::string&)’:
classes/../utility/utility.h:255:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  255 |         for (int i = 0; i < s.size(); i++) {
      |                         ~~^~~~~~~~~~
classes/Isoforms.cpp: In member function ‘void Isoforms::update_all_splice()’:
classes/Isoforms.cpp:233:35: warning: comparison of integer expressions of different signedness: ‘std::vector<StartEndPair>::size_type’ {aka ‘long unsigned int’} and ‘int’ [-Wsign-compare]
  233 |                 if (blocks.size() >= (int)this->Min_sup_cnt) {
      |                     ~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~~~~~~
classes/Isoforms.cpp: In member function ‘void Isoforms::filter_TSS_TES(std::ofstream&, DoubleJunctions, float)’:
classes/Isoforms.cpp:368:33: warning: comparison of integer expressions of different signedness: ‘std::unordered_map<int, int>::size_type’ {aka ‘long unsigned int’} and ‘int’ [-Wsign-compare]
  368 |         if ((left_counts.size() < (int)this->Min_sup_cnt) ||
      |              ~~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~~~~~
classes/Isoforms.cpp:369:38: warning: comparison of integer expressions of different signedness: ‘std::unordered_map<int, int>::size_type’ {aka ‘long unsigned int’} and ‘int’ [-Wsign-compare]
  369 |                 (right_counts.size() < (int)this->Min_sup_cnt)) {
      |                  ~~~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~~~~~
classes/Isoforms.cpp: In member function ‘void Isoforms::match_known_annotation(const std::unordered_map<std::__cxx11::basic_string<char>, Junctions>&, const std::unordered_map<std::__cxx11::basic_string<char>, Pos>&, const std::unordered_map<std::__cxx11::basic_string<char>, std::vector<StartEndPair> >&, GeneBlocks, std::unordered_map<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> >)’:
classes/Isoforms.cpp:662:51: warning: comparison of integer expressions of different signedness: ‘int’ and ‘const long unsigned int’ [-Wsign-compare]
  662 |                                 for (int j = 0; j < std::min(raw_iso_key.size() - 1, junction.junctions.size()); j++) {
      |                                                 ~~^~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
classes/Isoforms.cpp:713:43: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<int>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  713 |                         for (int i = 0; i < new_exons.size(); ++i) {
      |                                         ~~^~~~~~~~~~~~~~~~~~
classes/Isoforms.cpp:725:46: warning: comparisons like ‘X<=Y<=Z’ do not have their mathematical meaning [-Wparentheses]
  725 |                                 } else if (0 < i < raw_iso_key.size() - 1) { // a site from the middle somewhere
      |                                            ~~^~~
classes/Isoforms.cpp: In member function ‘std::string Isoforms::isoform_to_gff3(float)’:
classes/Isoforms.cpp:1140:43: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<int>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
 1140 |                         for (int i = 0; i < exons.size(); i+=2) {
      |                                         ~~^~~~~~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c classes/junctions.cpp -o classes/junctions.o
In file included from classes/junctions.cpp:12:
classes/../utility/utility.h: In function ‘std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > parseSpace(const std::string&)’:
classes/../utility/utility.h:255:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  255 |         for (int i = 0; i < s.size(); i++) {
      |                         ~~^~~~~~~~~~
classes/junctions.cpp: In function ‘std::unordered_map<std::__cxx11::basic_string<char>, std::vector<StartEndPair> > get_gene_flat(const std::unordered_map<std::__cxx11::basic_string<char>, std::vector<std::__cxx11::basic_string<char> > >&, const std::unordered_map<std::__cxx11::basic_string<char>, std::vector<StartEndPair> >&)’:
classes/junctions.cpp:166:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<StartEndPair>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  166 |         for (int i = 1; i < exons.size(); i++) {
      |                         ~~^~~~~~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c main-functions/find_isoform.cpp -o main-functions/find_isoform.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c main-functions/flexiplex.cpp -o main-functions/flexiplex.o
main-functions/flexiplex.cpp: In function ‘unsigned int edit_distance(const std::string&, const std::string&, unsigned int&, int)’:
main-functions/flexiplex.cpp:123:21: warning: comparison of integer expressions of different signedness: ‘unsigned int’ and ‘int’ [-Wsign-compare]
  123 |       if (min_value <= max_editd)
      |           ~~~~~~~~~~^~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘std::string get_umi(const std::string&, const std::vector<std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > >&, const std::vector<int>&, int, int, bool, int, int)’:
main-functions/flexiplex.cpp:164:22: warning: comparison of integer expressions of different signedness: ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} and ‘int’ [-Wsign-compare]
  164 |     if (seq.length() < umi_start + umi_length) {
      |         ~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:186:36: warning: comparison of integer expressions of different signedness: ‘std::vector<int>::size_type’ {aka ‘long unsigned int’} and ‘int’ [-Wsign-compare]
  186 |     if (read_to_subpatterns.size() > umi_index + 1) {
      |         ~~~~~~~~~~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘Barcode get_barcode(const std::string&, const std::unordered_set<std::__cxx11::basic_string<char> >&, int, int, const std::vector<std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > >&)’:
main-functions/flexiplex.cpp:299:19: warning: comparison of integer expressions of different signedness: ‘int’ and ‘__gnu_cxx::__alloc_traits<std::allocator<long unsigned int>, long unsigned int>::value_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  299 |     if (i_pattern >= subpattern_ends[i_subpattern]) {
main-functions/flexiplex.cpp:358:22: warning: comparison of integer expressions of different signedness: ‘unsigned int’ and ‘int’ [-Wsign-compare]
  358 |     if (editDistance == barcode.editd) {
      |         ~~~~~~~~~~~~~^~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:360:29: warning: comparison of integer expressions of different signedness: ‘unsigned int’ and ‘int’ [-Wsign-compare]
  360 |     } else if (editDistance < barcode.editd &&
      |                ~~~~~~~~~~~~~^~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:361:29: warning: comparison of integer expressions of different signedness: ‘unsigned int’ and ‘int’ [-Wsign-compare]
  361 |                editDistance <= barcode_max_editd) { // if best so far, update
      |                ~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘std::vector<Barcode> big_barcode_search(const std::string&, const std::unordered_set<std::__cxx11::basic_string<char> >&, int, int, const std::vector<std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > >&)’:
main-functions/flexiplex.cpp:400:29: warning: comparison of integer expressions of different signedness: ‘const int’ and ‘const std::__cxx11::basic_string<char>::size_type’ {aka ‘const long unsigned int’} [-Wsign-compare]
  400 |       if (barcode.flank_end == std::string::npos) {
      |           ~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘void write_bgzfstring(BGZF*, const std::string&)’:
main-functions/flexiplex.cpp:423:21: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  423 |   if (bytes_written != str.size()) {
      |       ~~~~~~~~~~~~~~^~~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘void print_stats(const std::string&, const std::vector<Barcode>&, BGZF*)’:
main-functions/flexiplex.cpp:438:28: warning: comparison of integer expressions of different signedness: ‘const int’ and ‘const std::__cxx11::basic_string<char>::size_type’ {aka ‘const long unsigned int’} [-Wsign-compare]
  438 |         (barcode.flank_end == std::string::npos ? "True" : "False") + "\n";
      |          ~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘void print_read(const std::string&, const std::string&, const std::string&, const std::vector<Barcode>&, BGZF*, std::unordered_set<std::__cxx11::basic_string<char> >&, bool, bool, bool)’:
main-functions/flexiplex.cpp:475:21: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<Barcode>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  475 |   for (int b = 0; b < vec_bc.size(); b++) {
      |                   ~~^~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:490:32: warning: comparison of integer expressions of different signedness: ‘const int’ and ‘const std::__cxx11::basic_string<char>::size_type’ {aka ‘const long unsigned int’} [-Wsign-compare]
  490 |     if (vec_bc.at(b).flank_end == std::string::npos) {
      |         ~~~~~~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:495:23: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<Barcode>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  495 |     for (int f = 0; f < vec_bc.size(); f++) {
      |                     ~~^~~~~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘Rcpp::IntegerVector flexiplex_cpp(Rcpp::StringVector, Rcpp::String, bool, int, int, Rcpp::StringVector, Rcpp::String, Rcpp::String, Rcpp::String, bool, int)’:
main-functions/flexiplex.cpp:754:25: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<std::vector<SearchResult> >::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  754 |       for (int t = 0; t < sr_v.size();
      |                       ~~^~~~~~~~~~~~~
main-functions/flexiplex.cpp:759:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<SearchResult>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  759 |         for (int r = 0; r < sr_v[t].size(); r++) { // loop over the reads
      |                         ~~^~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:761:29: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<Barcode>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  761 |           for (int b = 0; b < sr_v[t][r].vec_bc_for.size(); b++)
      |                           ~~^~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:763:29: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<Barcode>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  763 |           for (int b = 0; b < sr_v[t][r].vec_bc_rev.size(); b++)
      |                           ~~^~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c main-functions/get_transcript_seq.cpp -o main-functions/get_transcript_seq.o
main-functions/get_transcript_seq.cpp: In function ‘void write_fa(const std::string&, const std::string&, const std::string&, int)’:
main-functions/get_transcript_seq.cpp:87:14: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
   87 |     while (i < seq.size()) {
      |            ~~^~~~~~~~~~~~
main-functions/get_transcript_seq.cpp:88:26: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
   88 |         if (i + wrap_len > seq.size()) {
      |             ~~~~~~~~~~~~~^~~~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c main-functions/group_bam2isoform.cpp -o main-functions/group_bam2isoform.o
In file included from main-functions/group_bam2isoform.cpp:18:
main-functions/../utility/utility.h: In function ‘std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > parseSpace(const std::string&)’:
main-functions/../utility/utility.h:255:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  255 |         for (int i = 0; i < s.size(); i++) {
      |                         ~~^~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c main-functions/pileup_readid.cpp -o main-functions/pileup_readid.o
main-functions/pileup_readid.cpp: In function ‘Rcpp::NumericMatrix variant_count_matrix_cpp(Rcpp::String, Rcpp::String, int, bool, bool)’:
main-functions/pileup_readid.cpp:268:21: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  268 |   for (int i = 0; i < BASES.size(); i++) {
      |                   ~~^~~~~~~~~~~~~~
main-functions/pileup_readid.cpp: In instantiation of ‘std::unordered_map<std::__cxx11::basic_string<char>, std::vector<std::vector<std::__cxx11::basic_string<char> > > > group_umis(const TableType&, int) [with TableType = std::unordered_map<std::__cxx11::basic_string<char>, std::unordered_map<std::__cxx11::basic_string<char>, std::unordered_map<std::__cxx11::basic_string<char>, unsigned int> > >]’:
main-functions/pileup_readid.cpp:342:30:   required from here
main-functions/pileup_readid.cpp:86:16: warning: unused variable ‘end’ [-Wunused-variable]
   86 |   unsigned int end;
      |                ^~~
main-functions/pileup_readid.cpp: In instantiation of ‘std::unordered_map<std::__cxx11::basic_string<char>, std::vector<std::vector<std::__cxx11::basic_string<char> > > > group_umis(const TableType&, int) [with TableType = std::unordered_map<std::__cxx11::basic_string<char>, std::unordered_map<std::__cxx11::basic_string<char>, std::array<unsigned int, 5> > >]’:
main-functions/pileup_readid.cpp:344:30:   required from here
main-functions/pileup_readid.cpp:86:16: warning: unused variable ‘end’ [-Wunused-variable]
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c tests/test-junctions.cpp -o tests/test-junctions.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c tests/test-parsing.cpp -o tests/test-parsing.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c utility/cigars.cpp -o utility/cigars.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c utility/edlib-1.2.7/edlib.cpp -o utility/edlib-1.2.7/edlib.o
gcc -std=gnu2x -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security -c utility/bam.c -o utility/bam.o
g++ -std=gnu++17 -shared -L/home/biocbuild/bbs-3.22-bioc/R/lib -L/usr/local/lib -o FLAMES.so RcppExports.o RcppFunctions.o classes/BamRecord.o classes/GFFRecord.o classes/GeneAnnotationParser.o classes/Isoforms.o classes/junctions.o main-functions/find_isoform.o main-functions/flexiplex.o main-functions/get_transcript_seq.o main-functions/group_bam2isoform.o main-functions/pileup_readid.o tests/test-junctions.o tests/test-parsing.o utility/cigars.o utility/edlib-1.2.7/edlib.o utility/bam.o -pthread -lz /home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/usrlib/libhts.a -lcurl -lbz2 -llzma -lz -L/home/biocbuild/bbs-3.22-bioc/R/lib -lR
if test -e "/usr/bin/strip" & test -e "/bin/uname" & [[ `uname` == "Linux" ]] ; then /usr/bin/strip --strip-debug FLAMES.so; fi
Building for x86_64
(cd submodule/minimap2 && make -f Makefile CFLAGS="-g -O2  -Wall -Werror=format-security -Wno-unused-result" minimap2)
make[1]: Entering directory '/home/biocbuild/bbs-3.22-bioc/meat/FLAMES/src/submodule/minimap2'
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  main.c -o main.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  kthread.c -o kthread.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  kalloc.c -o kalloc.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  misc.c -o misc.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  bseq.c -o bseq.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  sketch.c -o sketch.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  sdust.c -o sdust.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  options.c -o options.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  index.c -o index.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  lchain.c -o lchain.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  align.c -o align.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  hit.c -o hit.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  seed.c -o seed.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  jump.c -o jump.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  map.c -o map.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  format.c -o format.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  pe.c -o pe.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  esterr.c -o esterr.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  splitidx.c -o splitidx.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse2 -DHAVE_KALLOC  ksw2_ll_sse.c -o ksw2_ll_sse.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH  ksw2_extz2_sse.c -o ksw2_extz2_sse41.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH  ksw2_extd2_sse.c -o ksw2_extd2_sse41.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH  ksw2_exts2_sse.c -o ksw2_exts2_sse41.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse2 -mno-sse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH -DKSW_SSE2_ONLY  ksw2_extz2_sse.c -o ksw2_extz2_sse2.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse2 -mno-sse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH -DKSW_SSE2_ONLY  ksw2_extd2_sse.c -o ksw2_extd2_sse2.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse2 -mno-sse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH -DKSW_SSE2_ONLY  ksw2_exts2_sse.c -o ksw2_exts2_sse2.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH  ksw2_dispatch.c -o ksw2_dispatch.o
ar -csru libminimap2.a kthread.o kalloc.o misc.o bseq.o sketch.o sdust.o options.o index.o lchain.o align.o hit.o seed.o jump.o map.o format.o pe.o esterr.o splitidx.o ksw2_ll_sse.o ksw2_extz2_sse41.o ksw2_extd2_sse41.o ksw2_exts2_sse41.o ksw2_extz2_sse2.o ksw2_extd2_sse2.o ksw2_exts2_sse2.o ksw2_dispatch.o
ar: `u' modifier ignored since `D' is the default (see `U')
cc -g -O2  -Wall -Werror=format-security -Wno-unused-result main.o -o minimap2 -L. -lminimap2 -lm -lz -lpthread
make[1]: Leaving directory '/home/biocbuild/bbs-3.22-bioc/meat/FLAMES/src/submodule/minimap2'
echo "Installing binary to /home/biocbuild/bbs-3.22-bioc/meat/FLAMES/src/../inst/bin"
Installing binary to /home/biocbuild/bbs-3.22-bioc/meat/FLAMES/src/../inst/bin
mkdir -p ../inst/bin
cp submodule/minimap2/minimap2 ../inst/bin/
Rust version too old for edition 2024, skipping oarfish installation.
installing to /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/libs
** R
** data
** inst
** byte-compile and prepare package for lazy loading
** help
*** installing help indices
*** copying figures
** building package indices
** installing vignettes
** testing if installed package can be loaded
* DONE (FLAMES)

R version 4.5.1 (2025-06-13) -- "Great Square Root"
Copyright (C) 2025 The R Foundation for Statistical Computing
Platform: x86_64-pc-linux-gnu

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

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

> # This file is part of the standard setup for testthat.
> # It is recommended that you do not modify it.
> #
> # Where should you do additional test configuration?
> # Learn more about the roles of various files in:
> # * https://r-pkgs.org/tests.html
> # * https://testthat.r-lib.org/reference/test_package.html#special-files
> 
> library(testthat)
> library(FLAMES)
> 
> test_check("FLAMES")
Writing configuration parameters to:  /tmp/Rtmpg78x8n/file187b5228191a5d/config_file_1604434.json 
Writing configuration parameters to:  /tmp/Rtmpg78x8n/file187b5228191a5d/config_file_1604434.json 
Writing configuration parameters to:  /tmp/Rtmpg78x8n/file187b5228191a5d/config_file_1604434.json 
Writing configuration parameters to:  /tmp/Rtmpg78x8n/file187b5269b1f24d/config_file_1604434.json 
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpg78x8n/file187b5246ac4c5b/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
Skipping TSO trimming...
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpg78x8n/file187b525c1e6d3b/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
Skipping TSO trimming...
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpg78x8n/file187b525c1e6d3b/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpg78x8n/file187b525af820be/musc_rps24_1.fastq
Searching for barcodes...
Processing file: /tmp/Rtmpg78x8n/file187b525af820be/musc_rps24_2.fastq
Searching for barcodes...
Processing file: /tmp/Rtmpg78x8n/file187b525af820be/musc_rps24_3.fastq
Searching for barcodes...
Processing file: /tmp/Rtmpg78x8n/file187b525af820be/musc_rps24_4.fastq
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
Skipping TSO trimming...
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpg78x8n/file187b52fd737ca/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
Skipping TSO trimming...
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpg78x8n/file187b5245d71fde/config_file_1604434.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 @ Sun Sep  7 23:47:28 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/Rtmpg78x8n/file187b5245d71fde/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/Rtmpg78x8n/file187b5245d71fde/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/Rtmpg78x8n/file187b5245d71fde/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Sun Sep  7 23:47:29 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

  |                                                                            
  |                                                                      |   0%[23:47:36] WARNING: src/learner.cc:553: 
  If you are loading a serialized model (like pickle in Python, RDS in R) generated by
  older XGBoost, please export the model by calling `Booster.save_model` from that version
  first, then load it back in current version. See:

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

  for more details about differences between saving model and serializing.

[23:47:36] WARNING: src/learner.cc:553: 
  If you are loading a serialized model (like pickle in Python, RDS in R) generated by
  older XGBoost, please export the model by calling `Booster.save_model` from that version
  first, then load it back in current version. See:

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

  for more details about differences between saving model and serializing.

[23:47:36] WARNING: src/learner.cc:553: 
  If you are loading a serialized model (like pickle in Python, RDS in R) generated by
  older XGBoost, please export the model by calling `Booster.save_model` from that version
  first, then load it back in current version. See:

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

  for more details about differences between saving model and serializing.

[23:47:36] WARNING: src/learner.cc:553: 
  If you are loading a serialized model (like pickle in Python, RDS in R) generated by
  older XGBoost, please export the model by calling `Booster.save_model` from that version
  first, then load it back in current version. See:

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

  for more details about differences between saving model and serializing.

[23:47:38] WARNING: src/learner.cc:553: 
  If you are loading a serialized model (like pickle in Python, RDS in R) generated by
  older XGBoost, please export the model by calling `Booster.save_model` from that version
  first, then load it back in current version. See:

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

  for more details about differences between saving model and serializing.

[23:47:38] WARNING: src/learner.cc:553: 
  If you are loading a serialized model (like pickle in Python, RDS in R) generated by
  older XGBoost, please export the model by calling `Booster.save_model` from that version
  first, then load it back in current version. See:

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

  for more details about differences between saving model and serializing.


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

Detected 4 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Sun Sep  7 23:47:56 2025 -------------------
Realigning sample sample1 -> /tmp/Rtmpg78x8n/file187b5245d71fde/sample1_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample2 -> /tmp/Rtmpg78x8n/file187b5245d71fde/sample2_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample3 -> /tmp/Rtmpg78x8n/file187b5245d71fde/sample3_realign2transcript.bam
Skipped sorting BAM files.

-- Running step: transcript_quantification @ Sun Sep  7 23:47:56 2025 ----------
2025-09-08T03:47:56.565738Z  INFO oarfish: setting user-provided filter parameters.
2025-09-08T03:47:56.566255Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpg78x8n/file187b5245d71fde/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-09-08T03:47:56.566268Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-08T03:47:56.566272Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-08T03:47:56.566347Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-08T03:47:56.566353Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-09-08T03:47:56.567866Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-09-08T03:47:56.568012Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 253   │
│ aligned fraction too low        │ 4     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 1     │
│ reads with valid best alignment │ 96    │
╰─────────────────────────────────┴───────╯

2025-09-08T03:47:56.568037Z  INFO oarfish::bulk: Total number of alignment records : 116
2025-09-08T03:47:56.568052Z  INFO oarfish::bulk: number of aligned reads : 96
2025-09-08T03:47:56.568054Z  INFO oarfish::bulk: number of unique alignments : 86
2025-09-08T03:47:56.568650Z  INFO oarfish: oarfish completed successfully.
2025-09-08T03:47:56.575985Z  INFO oarfish: setting user-provided filter parameters.
2025-09-08T03:47:56.576366Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpg78x8n/file187b5245d71fde/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-09-08T03:47:56.576375Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-08T03:47:56.576379Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-08T03:47:56.576444Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-08T03:47:56.576450Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-09-08T03:47:56.578093Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-09-08T03:47:56.578229Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 251   │
│ aligned fraction too low        │ 5     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 2     │
│ reads with valid best alignment │ 95    │
╰─────────────────────────────────┴───────╯

2025-09-08T03:47:56.578261Z  INFO oarfish::bulk: Total number of alignment records : 114
2025-09-08T03:47:56.578264Z  INFO oarfish::bulk: number of aligned reads : 95
2025-09-08T03:47:56.578266Z  INFO oarfish::bulk: number of unique alignments : 82
2025-09-08T03:47:56.578969Z  INFO oarfish: oarfish completed successfully.
2025-09-08T03:47:56.586259Z  INFO oarfish: setting user-provided filter parameters.
2025-09-08T03:47:56.586608Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpg78x8n/file187b5245d71fde/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-09-08T03:47:56.586616Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-08T03:47:56.586619Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-08T03:47:56.586673Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-08T03:47:56.586679Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-09-08T03:47:56.589244Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2025-09-08T03:47:56.589415Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 398   │
│ aligned fraction too low        │ 12    │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 5     │
│ reads with valid best alignment │ 179   │
╰─────────────────────────────────┴───────╯

2025-09-08T03:47:56.589455Z  INFO oarfish::bulk: Total number of alignment records : 239
2025-09-08T03:47:56.589458Z  INFO oarfish::bulk: number of aligned reads : 179
2025-09-08T03:47:56.589460Z  INFO oarfish::bulk: number of unique alignments : 143
2025-09-08T03:47:56.590137Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpg78x8n/file187b527e24162f/config_file_1604434.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 @ Sun Sep  7 23:47:56 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/Rtmpg78x8n/file187b527e24162f/sample1_align2genome.bam
sample2 ->/tmp/Rtmpg78x8n/file187b527e24162f/sample2_align2genome.bam
sample3 ->/tmp/Rtmpg78x8n/file187b527e24162f/sample3_align2genome.bam
-- Running step: isoform_identification @ Sun Sep  7 23:48:17 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 4 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Sun Sep  7 23:48:39 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/Rtmpg78x8n/file187b527e24162f/sample1_realign2transcript.bam
sample2 ->/tmp/Rtmpg78x8n/file187b527e24162f/sample2_realign2transcript.bam
sample3 ->/tmp/Rtmpg78x8n/file187b527e24162f/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Sun Sep  7 23:48:59 2025 ----------
2025-09-08T03:48:59.566440Z  INFO oarfish: setting user-provided filter parameters.
2025-09-08T03:48:59.567190Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpg78x8n/file187b527e24162f/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-09-08T03:48:59.567216Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-08T03:48:59.567220Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-08T03:48:59.567289Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-08T03:48:59.567295Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-09-08T03:48:59.569073Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-09-08T03:48:59.569219Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 253   │
│ aligned fraction too low        │ 4     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 1     │
│ reads with valid best alignment │ 96    │
╰─────────────────────────────────┴───────╯

2025-09-08T03:48:59.569241Z  INFO oarfish::bulk: Total number of alignment records : 116
2025-09-08T03:48:59.569243Z  INFO oarfish::bulk: number of aligned reads : 96
2025-09-08T03:48:59.569245Z  INFO oarfish::bulk: number of unique alignments : 86
2025-09-08T03:48:59.569907Z  INFO oarfish: oarfish completed successfully.
2025-09-08T03:48:59.581466Z  INFO oarfish: setting user-provided filter parameters.
2025-09-08T03:48:59.582078Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpg78x8n/file187b527e24162f/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-09-08T03:48:59.582091Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-08T03:48:59.582094Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-08T03:48:59.582159Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-08T03:48:59.582164Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-09-08T03:48:59.583928Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-09-08T03:48:59.584069Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 251   │
│ aligned fraction too low        │ 5     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 2     │
│ reads with valid best alignment │ 95    │
╰─────────────────────────────────┴───────╯

2025-09-08T03:48:59.584095Z  INFO oarfish::bulk: Total number of alignment records : 114
2025-09-08T03:48:59.584109Z  INFO oarfish::bulk: number of aligned reads : 95
2025-09-08T03:48:59.584111Z  INFO oarfish::bulk: number of unique alignments : 82
2025-09-08T03:48:59.584730Z  INFO oarfish: oarfish completed successfully.
2025-09-08T03:48:59.596284Z  INFO oarfish: setting user-provided filter parameters.
2025-09-08T03:48:59.596778Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpg78x8n/file187b527e24162f/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-09-08T03:48:59.596790Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-08T03:48:59.596794Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-08T03:48:59.596858Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-08T03:48:59.596863Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-09-08T03:48:59.599615Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2025-09-08T03:48:59.599802Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 398   │
│ aligned fraction too low        │ 12    │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 5     │
│ reads with valid best alignment │ 179   │
╰─────────────────────────────────┴───────╯

2025-09-08T03:48:59.599835Z  INFO oarfish::bulk: Total number of alignment records : 239
2025-09-08T03:48:59.599838Z  INFO oarfish::bulk: number of aligned reads : 179
2025-09-08T03:48:59.599840Z  INFO oarfish::bulk: number of unique alignments : 143
2025-09-08T03:48:59.600553Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpg78x8n/file187b52320f9b34/config_file_1604434.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 @ Sun Sep  7 23:48:59 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/Rtmpg78x8n/file187b52320f9b34/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/Rtmpg78x8n/file187b52320f9b34/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/Rtmpg78x8n/file187b52320f9b34/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Sun Sep  7 23:49:00 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 4 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Sun Sep  7 23:49:21 2025 -------------------
Realigning sample sample1 -> /tmp/Rtmpg78x8n/file187b52320f9b34/sample1_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample2 -> /tmp/Rtmpg78x8n/file187b52320f9b34/sample2_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample3 -> /tmp/Rtmpg78x8n/file187b52320f9b34/sample3_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Sun Sep  7 23:49:22 2025 ----------
23:49:22 Sun Sep 07 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpg78x8n/file187b527da701ef/config_file_1604434.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 @ Sun Sep  7 23:49:23 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/Rtmpg78x8n/file187b527da701ef/sample1_align2genome.bam
sample2 ->/tmp/Rtmpg78x8n/file187b527da701ef/sample2_align2genome.bam
sample3 ->/tmp/Rtmpg78x8n/file187b527da701ef/sample3_align2genome.bam
-- Running step: isoform_identification @ Sun Sep  7 23:49:43 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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Detected 4 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Sun Sep  7 23:50:03 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/Rtmpg78x8n/file187b527da701ef/sample1_realign2transcript.bam
sample2 ->/tmp/Rtmpg78x8n/file187b527da701ef/sample2_realign2transcript.bam
sample3 ->/tmp/Rtmpg78x8n/file187b527da701ef/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Sun Sep  7 23:50:23 2025 ----------
23:50:23 Sun Sep 07 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Inputs:  ['/tmp/Rtmpg78x8n/file187b52320f9b34/sample1_realign2transcript.bam', '/tmp/Rtmpg78x8n/file187b52320f9b34/sample2_realign2transcript.bam', '/tmp/Rtmpg78x8n/file187b52320f9b34/sample3_realign2transcript.bam'] /tmp/Rtmpg78x8n/file187b52320f9b34/transcript_assembly.fa.fai 5 0.4 0.4
	Counter({'counted_reads': 375, 'not_enough_coverage': 16, 'unmapped': 2})
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpg78x8n/file187b522afd30c1/config_file_1604434.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 @ Sun Sep  7 23:50:24 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/Rtmpg78x8n/file187b522afd30c1/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/Rtmpg78x8n/file187b522afd30c1/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/Rtmpg78x8n/file187b522afd30c1/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Sun Sep  7 23:50:24 2025 -------------
Inputs:  ['/tmp/Rtmpg78x8n/file187b527da701ef/sample1_realign2transcript.bam', '/tmp/Rtmpg78x8n/file187b527da701ef/sample2_realign2transcript.bam', '/tmp/Rtmpg78x8n/file187b527da701ef/sample3_realign2transcript.bam'] /tmp/Rtmpg78x8n/file187b527da701ef/transcript_assembly.fa.fai 5 0.4 0.4
	Counter({'counted_reads': 375, 'not_enough_coverage': 16, 'unmapped': 2})
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sun Sep  7 23:50:25 2025 -------------------
Realigning sample sample1 -> /tmp/Rtmpg78x8n/file187b522afd30c1/sample1_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample2 -> /tmp/Rtmpg78x8n/file187b522afd30c1/sample2_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample3 -> /tmp/Rtmpg78x8n/file187b522afd30c1/sample3_realign2transcript.bam
Skipped sorting BAM files.

-- Running step: transcript_quantification @ Sun Sep  7 23:50:26 2025 ----------
2025-09-08T03:50:26.220161Z  INFO oarfish: setting user-provided filter parameters.
2025-09-08T03:50:26.220678Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpg78x8n/file187b522afd30c1/sample1_realign2transcript.bam, contains 10 reference sequences.
2025-09-08T03:50:26.220691Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-08T03:50:26.220695Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-08T03:50:26.220791Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-08T03:50:26.220800Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-09-08T03:50:26.223420Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-09-08T03:50:26.223569Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 707   │
│ aligned fraction too low        │ 2     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 98    │
╰─────────────────────────────────┴───────╯

2025-09-08T03:50:26.223591Z  INFO oarfish::bulk: Total number of alignment records : 125
2025-09-08T03:50:26.223593Z  INFO oarfish::bulk: number of aligned reads : 98
2025-09-08T03:50:26.223595Z  INFO oarfish::bulk: number of unique alignments : 86
2025-09-08T03:50:26.224255Z  INFO oarfish: oarfish completed successfully.
2025-09-08T03:50:26.231942Z  INFO oarfish: setting user-provided filter parameters.
2025-09-08T03:50:26.232297Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpg78x8n/file187b522afd30c1/sample2_realign2transcript.bam, contains 10 reference sequences.
2025-09-08T03:50:26.232306Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-08T03:50:26.232309Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-08T03:50:26.232395Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-08T03:50:26.232402Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-09-08T03:50:26.235152Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-09-08T03:50:26.235304Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 701   │
│ aligned fraction too low        │ 3     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 97    │
╰─────────────────────────────────┴───────╯

2025-09-08T03:50:26.235329Z  INFO oarfish::bulk: Total number of alignment records : 136
2025-09-08T03:50:26.235332Z  INFO oarfish::bulk: number of aligned reads : 97
2025-09-08T03:50:26.235334Z  INFO oarfish::bulk: number of unique alignments : 79
2025-09-08T03:50:26.235984Z  INFO oarfish: oarfish completed successfully.
2025-09-08T03:50:26.243189Z  INFO oarfish: setting user-provided filter parameters.
2025-09-08T03:50:26.243666Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpg78x8n/file187b522afd30c1/sample3_realign2transcript.bam, contains 10 reference sequences.
2025-09-08T03:50:26.243674Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-08T03:50:26.243677Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-08T03:50:26.243754Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-08T03:50:26.243764Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-09-08T03:50:26.247944Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-09-08T03:50:26.248119Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 1060  │
│ aligned fraction too low        │ 6     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 187   │
╰─────────────────────────────────┴───────╯

2025-09-08T03:50:26.248148Z  INFO oarfish::bulk: Total number of alignment records : 272
2025-09-08T03:50:26.248150Z  INFO oarfish::bulk: number of aligned reads : 187
2025-09-08T03:50:26.248152Z  INFO oarfish::bulk: number of unique alignments : 140
2025-09-08T03:50:26.248886Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpg78x8n/file187b522029c3e2/config_file_1604434.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 @ Sun Sep  7 23:50:26 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/Rtmpg78x8n/file187b522029c3e2/sample1_align2genome.bam
sample2 ->/tmp/Rtmpg78x8n/file187b522029c3e2/sample2_align2genome.bam
sample3 ->/tmp/Rtmpg78x8n/file187b522029c3e2/sample3_align2genome.bam
-- Running step: isoform_identification @ Sun Sep  7 23:50:46 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sun Sep  7 23:50:46 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/Rtmpg78x8n/file187b522029c3e2/sample1_realign2transcript.bam
sample2 ->/tmp/Rtmpg78x8n/file187b522029c3e2/sample2_realign2transcript.bam
sample3 ->/tmp/Rtmpg78x8n/file187b522029c3e2/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Sun Sep  7 23:51:07 2025 ----------
2025-09-08T03:51:07.538504Z  INFO oarfish: setting user-provided filter parameters.
2025-09-08T03:51:07.539090Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpg78x8n/file187b522029c3e2/sample1_realign2transcript.bam, contains 10 reference sequences.
2025-09-08T03:51:07.539105Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-08T03:51:07.539119Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-08T03:51:07.539213Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-08T03:51:07.539220Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-09-08T03:51:07.541842Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-09-08T03:51:07.541987Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 707   │
│ aligned fraction too low        │ 2     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 98    │
╰─────────────────────────────────┴───────╯

2025-09-08T03:51:07.542012Z  INFO oarfish::bulk: Total number of alignment records : 125
2025-09-08T03:51:07.542015Z  INFO oarfish::bulk: number of aligned reads : 98
2025-09-08T03:51:07.542017Z  INFO oarfish::bulk: number of unique alignments : 86
2025-09-08T03:51:07.542641Z  INFO oarfish: oarfish completed successfully.
2025-09-08T03:51:07.555770Z  INFO oarfish: setting user-provided filter parameters.
2025-09-08T03:51:07.556286Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpg78x8n/file187b522029c3e2/sample2_realign2transcript.bam, contains 10 reference sequences.
2025-09-08T03:51:07.556295Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-08T03:51:07.556299Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-08T03:51:07.556377Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-08T03:51:07.556385Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-09-08T03:51:07.559033Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-09-08T03:51:07.559176Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 701   │
│ aligned fraction too low        │ 3     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 97    │
╰─────────────────────────────────┴───────╯

2025-09-08T03:51:07.559203Z  INFO oarfish::bulk: Total number of alignment records : 136
2025-09-08T03:51:07.559206Z  INFO oarfish::bulk: number of aligned reads : 97
2025-09-08T03:51:07.559220Z  INFO oarfish::bulk: number of unique alignments : 79
2025-09-08T03:51:07.559852Z  INFO oarfish: oarfish completed successfully.
2025-09-08T03:51:07.571805Z  INFO oarfish: setting user-provided filter parameters.
2025-09-08T03:51:07.572306Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpg78x8n/file187b522029c3e2/sample3_realign2transcript.bam, contains 10 reference sequences.
2025-09-08T03:51:07.572315Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-08T03:51:07.572318Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-08T03:51:07.572394Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-08T03:51:07.572402Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-09-08T03:51:07.576746Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-09-08T03:51:07.576910Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 1060  │
│ aligned fraction too low        │ 6     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 187   │
╰─────────────────────────────────┴───────╯

2025-09-08T03:51:07.576944Z  INFO oarfish::bulk: Total number of alignment records : 272
2025-09-08T03:51:07.576947Z  INFO oarfish::bulk: number of aligned reads : 187
2025-09-08T03:51:07.576949Z  INFO oarfish::bulk: number of unique alignments : 140
2025-09-08T03:51:07.577635Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpg78x8n/file187b521dcb2424/config_file_1604434.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 @ Sun Sep  7 23:51:07 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/Rtmpg78x8n/file187b521dcb2424/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/Rtmpg78x8n/file187b521dcb2424/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/Rtmpg78x8n/file187b521dcb2424/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Sun Sep  7 23:51:08 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sun Sep  7 23:51:09 2025 -------------------
Realigning sample sample1 -> /tmp/Rtmpg78x8n/file187b521dcb2424/sample1_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample2 -> /tmp/Rtmpg78x8n/file187b521dcb2424/sample2_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample3 -> /tmp/Rtmpg78x8n/file187b521dcb2424/sample3_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Sun Sep  7 23:51:09 2025 ----------
23:51:09 Sun Sep 07 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpg78x8n/file187b5217c01e66/config_file_1604434.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 @ Sun Sep  7 23:51:10 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/Rtmpg78x8n/file187b5217c01e66/sample1_align2genome.bam
sample2 ->/tmp/Rtmpg78x8n/file187b5217c01e66/sample2_align2genome.bam
sample3 ->/tmp/Rtmpg78x8n/file187b5217c01e66/sample3_align2genome.bam
-- Running step: isoform_identification @ Sun Sep  7 23:51:31 2025 -------------
Inputs:  ['/tmp/Rtmpg78x8n/file187b521dcb2424/sample1_realign2transcript.bam', '/tmp/Rtmpg78x8n/file187b521dcb2424/sample2_realign2transcript.bam', '/tmp/Rtmpg78x8n/file187b521dcb2424/sample3_realign2transcript.bam'] /tmp/Rtmpg78x8n/file187b521dcb2424/transcript_assembly.fa.fai 5 0.4 0.4
	Counter({'counted_reads': 391, 'not_enough_coverage': 2})
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sun Sep  7 23:51:31 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/Rtmpg78x8n/file187b5217c01e66/sample1_realign2transcript.bam
sample2 ->/tmp/Rtmpg78x8n/file187b5217c01e66/sample2_realign2transcript.bam
sample3 ->/tmp/Rtmpg78x8n/file187b5217c01e66/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Sun Sep  7 23:51:51 2025 ----------
23:51:51 Sun Sep 07 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpg78x8n/file187b5259d8f627/config_file_1604434.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Sun Sep  7 23:51:52 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpg78x8n/file187b5259d8f627/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Sun Sep  7 23:51:52 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmpg78x8n/file187b5259d8f627/matched_reads.fastq.gz -> /tmp/Rtmpg78x8n/file187b5259d8f627/align2genome.bam
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: isoform_identification @ Sun Sep  7 23:51:52 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---
Detected 2 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Sun Sep  7 23:52:03 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpg78x8n/file187b5259d8f627/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpg78x8n/file187b5259d8f627/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/Rtmpg78x8n/file187b5259d8f627/matched_reads.fastq.gz -> /tmp/Rtmpg78x8n/file187b5259d8f627/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 @ Sun Sep  7 23:52:03 2025 ----------
2025-09-08T03:52:03.232895Z  INFO oarfish: setting user-provided filter parameters.
2025-09-08T03:52:03.233306Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpg78x8n/file187b5259d8f627/realign2transcript.bam, contains 5 reference sequences.
2025-09-08T03:52:03.233317Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-08T03:52:03.233322Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-08T03:52:03.233384Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-08T03:52:03.233391Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-09-08T03:52:03.239467Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpg78x8n/file187b5223ec4a7a/config_file_1604434.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Sun Sep  7 23:52:03 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpg78x8n/file187b5223ec4a7a/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Sun Sep  7 23:52:03 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmpg78x8n/file187b5223ec4a7a/matched_reads.fastq.gz ->/tmp/Rtmpg78x8n/file187b5223ec4a7a/align2genome.bam
-- Running step: isoform_identification @ Sun Sep  7 23:52:22 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---
Detected 2 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Sun Sep  7 23:52:33 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpg78x8n/file187b5223ec4a7a/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpg78x8n/file187b5223ec4a7a/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/Rtmpg78x8n/file187b5223ec4a7a/matched_reads.fastq.gz ->/tmp/Rtmpg78x8n/file187b5223ec4a7a/realign2transcript.bam
-- Running step: transcript_quantification @ Sun Sep  7 23:52:53 2025 ----------
2025-09-08T03:52:53.041500Z  INFO oarfish: setting user-provided filter parameters.
2025-09-08T03:52:53.042074Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpg78x8n/file187b5223ec4a7a/realign2transcript.bam, contains 5 reference sequences.
2025-09-08T03:52:53.042091Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-08T03:52:53.042097Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-08T03:52:53.042189Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-08T03:52:53.042199Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-09-08T03:52:53.049876Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpg78x8n/file187b52386b430d/config_file_1604434.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Sun Sep  7 23:52:53 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpg78x8n/file187b52386b430d/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Sun Sep  7 23:52:53 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmpg78x8n/file187b52386b430d/matched_reads.fastq.gz -> /tmp/Rtmpg78x8n/file187b52386b430d/align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: isoform_identification @ Sun Sep  7 23:52:54 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---
Detected 2 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Sun Sep  7 23:53:04 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpg78x8n/file187b52386b430d/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpg78x8n/file187b52386b430d/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/Rtmpg78x8n/file187b52386b430d/matched_reads.fastq.gz -> /tmp/Rtmpg78x8n/file187b52386b430d/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 @ Sun Sep  7 23:53:04 2025 ----------
23:53:04 Sun Sep 07 2025 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Inputs:  ['/tmp/Rtmpg78x8n/file187b5217c01e66/sample1_realign2transcript.bam', '/tmp/Rtmpg78x8n/file187b5217c01e66/sample2_realign2transcript.bam', '/tmp/Rtmpg78x8n/file187b5217c01e66/sample3_realign2transcript.bam'] /tmp/Rtmpg78x8n/file187b5217c01e66/transcript_assembly.fa.fai 5 0.4 0.4
	Counter({'counted_reads': 391, 'not_enough_coverage': 2})
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpg78x8n/file187b526ce7501d/config_file_1604434.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Sun Sep  7 23:53:05 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpg78x8n/file187b526ce7501d/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Sun Sep  7 23:53:05 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmpg78x8n/file187b526ce7501d/matched_reads.fastq.gz ->/tmp/Rtmpg78x8n/file187b526ce7501d/align2genome.bam
-- Running step: isoform_identification @ Sun Sep  7 23:53:24 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---
Detected 2 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Sun Sep  7 23:53:35 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpg78x8n/file187b526ce7501d/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpg78x8n/file187b526ce7501d/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/Rtmpg78x8n/file187b526ce7501d/matched_reads.fastq.gz ->/tmp/Rtmpg78x8n/file187b526ce7501d/realign2transcript.bam
-- Running step: transcript_quantification @ Sun Sep  7 23:53:54 2025 ----------
23:53:54 Sun Sep 07 2025 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
	Counter({'counted_reads': 354, 'not_enough_coverage': 12, 'unmapped': 6})
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpg78x8n/file187b523c1bb3dd/config_file_1604434.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Sun Sep  7 23:53:55 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpg78x8n/file187b523c1bb3dd/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Sun Sep  7 23:53:55 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmpg78x8n/file187b523c1bb3dd/matched_reads.fastq.gz -> /tmp/Rtmpg78x8n/file187b523c1bb3dd/align2genome.bam
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: isoform_identification @ Sun Sep  7 23:53:55 2025 -------------
	Counter({'counted_reads': 354, 'not_enough_coverage': 12, 'unmapped': 6})
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sun Sep  7 23:53:56 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpg78x8n/file187b523c1bb3dd/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpg78x8n/file187b523c1bb3dd/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/Rtmpg78x8n/file187b523c1bb3dd/matched_reads.fastq.gz -> /tmp/Rtmpg78x8n/file187b523c1bb3dd/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 @ Sun Sep  7 23:53:56 2025 ----------
2025-09-08T03:53:56.335129Z  INFO oarfish: setting user-provided filter parameters.
2025-09-08T03:53:56.335642Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpg78x8n/file187b523c1bb3dd/realign2transcript.bam, contains 10 reference sequences.
2025-09-08T03:53:56.335652Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-08T03:53:56.335655Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-08T03:53:56.335742Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-08T03:53:56.335755Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-09-08T03:53:56.345592Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpg78x8n/file187b522912955c/config_file_1604434.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Sun Sep  7 23:53:57 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpg78x8n/file187b522912955c/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Sun Sep  7 23:53:57 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmpg78x8n/file187b522912955c/matched_reads.fastq.gz ->/tmp/Rtmpg78x8n/file187b522912955c/align2genome.bam
-- Running step: isoform_identification @ Sun Sep  7 23:54:15 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sun Sep  7 23:54:16 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpg78x8n/file187b522912955c/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpg78x8n/file187b522912955c/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/Rtmpg78x8n/file187b522912955c/matched_reads.fastq.gz ->/tmp/Rtmpg78x8n/file187b522912955c/realign2transcript.bam
-- Running step: transcript_quantification @ Sun Sep  7 23:54:34 2025 ----------
2025-09-08T03:54:34.539025Z  INFO oarfish: setting user-provided filter parameters.
2025-09-08T03:54:34.539513Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpg78x8n/file187b522912955c/realign2transcript.bam, contains 10 reference sequences.
2025-09-08T03:54:34.539526Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-08T03:54:34.539529Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-08T03:54:34.539611Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-08T03:54:34.539618Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-09-08T03:54:34.568921Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpg78x8n/file187b521a551407/config_file_1604434.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Sun Sep  7 23:54:35 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpg78x8n/file187b521a551407/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Sun Sep  7 23:54:35 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmpg78x8n/file187b521a551407/matched_reads.fastq.gz -> /tmp/Rtmpg78x8n/file187b521a551407/align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: isoform_identification @ Sun Sep  7 23:54:36 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sun Sep  7 23:54:36 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpg78x8n/file187b521a551407/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpg78x8n/file187b521a551407/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/Rtmpg78x8n/file187b521a551407/matched_reads.fastq.gz -> /tmp/Rtmpg78x8n/file187b521a551407/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 @ Sun Sep  7 23:54:36 2025 ----------
23:54:36 Sun Sep 07 2025 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpg78x8n/file187b5212d40f1/config_file_1604434.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Sun Sep  7 23:54:38 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpg78x8n/file187b5212d40f1/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Sun Sep  7 23:54:38 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmpg78x8n/file187b5212d40f1/matched_reads.fastq.gz ->/tmp/Rtmpg78x8n/file187b5212d40f1/align2genome.bam
-- Running step: isoform_identification @ Sun Sep  7 23:54:58 2025 -------------
	Counter({'counted_reads': 368, 'unmapped': 4})
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sun Sep  7 23:54:58 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpg78x8n/file187b5212d40f1/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpg78x8n/file187b5212d40f1/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/Rtmpg78x8n/file187b5212d40f1/matched_reads.fastq.gz ->/tmp/Rtmpg78x8n/file187b5212d40f1/realign2transcript.bam
-- Running step: transcript_quantification @ Sun Sep  7 23:55:17 2025 ----------
23:55:17 Sun Sep 07 2025 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpg78x8n/file187b52115c3d47/config_file_1604434.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 @ Sun Sep  7 23:55:18 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpg78x8n/file187b52115c3d47/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpg78x8n/file187b52115c3d47/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpg78x8n/file187b52115c3d47/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpg78x8n/file187b52115c3d47/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpg78x8n/file187b52115c3d47/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpg78x8n/file187b52115c3d47/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 92
Number of reads with exactly one barcode match: 91
Number of chimera reads: 1
All done!
Reads	Barcodes
4	1
3	9
2	9
1	44
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpg78x8n/file187b52115c3d47/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpg78x8n/file187b52115c3d47/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 95
Number of reads with exactly one barcode match: 94
Number of chimera reads: 0
All done!
Reads	Barcodes
4	2
3	3
2	16
1	47
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpg78x8n/file187b52115c3d47/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpg78x8n/file187b52115c3d47/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Sun Sep  7 23:55:19 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmpg78x8n/file187b52115c3d47/sampleA_matched_reads.fastq.gz -> /tmp/Rtmpg78x8n/file187b52115c3d47/sampleA_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmpg78x8n/file187b52115c3d47/sample1_matched_reads.fastq.gz -> /tmp/Rtmpg78x8n/file187b52115c3d47/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmpg78x8n/file187b52115c3d47/sample2_matched_reads.fastq.gz -> /tmp/Rtmpg78x8n/file187b52115c3d47/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmpg78x8n/file187b52115c3d47/sample3_matched_reads.fastq.gz -> /tmp/Rtmpg78x8n/file187b52115c3d47/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Sun Sep  7 23:55:20 2025 ----------------
23:55:20 Sun Sep 07 2025 quantify genes 
Using BAM(s): '/tmp/Rtmpg78x8n/file187b52115c3d47/sampleA_align2genome.bam',
'/tmp/Rtmpg78x8n/file187b52115c3d47/sample1_align2genome.bam',
'/tmp/Rtmpg78x8n/file187b52115c3d47/sample2_align2genome.bam', and
'/tmp/Rtmpg78x8n/file187b52115c3d47/sample3_align2genome.bam'
	Counter({'counted_reads': 368, 'unmapped': 4})
parsing /tmp/Rtmpg78x8n/file187b52115c3d47/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 20.96gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 425834.96Read/s]
parsing /tmp/Rtmpg78x8n/file187b52115c3d47/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 49.47gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1322456.80Read/s]
parsing /tmp/Rtmpg78x8n/file187b52115c3d47/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 49.81gene_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, 1314828.84Read/s]
parsing /tmp/Rtmpg78x8n/file187b52115c3d47/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 35.18gene_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, 671604.43Read/s]
-- Running step: isoform_identification @ Sun Sep  7 23:55:22 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 6 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Sun Sep  7 23:55:48 2025 -------------------
Checking for fastq file(s) /tmp/Rtmpg78x8n/file187b52115c3d47/fastq, /tmp/Rtmpg78x8n/file187b52115c3d47/fastq/sample1.fq.gz, /tmp/Rtmpg78x8n/file187b52115c3d47/fastq/sample2.fq.gz, /tmp/Rtmpg78x8n/file187b52115c3d47/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpg78x8n/file187b52115c3d47/sampleA_matched_reads.fastq.gz, /tmp/Rtmpg78x8n/file187b52115c3d47/sample1_matched_reads.fastq.gz, /tmp/Rtmpg78x8n/file187b52115c3d47/sample2_matched_reads.fastq.gz, /tmp/Rtmpg78x8n/file187b52115c3d47/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpg78x8n/file187b52115c3d47/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmpg78x8n/file187b52115c3d47/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmpg78x8n/file187b52115c3d47/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmpg78x8n/file187b52115c3d47/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/Rtmpg78x8n/file187b52115c3d47/sampleA_matched_reads_dedup.fastq.gz -> /tmp/Rtmpg78x8n/file187b52115c3d47/sampleA_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/Rtmpg78x8n/file187b52115c3d47/sample1_matched_reads_dedup.fastq.gz -> /tmp/Rtmpg78x8n/file187b52115c3d47/sample1_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/Rtmpg78x8n/file187b52115c3d47/sample2_matched_reads_dedup.fastq.gz -> /tmp/Rtmpg78x8n/file187b52115c3d47/sample2_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/Rtmpg78x8n/file187b52115c3d47/sample3_matched_reads_dedup.fastq.gz -> /tmp/Rtmpg78x8n/file187b52115c3d47/sample3_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

-- Running step: transcript_quantification @ Sun Sep  7 23:55:49 2025 ----------
2025-09-08T03:55:49.632144Z  INFO oarfish: setting user-provided filter parameters.
2025-09-08T03:55:49.632665Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpg78x8n/file187b52115c3d47/sampleA_realign2transcript.bam, contains 5 reference sequences.
2025-09-08T03:55:49.632688Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-08T03:55:49.632691Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-08T03:55:49.632765Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-08T03:55:49.632774Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-09-08T03:55:49.638357Z  INFO oarfish::single_cell: Processed 100 cells.
2025-09-08T03:55:49.952858Z  INFO oarfish: setting user-provided filter parameters.
2025-09-08T03:55:49.953358Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpg78x8n/file187b52115c3d47/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-09-08T03:55:49.953368Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-08T03:55:49.953371Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-08T03:55:49.953435Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-08T03:55:49.953441Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-09-08T03:55:50.237508Z  INFO oarfish: setting user-provided filter parameters.
2025-09-08T03:55:50.238005Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpg78x8n/file187b52115c3d47/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-09-08T03:55:50.238017Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-08T03:55:50.238021Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-08T03:55:50.238083Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-08T03:55:50.238089Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-09-08T03:55:50.526818Z  INFO oarfish: setting user-provided filter parameters.
2025-09-08T03:55:50.527192Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpg78x8n/file187b52115c3d47/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-09-08T03:55:50.527200Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-08T03:55:50.527203Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-08T03:55:50.527266Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-08T03:55:50.527272Z  INFO oarfish: parsed reference information for 5 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpg78x8n/file187b526fc01451/config_file_1604434.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 @ Sun Sep  7 23:55:51 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpg78x8n/file187b526fc01451/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpg78x8n/file187b526fc01451/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpg78x8n/file187b526fc01451/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpg78x8n/file187b526fc01451/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpg78x8n/file187b526fc01451/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpg78x8n/file187b526fc01451/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 92
Number of reads with exactly one barcode match: 91
Number of chimera reads: 1
All done!
Reads	Barcodes
4	1
3	9
2	9
1	44
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpg78x8n/file187b526fc01451/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpg78x8n/file187b526fc01451/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 95
Number of reads with exactly one barcode match: 94
Number of chimera reads: 0
All done!
Reads	Barcodes
4	2
3	3
2	16
1	47
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpg78x8n/file187b526fc01451/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpg78x8n/file187b526fc01451/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Sun Sep  7 23:55:51 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmpg78x8n/file187b526fc01451/sampleA_matched_reads.fastq.gz ->/tmp/Rtmpg78x8n/file187b526fc01451/sampleA_align2genome.bam
/tmp/Rtmpg78x8n/file187b526fc01451/sample1_matched_reads.fastq.gz ->/tmp/Rtmpg78x8n/file187b526fc01451/sample1_align2genome.bam
/tmp/Rtmpg78x8n/file187b526fc01451/sample2_matched_reads.fastq.gz ->/tmp/Rtmpg78x8n/file187b526fc01451/sample2_align2genome.bam
/tmp/Rtmpg78x8n/file187b526fc01451/sample3_matched_reads.fastq.gz ->/tmp/Rtmpg78x8n/file187b526fc01451/sample3_align2genome.bam
-- Running step: gene_quantification @ Sun Sep  7 23:56:10 2025 ----------------
23:56:10 Sun Sep 07 2025 quantify genes 
Using BAM(s): '/tmp/Rtmpg78x8n/file187b526fc01451/sampleA_align2genome.bam',
'/tmp/Rtmpg78x8n/file187b526fc01451/sample1_align2genome.bam',
'/tmp/Rtmpg78x8n/file187b526fc01451/sample2_align2genome.bam', and
'/tmp/Rtmpg78x8n/file187b526fc01451/sample3_align2genome.bam'
parsing /tmp/Rtmpg78x8n/file187b526fc01451/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 21.59gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 355040.29Read/s]
parsing /tmp/Rtmpg78x8n/file187b526fc01451/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 49.23gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1215176.73Read/s]
parsing /tmp/Rtmpg78x8n/file187b526fc01451/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 45.66gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1241947.18Read/s]
parsing /tmp/Rtmpg78x8n/file187b526fc01451/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 35.83gene_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, 594801.75Read/s]
-- Running step: isoform_identification @ Sun Sep  7 23:56:11 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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Detected 6 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Sun Sep  7 23:56:37 2025 -------------------
Checking for fastq file(s) /tmp/Rtmpg78x8n/file187b526fc01451/fastq, /tmp/Rtmpg78x8n/file187b526fc01451/fastq/sample1.fq.gz, /tmp/Rtmpg78x8n/file187b526fc01451/fastq/sample2.fq.gz, /tmp/Rtmpg78x8n/file187b526fc01451/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpg78x8n/file187b526fc01451/sampleA_matched_reads.fastq.gz, /tmp/Rtmpg78x8n/file187b526fc01451/sample1_matched_reads.fastq.gz, /tmp/Rtmpg78x8n/file187b526fc01451/sample2_matched_reads.fastq.gz, /tmp/Rtmpg78x8n/file187b526fc01451/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpg78x8n/file187b526fc01451/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmpg78x8n/file187b526fc01451/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmpg78x8n/file187b526fc01451/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmpg78x8n/file187b526fc01451/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/Rtmpg78x8n/file187b526fc01451/sampleA_matched_reads_dedup.fastq.gz ->/tmp/Rtmpg78x8n/file187b526fc01451/sampleA_realign2transcript.bam
/tmp/Rtmpg78x8n/file187b526fc01451/sample1_matched_reads_dedup.fastq.gz ->/tmp/Rtmpg78x8n/file187b526fc01451/sample1_realign2transcript.bam
/tmp/Rtmpg78x8n/file187b526fc01451/sample2_matched_reads_dedup.fastq.gz ->/tmp/Rtmpg78x8n/file187b526fc01451/sample2_realign2transcript.bam
/tmp/Rtmpg78x8n/file187b526fc01451/sample3_matched_reads_dedup.fastq.gz ->/tmp/Rtmpg78x8n/file187b526fc01451/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Sun Sep  7 23:56:56 2025 ----------
2025-09-08T03:56:56.628691Z  INFO oarfish: setting user-provided filter parameters.
2025-09-08T03:56:56.629074Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpg78x8n/file187b526fc01451/sampleA_realign2transcript.bam, contains 5 reference sequences.
2025-09-08T03:56:56.629087Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-08T03:56:56.629091Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-08T03:56:56.629157Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-08T03:56:56.629164Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-09-08T03:56:56.635191Z  INFO oarfish::single_cell: Processed 100 cells.
2025-09-08T03:56:57.011006Z  INFO oarfish: setting user-provided filter parameters.
2025-09-08T03:56:57.011366Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpg78x8n/file187b526fc01451/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-09-08T03:56:57.011376Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-08T03:56:57.011380Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-08T03:56:57.011452Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-08T03:56:57.011459Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-09-08T03:56:57.375394Z  INFO oarfish: setting user-provided filter parameters.
2025-09-08T03:56:57.375965Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpg78x8n/file187b526fc01451/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-09-08T03:56:57.375979Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-08T03:56:57.375984Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-08T03:56:57.376050Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-08T03:56:57.376057Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-09-08T03:56:57.733361Z  INFO oarfish: setting user-provided filter parameters.
2025-09-08T03:56:57.733775Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpg78x8n/file187b526fc01451/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-09-08T03:56:57.733789Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-08T03:56:57.733793Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-08T03:56:57.733864Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-08T03:56:57.733885Z  INFO oarfish: parsed reference information for 5 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpg78x8n/file187b525877f969/config_file_1604434.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 @ Sun Sep  7 23:56:58 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpg78x8n/file187b525877f969/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpg78x8n/file187b525877f969/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpg78x8n/file187b525877f969/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpg78x8n/file187b525877f969/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpg78x8n/file187b525877f969/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpg78x8n/file187b525877f969/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 92
Number of reads with exactly one barcode match: 91
Number of chimera reads: 1
All done!
Reads	Barcodes
4	1
3	9
2	9
1	44
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpg78x8n/file187b525877f969/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpg78x8n/file187b525877f969/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 95
Number of reads with exactly one barcode match: 94
Number of chimera reads: 0
All done!
Reads	Barcodes
4	2
3	3
2	16
1	47
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpg78x8n/file187b525877f969/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpg78x8n/file187b525877f969/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Sun Sep  7 23:56:59 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmpg78x8n/file187b525877f969/sampleA_matched_reads.fastq.gz -> /tmp/Rtmpg78x8n/file187b525877f969/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/Rtmpg78x8n/file187b525877f969/sample1_matched_reads.fastq.gz -> /tmp/Rtmpg78x8n/file187b525877f969/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/Rtmpg78x8n/file187b525877f969/sample2_matched_reads.fastq.gz -> /tmp/Rtmpg78x8n/file187b525877f969/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/Rtmpg78x8n/file187b525877f969/sample3_matched_reads.fastq.gz -> /tmp/Rtmpg78x8n/file187b525877f969/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 @ Sun Sep  7 23:57:00 2025 ----------------
23:57:00 Sun Sep 07 2025 quantify genes 
Using BAM(s): '/tmp/Rtmpg78x8n/file187b525877f969/sampleA_align2genome.bam',
'/tmp/Rtmpg78x8n/file187b525877f969/sample1_align2genome.bam',
'/tmp/Rtmpg78x8n/file187b525877f969/sample2_align2genome.bam', and
'/tmp/Rtmpg78x8n/file187b525877f969/sample3_align2genome.bam'
parsing /tmp/Rtmpg78x8n/file187b525877f969/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 21.12gene_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, 388289.58Read/s]
parsing /tmp/Rtmpg78x8n/file187b525877f969/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 51.49gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1098675.61Read/s]
parsing /tmp/Rtmpg78x8n/file187b525877f969/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 48.93gene_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, 1300640.04Read/s]
parsing /tmp/Rtmpg78x8n/file187b525877f969/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 34.07gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 731785.89Read/s]
-- Running step: isoform_identification @ Sun Sep  7 23:57:01 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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Detected 6 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Sun Sep  7 23:57:28 2025 -------------------
Checking for fastq file(s) /tmp/Rtmpg78x8n/file187b525877f969/fastq, /tmp/Rtmpg78x8n/file187b525877f969/fastq/sample1.fq.gz, /tmp/Rtmpg78x8n/file187b525877f969/fastq/sample2.fq.gz, /tmp/Rtmpg78x8n/file187b525877f969/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpg78x8n/file187b525877f969/sampleA_matched_reads.fastq.gz, /tmp/Rtmpg78x8n/file187b525877f969/sample1_matched_reads.fastq.gz, /tmp/Rtmpg78x8n/file187b525877f969/sample2_matched_reads.fastq.gz, /tmp/Rtmpg78x8n/file187b525877f969/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpg78x8n/file187b525877f969/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmpg78x8n/file187b525877f969/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmpg78x8n/file187b525877f969/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmpg78x8n/file187b525877f969/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/Rtmpg78x8n/file187b525877f969/sampleA_matched_reads_dedup.fastq.gz -> /tmp/Rtmpg78x8n/file187b525877f969/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/Rtmpg78x8n/file187b525877f969/sample1_matched_reads_dedup.fastq.gz -> /tmp/Rtmpg78x8n/file187b525877f969/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/Rtmpg78x8n/file187b525877f969/sample2_matched_reads_dedup.fastq.gz -> /tmp/Rtmpg78x8n/file187b525877f969/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/Rtmpg78x8n/file187b525877f969/sample3_matched_reads_dedup.fastq.gz -> /tmp/Rtmpg78x8n/file187b525877f969/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 @ Sun Sep  7 23:57:29 2025 ----------
23:57:29 Sun Sep 07 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/Rtmpg78x8n/file187b525877f969/sampleA_realign2transcript.bam...
parsing /tmp/Rtmpg78x8n/file187b525877f969/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpg78x8n/file187b525877f969/sampleA_realign2transcript.bamdone
parsing /tmp/Rtmpg78x8n/file187b525877f969/sample1_realign2transcript.bam...
parsing /tmp/Rtmpg78x8n/file187b525877f969/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpg78x8n/file187b525877f969/sample1_realign2transcript.bamdone
parsing /tmp/Rtmpg78x8n/file187b525877f969/sample2_realign2transcript.bam...
parsing /tmp/Rtmpg78x8n/file187b525877f969/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpg78x8n/file187b525877f969/sample2_realign2transcript.bamdone
parsing /tmp/Rtmpg78x8n/file187b525877f969/sample3_realign2transcript.bam...
parsing /tmp/Rtmpg78x8n/file187b525877f969/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpg78x8n/file187b525877f969/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpg78x8n/file187b5241684d8/config_file_1604434.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 @ Sun Sep  7 23:57:31 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpg78x8n/file187b5241684d8/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpg78x8n/file187b5241684d8/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpg78x8n/file187b5241684d8/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpg78x8n/file187b5241684d8/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpg78x8n/file187b5241684d8/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpg78x8n/file187b5241684d8/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 92
Number of reads with exactly one barcode match: 91
Number of chimera reads: 1
All done!
Reads	Barcodes
4	1
3	9
2	9
1	44
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpg78x8n/file187b5241684d8/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpg78x8n/file187b5241684d8/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 95
Number of reads with exactly one barcode match: 94
Number of chimera reads: 0
All done!
Reads	Barcodes
4	2
3	3
2	16
1	47
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpg78x8n/file187b5241684d8/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpg78x8n/file187b5241684d8/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Sun Sep  7 23:57:32 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmpg78x8n/file187b5241684d8/sampleA_matched_reads.fastq.gz ->/tmp/Rtmpg78x8n/file187b5241684d8/sampleA_align2genome.bam
/tmp/Rtmpg78x8n/file187b5241684d8/sample1_matched_reads.fastq.gz ->/tmp/Rtmpg78x8n/file187b5241684d8/sample1_align2genome.bam
/tmp/Rtmpg78x8n/file187b5241684d8/sample2_matched_reads.fastq.gz ->/tmp/Rtmpg78x8n/file187b5241684d8/sample2_align2genome.bam
/tmp/Rtmpg78x8n/file187b5241684d8/sample3_matched_reads.fastq.gz ->/tmp/Rtmpg78x8n/file187b5241684d8/sample3_align2genome.bam
-- Running step: gene_quantification @ Sun Sep  7 23:57:52 2025 ----------------
23:57:52 Sun Sep 07 2025 quantify genes 
Using BAM(s): '/tmp/Rtmpg78x8n/file187b5241684d8/sampleA_align2genome.bam',
'/tmp/Rtmpg78x8n/file187b5241684d8/sample1_align2genome.bam',
'/tmp/Rtmpg78x8n/file187b5241684d8/sample2_align2genome.bam', and
'/tmp/Rtmpg78x8n/file187b5241684d8/sample3_align2genome.bam'
	Counter({'counted_reads': 347, 'not_enough_coverage': 9, 'unmapped': 2})
	Counter({'counted_reads': 89, 'not_enough_coverage': 2})
	Counter({'counted_reads': 92, 'not_enough_coverage': 3})
	Counter({'counted_reads': 168, 'not_enough_coverage': 6, 'unmapped': 2})
parsing /tmp/Rtmpg78x8n/file187b5241684d8/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 21.50gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 361278.94Read/s]
parsing /tmp/Rtmpg78x8n/file187b5241684d8/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 33.80gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1304523.51Read/s]
parsing /tmp/Rtmpg78x8n/file187b5241684d8/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 47.36gene_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, 1260762.29Read/s]
parsing /tmp/Rtmpg78x8n/file187b5241684d8/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 31.59gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 726815.00Read/s]
-- Running step: isoform_identification @ Sun Sep  7 23:57:53 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 6 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Sun Sep  7 23:58:18 2025 -------------------
Checking for fastq file(s) /tmp/Rtmpg78x8n/file187b5241684d8/fastq, /tmp/Rtmpg78x8n/file187b5241684d8/fastq/sample1.fq.gz, /tmp/Rtmpg78x8n/file187b5241684d8/fastq/sample2.fq.gz, /tmp/Rtmpg78x8n/file187b5241684d8/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpg78x8n/file187b5241684d8/sampleA_matched_reads.fastq.gz, /tmp/Rtmpg78x8n/file187b5241684d8/sample1_matched_reads.fastq.gz, /tmp/Rtmpg78x8n/file187b5241684d8/sample2_matched_reads.fastq.gz, /tmp/Rtmpg78x8n/file187b5241684d8/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpg78x8n/file187b5241684d8/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmpg78x8n/file187b5241684d8/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmpg78x8n/file187b5241684d8/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmpg78x8n/file187b5241684d8/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/Rtmpg78x8n/file187b5241684d8/sampleA_matched_reads_dedup.fastq.gz ->/tmp/Rtmpg78x8n/file187b5241684d8/sampleA_realign2transcript.bam
/tmp/Rtmpg78x8n/file187b5241684d8/sample1_matched_reads_dedup.fastq.gz ->/tmp/Rtmpg78x8n/file187b5241684d8/sample1_realign2transcript.bam
/tmp/Rtmpg78x8n/file187b5241684d8/sample2_matched_reads_dedup.fastq.gz ->/tmp/Rtmpg78x8n/file187b5241684d8/sample2_realign2transcript.bam
/tmp/Rtmpg78x8n/file187b5241684d8/sample3_matched_reads_dedup.fastq.gz ->/tmp/Rtmpg78x8n/file187b5241684d8/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Sun Sep  7 23:58:37 2025 ----------
23:58:37 Sun Sep 07 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/Rtmpg78x8n/file187b5241684d8/sampleA_realign2transcript.bam...
parsing /tmp/Rtmpg78x8n/file187b5241684d8/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpg78x8n/file187b5241684d8/sampleA_realign2transcript.bamdone
parsing /tmp/Rtmpg78x8n/file187b5241684d8/sample1_realign2transcript.bam...
parsing /tmp/Rtmpg78x8n/file187b5241684d8/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpg78x8n/file187b5241684d8/sample1_realign2transcript.bamdone
parsing /tmp/Rtmpg78x8n/file187b5241684d8/sample2_realign2transcript.bam...
parsing /tmp/Rtmpg78x8n/file187b5241684d8/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpg78x8n/file187b5241684d8/sample2_realign2transcript.bamdone
parsing /tmp/Rtmpg78x8n/file187b5241684d8/sample3_realign2transcript.bam...
parsing /tmp/Rtmpg78x8n/file187b5241684d8/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpg78x8n/file187b5241684d8/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpg78x8n/file187b52554eae91/config_file_1604434.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 @ Sun Sep  7 23:58:39 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpg78x8n/file187b52554eae91/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpg78x8n/file187b52554eae91/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpg78x8n/file187b52554eae91/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpg78x8n/file187b52554eae91/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpg78x8n/file187b52554eae91/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpg78x8n/file187b52554eae91/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 92
Number of reads with exactly one barcode match: 91
Number of chimera reads: 1
All done!
Reads	Barcodes
4	1
3	9
2	9
1	44
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpg78x8n/file187b52554eae91/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpg78x8n/file187b52554eae91/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 95
Number of reads with exactly one barcode match: 94
Number of chimera reads: 0
All done!
Reads	Barcodes
4	2
3	3
2	16
1	47
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpg78x8n/file187b52554eae91/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpg78x8n/file187b52554eae91/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Sun Sep  7 23:58:40 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmpg78x8n/file187b52554eae91/sampleA_matched_reads.fastq.gz -> /tmp/Rtmpg78x8n/file187b52554eae91/sampleA_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmpg78x8n/file187b52554eae91/sample1_matched_reads.fastq.gz -> /tmp/Rtmpg78x8n/file187b52554eae91/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmpg78x8n/file187b52554eae91/sample2_matched_reads.fastq.gz -> /tmp/Rtmpg78x8n/file187b52554eae91/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmpg78x8n/file187b52554eae91/sample3_matched_reads.fastq.gz -> /tmp/Rtmpg78x8n/file187b52554eae91/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Sun Sep  7 23:58:42 2025 ----------------
23:58:42 Sun Sep 07 2025 quantify genes 
Using BAM(s): '/tmp/Rtmpg78x8n/file187b52554eae91/sampleA_align2genome.bam',
'/tmp/Rtmpg78x8n/file187b52554eae91/sample1_align2genome.bam',
'/tmp/Rtmpg78x8n/file187b52554eae91/sample2_align2genome.bam', and
'/tmp/Rtmpg78x8n/file187b52554eae91/sample3_align2genome.bam'
	Counter({'counted_reads': 347, 'not_enough_coverage': 9, 'unmapped': 2})
	Counter({'counted_reads': 89, 'not_enough_coverage': 2})
	Counter({'counted_reads': 92, 'not_enough_coverage': 3})
	Counter({'counted_reads': 168, 'not_enough_coverage': 6, 'unmapped': 2})
parsing /tmp/Rtmpg78x8n/file187b52554eae91/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 22.36gene_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, 389125.32Read/s]
parsing /tmp/Rtmpg78x8n/file187b52554eae91/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 50.57gene_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, 1389210.39Read/s]
parsing /tmp/Rtmpg78x8n/file187b52554eae91/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 50.63gene_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, 1325130.80Read/s]
parsing /tmp/Rtmpg78x8n/file187b52554eae91/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 35.68gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 767906.26Read/s]
-- Running step: isoform_identification @ Sun Sep  7 23:58:43 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sun Sep  7 23:58:43 2025 -------------------
Checking for fastq file(s) /tmp/Rtmpg78x8n/file187b52554eae91/fastq, /tmp/Rtmpg78x8n/file187b52554eae91/fastq/sample1.fq.gz, /tmp/Rtmpg78x8n/file187b52554eae91/fastq/sample2.fq.gz, /tmp/Rtmpg78x8n/file187b52554eae91/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpg78x8n/file187b52554eae91/sampleA_matched_reads.fastq.gz, /tmp/Rtmpg78x8n/file187b52554eae91/sample1_matched_reads.fastq.gz, /tmp/Rtmpg78x8n/file187b52554eae91/sample2_matched_reads.fastq.gz, /tmp/Rtmpg78x8n/file187b52554eae91/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpg78x8n/file187b52554eae91/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmpg78x8n/file187b52554eae91/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmpg78x8n/file187b52554eae91/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmpg78x8n/file187b52554eae91/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/Rtmpg78x8n/file187b52554eae91/sampleA_matched_reads_dedup.fastq.gz -> /tmp/Rtmpg78x8n/file187b52554eae91/sampleA_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/Rtmpg78x8n/file187b52554eae91/sample1_matched_reads_dedup.fastq.gz -> /tmp/Rtmpg78x8n/file187b52554eae91/sample1_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/Rtmpg78x8n/file187b52554eae91/sample2_matched_reads_dedup.fastq.gz -> /tmp/Rtmpg78x8n/file187b52554eae91/sample2_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/Rtmpg78x8n/file187b52554eae91/sample3_matched_reads_dedup.fastq.gz -> /tmp/Rtmpg78x8n/file187b52554eae91/sample3_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

-- Running step: transcript_quantification @ Sun Sep  7 23:58:45 2025 ----------
2025-09-08T03:58:45.493442Z  INFO oarfish: setting user-provided filter parameters.
2025-09-08T03:58:45.493878Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpg78x8n/file187b52554eae91/sampleA_realign2transcript.bam, contains 14 reference sequences.
2025-09-08T03:58:45.493890Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-08T03:58:45.493893Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-08T03:58:45.493997Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-08T03:58:45.494004Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-09-08T03:58:45.505890Z  INFO oarfish::single_cell: Processed 100 cells.
2025-09-08T03:58:46.043647Z  INFO oarfish: setting user-provided filter parameters.
2025-09-08T03:58:46.044080Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpg78x8n/file187b52554eae91/sample1_realign2transcript.bam, contains 14 reference sequences.
2025-09-08T03:58:46.044092Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-08T03:58:46.044096Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-08T03:58:46.044190Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-08T03:58:46.044198Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-09-08T03:58:46.607178Z  INFO oarfish: setting user-provided filter parameters.
2025-09-08T03:58:46.607561Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpg78x8n/file187b52554eae91/sample2_realign2transcript.bam, contains 14 reference sequences.
2025-09-08T03:58:46.607570Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-08T03:58:46.607573Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-08T03:58:46.607662Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-08T03:58:46.607670Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-09-08T03:58:47.173437Z  INFO oarfish: setting user-provided filter parameters.
2025-09-08T03:58:47.173820Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpg78x8n/file187b52554eae91/sample3_realign2transcript.bam, contains 14 reference sequences.
2025-09-08T03:58:47.173831Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-08T03:58:47.173834Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-08T03:58:47.173921Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-08T03:58:47.173929Z  INFO oarfish: parsed reference information for 14 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpg78x8n/file187b527e03918a/config_file_1604434.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 @ Sun Sep  7 23:58:47 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpg78x8n/file187b527e03918a/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpg78x8n/file187b527e03918a/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpg78x8n/file187b527e03918a/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpg78x8n/file187b527e03918a/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpg78x8n/file187b527e03918a/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpg78x8n/file187b527e03918a/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 92
Number of reads with exactly one barcode match: 91
Number of chimera reads: 1
All done!
Reads	Barcodes
4	1
3	9
2	9
1	44
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpg78x8n/file187b527e03918a/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpg78x8n/file187b527e03918a/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 95
Number of reads with exactly one barcode match: 94
Number of chimera reads: 0
All done!
Reads	Barcodes
4	2
3	3
2	16
1	47
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpg78x8n/file187b527e03918a/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpg78x8n/file187b527e03918a/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Sun Sep  7 23:58:48 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmpg78x8n/file187b527e03918a/sampleA_matched_reads.fastq.gz ->/tmp/Rtmpg78x8n/file187b527e03918a/sampleA_align2genome.bam
/tmp/Rtmpg78x8n/file187b527e03918a/sample1_matched_reads.fastq.gz ->/tmp/Rtmpg78x8n/file187b527e03918a/sample1_align2genome.bam
/tmp/Rtmpg78x8n/file187b527e03918a/sample2_matched_reads.fastq.gz ->/tmp/Rtmpg78x8n/file187b527e03918a/sample2_align2genome.bam
/tmp/Rtmpg78x8n/file187b527e03918a/sample3_matched_reads.fastq.gz ->/tmp/Rtmpg78x8n/file187b527e03918a/sample3_align2genome.bam
-- Running step: gene_quantification @ Sun Sep  7 23:59:09 2025 ----------------
23:59:09 Sun Sep 07 2025 quantify genes 
Using BAM(s): '/tmp/Rtmpg78x8n/file187b527e03918a/sampleA_align2genome.bam',
'/tmp/Rtmpg78x8n/file187b527e03918a/sample1_align2genome.bam',
'/tmp/Rtmpg78x8n/file187b527e03918a/sample2_align2genome.bam', and
'/tmp/Rtmpg78x8n/file187b527e03918a/sample3_align2genome.bam'
parsing /tmp/Rtmpg78x8n/file187b527e03918a/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 22.41gene_group/s]
/home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/python/count_gene.py:705: RuntimeWarning: More than 20 figures have been opened. Figures created through the pyplot interface (`matplotlib.pyplot.figure`) are retained until explicitly closed and may consume too much memory. (To control this warning, see the rcParam `figure.max_open_warning`). Consider using `matplotlib.pyplot.close()`.
  plt.figure(figsize=(8, 6))
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 342380.98Read/s]
parsing /tmp/Rtmpg78x8n/file187b527e03918a/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 51.47gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1172248.18Read/s]
parsing /tmp/Rtmpg78x8n/file187b527e03918a/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 50.54gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1216304.37Read/s]
parsing /tmp/Rtmpg78x8n/file187b527e03918a/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 35.01gene_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, 635154.16Read/s]
-- Running step: isoform_identification @ Sun Sep  7 23:59:10 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sun Sep  7 23:59:10 2025 -------------------
Checking for fastq file(s) /tmp/Rtmpg78x8n/file187b527e03918a/fastq, /tmp/Rtmpg78x8n/file187b527e03918a/fastq/sample1.fq.gz, /tmp/Rtmpg78x8n/file187b527e03918a/fastq/sample2.fq.gz, /tmp/Rtmpg78x8n/file187b527e03918a/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpg78x8n/file187b527e03918a/sampleA_matched_reads.fastq.gz, /tmp/Rtmpg78x8n/file187b527e03918a/sample1_matched_reads.fastq.gz, /tmp/Rtmpg78x8n/file187b527e03918a/sample2_matched_reads.fastq.gz, /tmp/Rtmpg78x8n/file187b527e03918a/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpg78x8n/file187b527e03918a/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmpg78x8n/file187b527e03918a/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmpg78x8n/file187b527e03918a/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmpg78x8n/file187b527e03918a/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/Rtmpg78x8n/file187b527e03918a/sampleA_matched_reads_dedup.fastq.gz ->/tmp/Rtmpg78x8n/file187b527e03918a/sampleA_realign2transcript.bam
/tmp/Rtmpg78x8n/file187b527e03918a/sample1_matched_reads_dedup.fastq.gz ->/tmp/Rtmpg78x8n/file187b527e03918a/sample1_realign2transcript.bam
/tmp/Rtmpg78x8n/file187b527e03918a/sample2_matched_reads_dedup.fastq.gz ->/tmp/Rtmpg78x8n/file187b527e03918a/sample2_realign2transcript.bam
/tmp/Rtmpg78x8n/file187b527e03918a/sample3_matched_reads_dedup.fastq.gz ->/tmp/Rtmpg78x8n/file187b527e03918a/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Sun Sep  7 23:59:31 2025 ----------
2025-09-08T03:59:31.198914Z  INFO oarfish: setting user-provided filter parameters.
2025-09-08T03:59:31.199459Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpg78x8n/file187b527e03918a/sampleA_realign2transcript.bam, contains 14 reference sequences.
2025-09-08T03:59:31.199480Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-08T03:59:31.199483Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-08T03:59:31.199566Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-08T03:59:31.199574Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-09-08T03:59:31.211029Z  INFO oarfish::single_cell: Processed 100 cells.
2025-09-08T03:59:31.905095Z  INFO oarfish: setting user-provided filter parameters.
2025-09-08T03:59:31.905474Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpg78x8n/file187b527e03918a/sample1_realign2transcript.bam, contains 14 reference sequences.
2025-09-08T03:59:31.905484Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-08T03:59:31.905487Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-08T03:59:31.905579Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-08T03:59:31.905587Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-09-08T03:59:32.570360Z  INFO oarfish: setting user-provided filter parameters.
2025-09-08T03:59:32.570744Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpg78x8n/file187b527e03918a/sample2_realign2transcript.bam, contains 14 reference sequences.
2025-09-08T03:59:32.570757Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-08T03:59:32.570760Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-08T03:59:32.570852Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-08T03:59:32.570861Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-09-08T03:59:33.204235Z  INFO oarfish: setting user-provided filter parameters.
2025-09-08T03:59:33.204698Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpg78x8n/file187b527e03918a/sample3_realign2transcript.bam, contains 14 reference sequences.
2025-09-08T03:59:33.204707Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-08T03:59:33.204710Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-08T03:59:33.204838Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-08T03:59:33.204850Z  INFO oarfish: parsed reference information for 14 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpg78x8n/file187b523a0d2e4c/config_file_1604434.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 @ Sun Sep  7 23:59:34 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpg78x8n/file187b523a0d2e4c/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpg78x8n/file187b523a0d2e4c/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpg78x8n/file187b523a0d2e4c/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpg78x8n/file187b523a0d2e4c/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpg78x8n/file187b523a0d2e4c/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpg78x8n/file187b523a0d2e4c/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 92
Number of reads with exactly one barcode match: 91
Number of chimera reads: 1
All done!
Reads	Barcodes
4	1
3	9
2	9
1	44
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpg78x8n/file187b523a0d2e4c/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpg78x8n/file187b523a0d2e4c/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 95
Number of reads with exactly one barcode match: 94
Number of chimera reads: 0
All done!
Reads	Barcodes
4	2
3	3
2	16
1	47
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpg78x8n/file187b523a0d2e4c/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpg78x8n/file187b523a0d2e4c/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Sun Sep  7 23:59:34 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmpg78x8n/file187b523a0d2e4c/sampleA_matched_reads.fastq.gz -> /tmp/Rtmpg78x8n/file187b523a0d2e4c/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/Rtmpg78x8n/file187b523a0d2e4c/sample1_matched_reads.fastq.gz -> /tmp/Rtmpg78x8n/file187b523a0d2e4c/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/Rtmpg78x8n/file187b523a0d2e4c/sample2_matched_reads.fastq.gz -> /tmp/Rtmpg78x8n/file187b523a0d2e4c/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/Rtmpg78x8n/file187b523a0d2e4c/sample3_matched_reads.fastq.gz -> /tmp/Rtmpg78x8n/file187b523a0d2e4c/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 @ Sun Sep  7 23:59:36 2025 ----------------
23:59:36 Sun Sep 07 2025 quantify genes 
Using BAM(s): '/tmp/Rtmpg78x8n/file187b523a0d2e4c/sampleA_align2genome.bam',
'/tmp/Rtmpg78x8n/file187b523a0d2e4c/sample1_align2genome.bam',
'/tmp/Rtmpg78x8n/file187b523a0d2e4c/sample2_align2genome.bam', and
'/tmp/Rtmpg78x8n/file187b523a0d2e4c/sample3_align2genome.bam'
parsing /tmp/Rtmpg78x8n/file187b523a0d2e4c/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 22.12gene_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, 384150.06Read/s]
parsing /tmp/Rtmpg78x8n/file187b523a0d2e4c/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  8.44gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  8.42gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1260004.81Read/s]
parsing /tmp/Rtmpg78x8n/file187b523a0d2e4c/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 52.15gene_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, 1224685.82Read/s]
parsing /tmp/Rtmpg78x8n/file187b523a0d2e4c/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 36.19gene_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, 691764.08Read/s]
-- Running step: isoform_identification @ Sun Sep  7 23:59:37 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sun Sep  7 23:59:37 2025 -------------------
Checking for fastq file(s) /tmp/Rtmpg78x8n/file187b523a0d2e4c/fastq, /tmp/Rtmpg78x8n/file187b523a0d2e4c/fastq/sample1.fq.gz, /tmp/Rtmpg78x8n/file187b523a0d2e4c/fastq/sample2.fq.gz, /tmp/Rtmpg78x8n/file187b523a0d2e4c/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpg78x8n/file187b523a0d2e4c/sampleA_matched_reads.fastq.gz, /tmp/Rtmpg78x8n/file187b523a0d2e4c/sample1_matched_reads.fastq.gz, /tmp/Rtmpg78x8n/file187b523a0d2e4c/sample2_matched_reads.fastq.gz, /tmp/Rtmpg78x8n/file187b523a0d2e4c/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpg78x8n/file187b523a0d2e4c/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmpg78x8n/file187b523a0d2e4c/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmpg78x8n/file187b523a0d2e4c/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmpg78x8n/file187b523a0d2e4c/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/Rtmpg78x8n/file187b523a0d2e4c/sampleA_matched_reads_dedup.fastq.gz -> /tmp/Rtmpg78x8n/file187b523a0d2e4c/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/Rtmpg78x8n/file187b523a0d2e4c/sample1_matched_reads_dedup.fastq.gz -> /tmp/Rtmpg78x8n/file187b523a0d2e4c/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/Rtmpg78x8n/file187b523a0d2e4c/sample2_matched_reads_dedup.fastq.gz -> /tmp/Rtmpg78x8n/file187b523a0d2e4c/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/Rtmpg78x8n/file187b523a0d2e4c/sample3_matched_reads_dedup.fastq.gz -> /tmp/Rtmpg78x8n/file187b523a0d2e4c/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 @ Sun Sep  7 23:59:38 2025 ----------
23:59:38 Sun Sep 07 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/Rtmpg78x8n/file187b523a0d2e4c/sampleA_realign2transcript.bam...
parsing /tmp/Rtmpg78x8n/file187b523a0d2e4c/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpg78x8n/file187b523a0d2e4c/sampleA_realign2transcript.bamdone
parsing /tmp/Rtmpg78x8n/file187b523a0d2e4c/sample1_realign2transcript.bam...
parsing /tmp/Rtmpg78x8n/file187b523a0d2e4c/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpg78x8n/file187b523a0d2e4c/sample1_realign2transcript.bamdone
parsing /tmp/Rtmpg78x8n/file187b523a0d2e4c/sample2_realign2transcript.bam...
parsing /tmp/Rtmpg78x8n/file187b523a0d2e4c/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpg78x8n/file187b523a0d2e4c/sample2_realign2transcript.bamdone
parsing /tmp/Rtmpg78x8n/file187b523a0d2e4c/sample3_realign2transcript.bam...
parsing /tmp/Rtmpg78x8n/file187b523a0d2e4c/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpg78x8n/file187b523a0d2e4c/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpg78x8n/file187b523912ac3a/config_file_1604434.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 @ Sun Sep  7 23:59:41 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpg78x8n/file187b523912ac3a/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpg78x8n/file187b523912ac3a/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpg78x8n/file187b523912ac3a/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpg78x8n/file187b523912ac3a/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpg78x8n/file187b523912ac3a/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpg78x8n/file187b523912ac3a/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 92
Number of reads with exactly one barcode match: 91
Number of chimera reads: 1
All done!
Reads	Barcodes
4	1
3	9
2	9
1	44
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpg78x8n/file187b523912ac3a/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpg78x8n/file187b523912ac3a/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 95
Number of reads with exactly one barcode match: 94
Number of chimera reads: 0
All done!
Reads	Barcodes
4	2
3	3
2	16
1	47
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpg78x8n/file187b523912ac3a/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpg78x8n/file187b523912ac3a/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Sun Sep  7 23:59:42 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmpg78x8n/file187b523912ac3a/sampleA_matched_reads.fastq.gz ->/tmp/Rtmpg78x8n/file187b523912ac3a/sampleA_align2genome.bam
/tmp/Rtmpg78x8n/file187b523912ac3a/sample1_matched_reads.fastq.gz ->/tmp/Rtmpg78x8n/file187b523912ac3a/sample1_align2genome.bam
/tmp/Rtmpg78x8n/file187b523912ac3a/sample2_matched_reads.fastq.gz ->/tmp/Rtmpg78x8n/file187b523912ac3a/sample2_align2genome.bam
/tmp/Rtmpg78x8n/file187b523912ac3a/sample3_matched_reads.fastq.gz ->/tmp/Rtmpg78x8n/file187b523912ac3a/sample3_align2genome.bam
-- Running step: gene_quantification @ Mon Sep  8 00:00:03 2025 ----------------
00:00:03 Mon Sep 08 2025 quantify genes 
Using BAM(s): '/tmp/Rtmpg78x8n/file187b523912ac3a/sampleA_align2genome.bam',
'/tmp/Rtmpg78x8n/file187b523912ac3a/sample1_align2genome.bam',
'/tmp/Rtmpg78x8n/file187b523912ac3a/sample2_align2genome.bam', and
'/tmp/Rtmpg78x8n/file187b523912ac3a/sample3_align2genome.bam'
	Counter({'counted_reads': 358})
	Counter({'counted_reads': 91})
	Counter({'counted_reads': 95})
	Counter({'counted_reads': 176})
parsing /tmp/Rtmpg78x8n/file187b523912ac3a/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 22.28gene_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, 425956.05Read/s]
parsing /tmp/Rtmpg78x8n/file187b523912ac3a/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 53.10gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1447110.13Read/s]
parsing /tmp/Rtmpg78x8n/file187b523912ac3a/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 51.62gene_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, 1354749.35Read/s]
parsing /tmp/Rtmpg78x8n/file187b523912ac3a/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 36.22gene_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, 745839.68Read/s]
-- Running step: isoform_identification @ Mon Sep  8 00:00:04 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Mon Sep  8 00:00:04 2025 -------------------
Checking for fastq file(s) /tmp/Rtmpg78x8n/file187b523912ac3a/fastq, /tmp/Rtmpg78x8n/file187b523912ac3a/fastq/sample1.fq.gz, /tmp/Rtmpg78x8n/file187b523912ac3a/fastq/sample2.fq.gz, /tmp/Rtmpg78x8n/file187b523912ac3a/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpg78x8n/file187b523912ac3a/sampleA_matched_reads.fastq.gz, /tmp/Rtmpg78x8n/file187b523912ac3a/sample1_matched_reads.fastq.gz, /tmp/Rtmpg78x8n/file187b523912ac3a/sample2_matched_reads.fastq.gz, /tmp/Rtmpg78x8n/file187b523912ac3a/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpg78x8n/file187b523912ac3a/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmpg78x8n/file187b523912ac3a/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmpg78x8n/file187b523912ac3a/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmpg78x8n/file187b523912ac3a/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/Rtmpg78x8n/file187b523912ac3a/sampleA_matched_reads_dedup.fastq.gz ->/tmp/Rtmpg78x8n/file187b523912ac3a/sampleA_realign2transcript.bam
/tmp/Rtmpg78x8n/file187b523912ac3a/sample1_matched_reads_dedup.fastq.gz ->/tmp/Rtmpg78x8n/file187b523912ac3a/sample1_realign2transcript.bam
/tmp/Rtmpg78x8n/file187b523912ac3a/sample2_matched_reads_dedup.fastq.gz ->/tmp/Rtmpg78x8n/file187b523912ac3a/sample2_realign2transcript.bam
/tmp/Rtmpg78x8n/file187b523912ac3a/sample3_matched_reads_dedup.fastq.gz ->/tmp/Rtmpg78x8n/file187b523912ac3a/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Mon Sep  8 00:00:24 2025 ----------
00:00:24 Mon Sep 08 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/Rtmpg78x8n/file187b523912ac3a/sampleA_realign2transcript.bam...
parsing /tmp/Rtmpg78x8n/file187b523912ac3a/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpg78x8n/file187b523912ac3a/sampleA_realign2transcript.bamdone
parsing /tmp/Rtmpg78x8n/file187b523912ac3a/sample1_realign2transcript.bam...
parsing /tmp/Rtmpg78x8n/file187b523912ac3a/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpg78x8n/file187b523912ac3a/sample1_realign2transcript.bamdone
parsing /tmp/Rtmpg78x8n/file187b523912ac3a/sample2_realign2transcript.bam...
parsing /tmp/Rtmpg78x8n/file187b523912ac3a/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpg78x8n/file187b523912ac3a/sample2_realign2transcript.bamdone
parsing /tmp/Rtmpg78x8n/file187b523912ac3a/sample3_realign2transcript.bam...
parsing /tmp/Rtmpg78x8n/file187b523912ac3a/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpg78x8n/file187b523912ac3a/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
[ FAIL 0 | WARN 183 | SKIP 0 | PASS 59 ]

[ FAIL 0 | WARN 183 | SKIP 0 | PASS 59 ]
	Counter({'counted_reads': 358})
	Counter({'counted_reads': 91})
	Counter({'counted_reads': 95})
	Counter({'counted_reads': 176})
> 
> proc.time()
    user   system  elapsed 
1716.479   49.650  797.458