Back to Multiple platform build/check report for BioC 3.22:   simplified   long
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This page was generated on 2025-08-30 12:04 -0400 (Sat, 30 Aug 2025).

HostnameOSArch (*)R versionInstalled pkgs
nebbiolo2Linux (Ubuntu 24.04.3 LTS)x86_644.5.1 (2025-06-13) -- "Great Square Root" 4824
lconwaymacOS 12.7.1 Montereyx86_644.5.1 (2025-06-13) -- "Great Square Root" 4615
kjohnson3macOS 13.7.7 Venturaarm644.5.1 Patched (2025-06-14 r88325) -- "Great Square Root" 4562
taishanLinux (openEuler 24.03 LTS)aarch644.5.0 (2025-04-11) -- "How About a Twenty-Six" 4541
Click on any hostname to see more info about the system (e.g. compilers)      (*) as reported by 'uname -p', except on Windows and Mac OS X

Package 730/2320HostnameOS / ArchINSTALLBUILDCHECKBUILD BIN
FLAMES 2.3.5  (landing page)
Changqing Wang
Snapshot Date: 2025-08-29 13:45 -0400 (Fri, 29 Aug 2025)
git_url: https://git.bioconductor.org/packages/FLAMES
git_branch: devel
git_last_commit: 1f17e9d
git_last_commit_date: 2025-08-25 02:02:57 -0400 (Mon, 25 Aug 2025)
nebbiolo2Linux (Ubuntu 24.04.3 LTS) / x86_64  OK    OK    OK  UNNEEDED, same version is already published
lconwaymacOS 12.7.1 Monterey / x86_64  OK    OK    OK    OK  UNNEEDED, same version is already published
kjohnson3macOS 13.7.7 Ventura / arm64  OK    OK    OK    OK  UNNEEDED, same version is already published
taishanLinux (openEuler 24.03 LTS) / aarch64  ERROR    ERROR  skipped


CHECK results for FLAMES on nebbiolo2

To the developers/maintainers of the FLAMES package:
- Allow up to 24 hours (and sometimes 48 hours) for your latest push to git@git.bioconductor.org:packages/FLAMES.git to reflect on this report. See Troubleshooting Build Report for more information.
- Use the following Renviron settings to reproduce errors and warnings.
- If 'R CMD check' started to fail recently on the Linux builder(s) over a missing dependency, add the missing dependency to 'Suggests:' in your DESCRIPTION file. See Renviron.bioc for more information.

raw results


Summary

Package: FLAMES
Version: 2.3.5
Command: /home/biocbuild/bbs-3.22-bioc/R/bin/R CMD check --install=check:FLAMES.install-out.txt --library=/home/biocbuild/bbs-3.22-bioc/R/site-library --timings FLAMES_2.3.5.tar.gz
StartedAt: 2025-08-29 22:45:15 -0400 (Fri, 29 Aug 2025)
EndedAt: 2025-08-29 23:06:59 -0400 (Fri, 29 Aug 2025)
EllapsedTime: 1304.4 seconds
RetCode: 0
Status:   OK  
CheckDir: FLAMES.Rcheck
Warnings: 0

Command output

##############################################################################
##############################################################################
###
### Running command:
###
###   /home/biocbuild/bbs-3.22-bioc/R/bin/R CMD check --install=check:FLAMES.install-out.txt --library=/home/biocbuild/bbs-3.22-bioc/R/site-library --timings FLAMES_2.3.5.tar.gz
###
##############################################################################
##############################################################################


* using log directory ‘/home/biocbuild/bbs-3.22-bioc/meat/FLAMES.Rcheck’
* using R version 4.5.1 (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
find_variants                21.997  1.771  23.159
plot_isoform_reduced_dim     22.751  0.709  23.461
blaze                         4.417 14.184  10.590
bulk_long_pipeline            2.366 11.047   2.573
sc_long_multisample_pipeline  7.963  5.219   7.592
MultiSampleSCPipeline        10.123  0.711  11.361
sc_plot_genotype             10.015  0.238   9.112
sc_DTU_analysis               7.010  1.973   6.813
plot_isoform_heatmap          6.655  0.289   6.943
create_sce_from_dir           3.533  2.182   3.650
* checking for unstated dependencies in ‘tests’ ... OK
* checking tests ...
  Running ‘testthat.R’
 OK
* checking for unstated dependencies in vignettes ... OK
* checking package vignettes ... OK
* checking re-building of vignette outputs ... OK
* checking PDF version of manual ... OK
* DONE

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


Installation output

FLAMES.Rcheck/00install.out

##############################################################################
##############################################################################
###
### Running command:
###
###   /home/biocbuild/bbs-3.22-bioc/R/bin/R CMD INSTALL FLAMES
###
##############################################################################
##############################################################################


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

Tests output

FLAMES.Rcheck/tests/testthat.Rout


R version 4.5.1 (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.

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

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

> # This file is part of the standard setup for testthat.
> # It is recommended that you do not modify it.
> #
> # Where should you do additional test configuration?
> # Learn more about the roles of various files in:
> # * https://r-pkgs.org/tests.html
> # * https://testthat.r-lib.org/reference/test_package.html#special-files
> 
> library(testthat)
> library(FLAMES)
> 
> test_check("FLAMES")
Writing configuration parameters to:  /tmp/RtmpHV1zTz/file36fe2a48db89ca/config_file_3604010.json 
Writing configuration parameters to:  /tmp/RtmpHV1zTz/file36fe2a48db89ca/config_file_3604010.json 
Writing configuration parameters to:  /tmp/RtmpHV1zTz/file36fe2a48db89ca/config_file_3604010.json 
Writing configuration parameters to:  /tmp/RtmpHV1zTz/file36fe2a5b5eb218/config_file_3604010.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/RtmpHV1zTz/file36fe2a903b9cd/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/RtmpHV1zTz/file36fe2a766f6626/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/RtmpHV1zTz/file36fe2a766f6626/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpHV1zTz/file36fe2a42e0262b/musc_rps24_1.fastq
Searching for barcodes...
Processing file: /tmp/RtmpHV1zTz/file36fe2a42e0262b/musc_rps24_2.fastq
Searching for barcodes...
Processing file: /tmp/RtmpHV1zTz/file36fe2a42e0262b/musc_rps24_3.fastq
Searching for barcodes...
Processing file: /tmp/RtmpHV1zTz/file36fe2a42e0262b/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/RtmpHV1zTz/file36fe2a4ea34cf0/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/RtmpHV1zTz/file36fe2a30513693/config_file_3604010.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Fri Aug 29 22:53:46 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpHV1zTz/file36fe2a30513693/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpHV1zTz/file36fe2a30513693/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpHV1zTz/file36fe2a30513693/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Fri Aug 29 22:53:47 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

  |                                                                            
  |                                                                      |   0%[22:53:53] 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.

[22:53:53] 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.

[22:53:53] 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.

[22:53:53] 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.

[22:53:55] 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.

[22:53:55] WARNING: src/learner.cc:553: 
  If you are loading a serialized model (like pickle in Python, RDS in R) generated by
  older XGBoost, please export the model by calling `Booster.save_model` from that version
  first, then load it back in current version. See:

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

  for more details about differences between saving model and serializing.


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

Detected 4 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Fri Aug 29 22:54:10 2025 -------------------
Realigning sample sample1 -> /tmp/RtmpHV1zTz/file36fe2a30513693/sample1_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample2 -> /tmp/RtmpHV1zTz/file36fe2a30513693/sample2_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample3 -> /tmp/RtmpHV1zTz/file36fe2a30513693/sample3_realign2transcript.bam
Skipped sorting BAM files.

-- Running step: transcript_quantification @ Fri Aug 29 22:54:10 2025 ----------
2025-08-30T02:54:10.693144Z  INFO oarfish: setting user-provided filter parameters.
2025-08-30T02:54:10.693501Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpHV1zTz/file36fe2a30513693/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-08-30T02:54:10.693513Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-30T02:54:10.693516Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-30T02:54:10.693572Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-30T02:54:10.693578Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-08-30T02:54:10.695056Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-08-30T02:54:10.695182Z  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    │
╰─────────────────────────────────┴───────╯

2025-08-30T02:54:10.695213Z  INFO oarfish::bulk: Total number of alignment records : 116
2025-08-30T02:54:10.695225Z  INFO oarfish::bulk: number of aligned reads : 96
2025-08-30T02:54:10.695227Z  INFO oarfish::bulk: number of unique alignments : 86
2025-08-30T02:54:10.695810Z  INFO oarfish: oarfish completed successfully.
2025-08-30T02:54:10.702646Z  INFO oarfish: setting user-provided filter parameters.
2025-08-30T02:54:10.702960Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpHV1zTz/file36fe2a30513693/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-08-30T02:54:10.702968Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-30T02:54:10.702970Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-30T02:54:10.703017Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-30T02:54:10.703021Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-08-30T02:54:10.704549Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-08-30T02:54:10.704665Z  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    │
╰─────────────────────────────────┴───────╯

2025-08-30T02:54:10.704688Z  INFO oarfish::bulk: Total number of alignment records : 114
2025-08-30T02:54:10.704691Z  INFO oarfish::bulk: number of aligned reads : 95
2025-08-30T02:54:10.704693Z  INFO oarfish::bulk: number of unique alignments : 82
2025-08-30T02:54:10.705277Z  INFO oarfish: oarfish completed successfully.
2025-08-30T02:54:10.711981Z  INFO oarfish: setting user-provided filter parameters.
2025-08-30T02:54:10.712412Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpHV1zTz/file36fe2a30513693/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-08-30T02:54:10.712422Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-30T02:54:10.712425Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-30T02:54:10.712475Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-30T02:54:10.712480Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-08-30T02:54:10.715220Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2025-08-30T02:54:10.715367Z  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   │
╰─────────────────────────────────┴───────╯

2025-08-30T02:54:10.715402Z  INFO oarfish::bulk: Total number of alignment records : 239
2025-08-30T02:54:10.715405Z  INFO oarfish::bulk: number of aligned reads : 179
2025-08-30T02:54:10.715407Z  INFO oarfish::bulk: number of unique alignments : 143
2025-08-30T02:54:10.716084Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpHV1zTz/file36fe2a2b525b91/config_file_3604010.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Fri Aug 29 22:54:10 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpHV1zTz/file36fe2a2b525b91/sample1_align2genome.bam
sample2 ->/tmp/RtmpHV1zTz/file36fe2a2b525b91/sample2_align2genome.bam
sample3 ->/tmp/RtmpHV1zTz/file36fe2a2b525b91/sample3_align2genome.bam
-- Running step: isoform_identification @ Fri Aug 29 22:54:30 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 4 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Fri Aug 29 22:54:49 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpHV1zTz/file36fe2a2b525b91/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpHV1zTz/file36fe2a2b525b91/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpHV1zTz/file36fe2a2b525b91/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Fri Aug 29 22:55:09 2025 ----------
2025-08-30T02:55:09.746447Z  INFO oarfish: setting user-provided filter parameters.
2025-08-30T02:55:09.747164Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpHV1zTz/file36fe2a2b525b91/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-08-30T02:55:09.747204Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-30T02:55:09.747208Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-30T02:55:09.747278Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-30T02:55:09.747284Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-08-30T02:55:09.749204Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-08-30T02:55:09.749351Z  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    │
╰─────────────────────────────────┴───────╯

2025-08-30T02:55:09.749374Z  INFO oarfish::bulk: Total number of alignment records : 116
2025-08-30T02:55:09.749377Z  INFO oarfish::bulk: number of aligned reads : 96
2025-08-30T02:55:09.749380Z  INFO oarfish::bulk: number of unique alignments : 86
2025-08-30T02:55:09.750017Z  INFO oarfish: oarfish completed successfully.
2025-08-30T02:55:09.759432Z  INFO oarfish: setting user-provided filter parameters.
2025-08-30T02:55:09.760009Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpHV1zTz/file36fe2a2b525b91/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-08-30T02:55:09.760019Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-30T02:55:09.760024Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-30T02:55:09.760090Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-30T02:55:09.760096Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-08-30T02:55:09.761962Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-08-30T02:55:09.762112Z  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    │
╰─────────────────────────────────┴───────╯

2025-08-30T02:55:09.762138Z  INFO oarfish::bulk: Total number of alignment records : 114
2025-08-30T02:55:09.762153Z  INFO oarfish::bulk: number of aligned reads : 95
2025-08-30T02:55:09.762155Z  INFO oarfish::bulk: number of unique alignments : 82
2025-08-30T02:55:09.762807Z  INFO oarfish: oarfish completed successfully.
2025-08-30T02:55:09.770192Z  INFO oarfish: setting user-provided filter parameters.
2025-08-30T02:55:09.770584Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpHV1zTz/file36fe2a2b525b91/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-08-30T02:55:09.770594Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-30T02:55:09.770597Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-30T02:55:09.770651Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-30T02:55:09.770657Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-08-30T02:55:09.773375Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2025-08-30T02:55:09.773537Z  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   │
╰─────────────────────────────────┴───────╯

2025-08-30T02:55:09.773564Z  INFO oarfish::bulk: Total number of alignment records : 239
2025-08-30T02:55:09.773567Z  INFO oarfish::bulk: number of aligned reads : 179
2025-08-30T02:55:09.773570Z  INFO oarfish::bulk: number of unique alignments : 143
2025-08-30T02:55:09.774229Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpHV1zTz/file36fe2a9b38fb6/config_file_3604010.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Fri Aug 29 22:55:09 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpHV1zTz/file36fe2a9b38fb6/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpHV1zTz/file36fe2a9b38fb6/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpHV1zTz/file36fe2a9b38fb6/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Fri Aug 29 22:55:10 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 4 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Fri Aug 29 22:55:28 2025 -------------------
Realigning sample sample1 -> /tmp/RtmpHV1zTz/file36fe2a9b38fb6/sample1_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample2 -> /tmp/RtmpHV1zTz/file36fe2a9b38fb6/sample2_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample3 -> /tmp/RtmpHV1zTz/file36fe2a9b38fb6/sample3_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Fri Aug 29 22:55:29 2025 ----------
22:55:29 Fri Aug 29 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/RtmpHV1zTz/file36fe2a7cdb69f5/config_file_3604010.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Fri Aug 29 22:55:30 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpHV1zTz/file36fe2a7cdb69f5/sample1_align2genome.bam
sample2 ->/tmp/RtmpHV1zTz/file36fe2a7cdb69f5/sample2_align2genome.bam
sample3 ->/tmp/RtmpHV1zTz/file36fe2a7cdb69f5/sample3_align2genome.bam
-- Running step: isoform_identification @ Fri Aug 29 22:55:50 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 4 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Fri Aug 29 22:56:07 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpHV1zTz/file36fe2a7cdb69f5/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpHV1zTz/file36fe2a7cdb69f5/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpHV1zTz/file36fe2a7cdb69f5/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Fri Aug 29 22:56:27 2025 ----------
22:56:27 Fri Aug 29 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Inputs:  ['/tmp/RtmpHV1zTz/file36fe2a9b38fb6/sample1_realign2transcript.bam', '/tmp/RtmpHV1zTz/file36fe2a9b38fb6/sample2_realign2transcript.bam', '/tmp/RtmpHV1zTz/file36fe2a9b38fb6/sample3_realign2transcript.bam'] /tmp/RtmpHV1zTz/file36fe2a9b38fb6/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/RtmpHV1zTz/file36fe2a26b59f1e/config_file_3604010.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Fri Aug 29 22:56:28 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpHV1zTz/file36fe2a26b59f1e/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpHV1zTz/file36fe2a26b59f1e/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpHV1zTz/file36fe2a26b59f1e/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Fri Aug 29 22:56:29 2025 -------------
Inputs:  ['/tmp/RtmpHV1zTz/file36fe2a7cdb69f5/sample1_realign2transcript.bam', '/tmp/RtmpHV1zTz/file36fe2a7cdb69f5/sample2_realign2transcript.bam', '/tmp/RtmpHV1zTz/file36fe2a7cdb69f5/sample3_realign2transcript.bam'] /tmp/RtmpHV1zTz/file36fe2a7cdb69f5/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 @ Fri Aug 29 22:56:29 2025 -------------------
Realigning sample sample1 -> /tmp/RtmpHV1zTz/file36fe2a26b59f1e/sample1_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample2 -> /tmp/RtmpHV1zTz/file36fe2a26b59f1e/sample2_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample3 -> /tmp/RtmpHV1zTz/file36fe2a26b59f1e/sample3_realign2transcript.bam
Skipped sorting BAM files.

-- Running step: transcript_quantification @ Fri Aug 29 22:56:30 2025 ----------
2025-08-30T02:56:30.248719Z  INFO oarfish: setting user-provided filter parameters.
2025-08-30T02:56:30.249130Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpHV1zTz/file36fe2a26b59f1e/sample1_realign2transcript.bam, contains 10 reference sequences.
2025-08-30T02:56:30.249141Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-30T02:56:30.249145Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-30T02:56:30.249223Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-30T02:56:30.249232Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-08-30T02:56:30.251891Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-08-30T02:56:30.252041Z  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    │
╰─────────────────────────────────┴───────╯

2025-08-30T02:56:30.252062Z  INFO oarfish::bulk: Total number of alignment records : 125
2025-08-30T02:56:30.252064Z  INFO oarfish::bulk: number of aligned reads : 98
2025-08-30T02:56:30.252067Z  INFO oarfish::bulk: number of unique alignments : 86
2025-08-30T02:56:30.252688Z  INFO oarfish: oarfish completed successfully.
2025-08-30T02:56:30.261736Z  INFO oarfish: setting user-provided filter parameters.
2025-08-30T02:56:30.262153Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpHV1zTz/file36fe2a26b59f1e/sample2_realign2transcript.bam, contains 10 reference sequences.
2025-08-30T02:56:30.262161Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-30T02:56:30.262164Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-30T02:56:30.262241Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-30T02:56:30.262252Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-08-30T02:56:30.265091Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-08-30T02:56:30.265251Z  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    │
╰─────────────────────────────────┴───────╯

2025-08-30T02:56:30.265283Z  INFO oarfish::bulk: Total number of alignment records : 136
2025-08-30T02:56:30.265286Z  INFO oarfish::bulk: number of aligned reads : 97
2025-08-30T02:56:30.265288Z  INFO oarfish::bulk: number of unique alignments : 79
2025-08-30T02:56:30.265925Z  INFO oarfish: oarfish completed successfully.
2025-08-30T02:56:30.273597Z  INFO oarfish: setting user-provided filter parameters.
2025-08-30T02:56:30.274079Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpHV1zTz/file36fe2a26b59f1e/sample3_realign2transcript.bam, contains 10 reference sequences.
2025-08-30T02:56:30.274087Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-30T02:56:30.274090Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-30T02:56:30.274158Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-30T02:56:30.274165Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-08-30T02:56:30.278729Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-08-30T02:56:30.278895Z  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   │
╰─────────────────────────────────┴───────╯

2025-08-30T02:56:30.278923Z  INFO oarfish::bulk: Total number of alignment records : 272
2025-08-30T02:56:30.278925Z  INFO oarfish::bulk: number of aligned reads : 187
2025-08-30T02:56:30.278927Z  INFO oarfish::bulk: number of unique alignments : 140
2025-08-30T02:56:30.279670Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpHV1zTz/file36fe2af1ee2f0/config_file_3604010.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Fri Aug 29 22:56:30 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpHV1zTz/file36fe2af1ee2f0/sample1_align2genome.bam
sample2 ->/tmp/RtmpHV1zTz/file36fe2af1ee2f0/sample2_align2genome.bam
sample3 ->/tmp/RtmpHV1zTz/file36fe2af1ee2f0/sample3_align2genome.bam
-- Running step: isoform_identification @ Fri Aug 29 22:56:49 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri Aug 29 22:56:50 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpHV1zTz/file36fe2af1ee2f0/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpHV1zTz/file36fe2af1ee2f0/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpHV1zTz/file36fe2af1ee2f0/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Fri Aug 29 22:57:09 2025 ----------
2025-08-30T02:57:09.674360Z  INFO oarfish: setting user-provided filter parameters.
2025-08-30T02:57:09.674834Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpHV1zTz/file36fe2af1ee2f0/sample1_realign2transcript.bam, contains 10 reference sequences.
2025-08-30T02:57:09.674845Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-30T02:57:09.674857Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-30T02:57:09.674939Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-30T02:57:09.674946Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-08-30T02:57:09.677498Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-08-30T02:57:09.677619Z  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    │
╰─────────────────────────────────┴───────╯

2025-08-30T02:57:09.677641Z  INFO oarfish::bulk: Total number of alignment records : 125
2025-08-30T02:57:09.677644Z  INFO oarfish::bulk: number of aligned reads : 98
2025-08-30T02:57:09.677646Z  INFO oarfish::bulk: number of unique alignments : 86
2025-08-30T02:57:09.678266Z  INFO oarfish: oarfish completed successfully.
2025-08-30T02:57:09.689507Z  INFO oarfish: setting user-provided filter parameters.
2025-08-30T02:57:09.689853Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpHV1zTz/file36fe2af1ee2f0/sample2_realign2transcript.bam, contains 10 reference sequences.
2025-08-30T02:57:09.689861Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-30T02:57:09.689864Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-30T02:57:09.689926Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-30T02:57:09.689932Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-08-30T02:57:09.692431Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-08-30T02:57:09.692565Z  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    │
╰─────────────────────────────────┴───────╯

2025-08-30T02:57:09.692590Z  INFO oarfish::bulk: Total number of alignment records : 136
2025-08-30T02:57:09.692592Z  INFO oarfish::bulk: number of aligned reads : 97
2025-08-30T02:57:09.692600Z  INFO oarfish::bulk: number of unique alignments : 79
2025-08-30T02:57:09.693240Z  INFO oarfish: oarfish completed successfully.
2025-08-30T02:57:09.705433Z  INFO oarfish: setting user-provided filter parameters.
2025-08-30T02:57:09.705797Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpHV1zTz/file36fe2af1ee2f0/sample3_realign2transcript.bam, contains 10 reference sequences.
2025-08-30T02:57:09.705807Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-30T02:57:09.705810Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-30T02:57:09.705881Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-30T02:57:09.705887Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-08-30T02:57:09.710371Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-08-30T02:57:09.710545Z  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   │
╰─────────────────────────────────┴───────╯

2025-08-30T02:57:09.710573Z  INFO oarfish::bulk: Total number of alignment records : 272
2025-08-30T02:57:09.710576Z  INFO oarfish::bulk: number of aligned reads : 187
2025-08-30T02:57:09.710578Z  INFO oarfish::bulk: number of unique alignments : 140
2025-08-30T02:57:09.711281Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpHV1zTz/file36fe2a4138480e/config_file_3604010.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Fri Aug 29 22:57:09 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpHV1zTz/file36fe2a4138480e/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpHV1zTz/file36fe2a4138480e/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpHV1zTz/file36fe2a4138480e/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Fri Aug 29 22:57:10 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri Aug 29 22:57:11 2025 -------------------
Realigning sample sample1 -> /tmp/RtmpHV1zTz/file36fe2a4138480e/sample1_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample2 -> /tmp/RtmpHV1zTz/file36fe2a4138480e/sample2_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample3 -> /tmp/RtmpHV1zTz/file36fe2a4138480e/sample3_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Fri Aug 29 22:57:11 2025 ----------
22:57:11 Fri Aug 29 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/RtmpHV1zTz/file36fe2a41869f4f/config_file_3604010.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Fri Aug 29 22:57:12 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpHV1zTz/file36fe2a41869f4f/sample1_align2genome.bam
sample2 ->/tmp/RtmpHV1zTz/file36fe2a41869f4f/sample2_align2genome.bam
sample3 ->/tmp/RtmpHV1zTz/file36fe2a41869f4f/sample3_align2genome.bam
-- Running step: isoform_identification @ Fri Aug 29 22:57:33 2025 -------------
Inputs:  ['/tmp/RtmpHV1zTz/file36fe2a4138480e/sample1_realign2transcript.bam', '/tmp/RtmpHV1zTz/file36fe2a4138480e/sample2_realign2transcript.bam', '/tmp/RtmpHV1zTz/file36fe2a4138480e/sample3_realign2transcript.bam'] /tmp/RtmpHV1zTz/file36fe2a4138480e/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 @ Fri Aug 29 22:57:34 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpHV1zTz/file36fe2a41869f4f/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpHV1zTz/file36fe2a41869f4f/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpHV1zTz/file36fe2a41869f4f/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Fri Aug 29 22:57:53 2025 ----------
22:57:53 Fri Aug 29 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/RtmpHV1zTz/file36fe2a3dc84159/config_file_3604010.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 @ Fri Aug 29 22:57:54 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/RtmpHV1zTz/file36fe2a3dc84159/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 @ Fri Aug 29 22:57:54 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpHV1zTz/file36fe2a3dc84159/matched_reads.fastq.gz -> /tmp/RtmpHV1zTz/file36fe2a3dc84159/align2genome.bam
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: isoform_identification @ Fri Aug 29 22:57:54 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 @ Fri Aug 29 22:58:04 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/RtmpHV1zTz/file36fe2a3dc84159/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpHV1zTz/file36fe2a3dc84159/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/RtmpHV1zTz/file36fe2a3dc84159/matched_reads.fastq.gz -> /tmp/RtmpHV1zTz/file36fe2a3dc84159/realign2transcript.bam
Sorting BAM files by 8 with CB threads...

[bam_sort_core] merging from 0 files and 8 in-memory blocks...
-- Running step: transcript_quantification @ Fri Aug 29 22:58:04 2025 ----------
2025-08-30T02:58:04.778248Z  INFO oarfish: setting user-provided filter parameters.
2025-08-30T02:58:04.778816Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpHV1zTz/file36fe2a3dc84159/realign2transcript.bam, contains 5 reference sequences.
2025-08-30T02:58:04.778829Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-30T02:58:04.778833Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-30T02:58:04.778892Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-30T02:58:04.778898Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-08-30T02:58:04.786144Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpHV1zTz/file36fe2a66aa2c00/config_file_3604010.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 @ Fri Aug 29 22:58:05 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/RtmpHV1zTz/file36fe2a66aa2c00/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 @ Fri Aug 29 22:58:05 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpHV1zTz/file36fe2a66aa2c00/matched_reads.fastq.gz ->/tmp/RtmpHV1zTz/file36fe2a66aa2c00/align2genome.bam
-- Running step: isoform_identification @ Fri Aug 29 22:58:25 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 @ Fri Aug 29 22:58:35 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/RtmpHV1zTz/file36fe2a66aa2c00/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpHV1zTz/file36fe2a66aa2c00/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/RtmpHV1zTz/file36fe2a66aa2c00/matched_reads.fastq.gz ->/tmp/RtmpHV1zTz/file36fe2a66aa2c00/realign2transcript.bam
-- Running step: transcript_quantification @ Fri Aug 29 22:58:55 2025 ----------
2025-08-30T02:58:55.555533Z  INFO oarfish: setting user-provided filter parameters.
2025-08-30T02:58:55.556003Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpHV1zTz/file36fe2a66aa2c00/realign2transcript.bam, contains 5 reference sequences.
2025-08-30T02:58:55.556015Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-30T02:58:55.556020Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-30T02:58:55.556084Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-30T02:58:55.556091Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-08-30T02:58:55.563158Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpHV1zTz/file36fe2a1baa028d/config_file_3604010.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 @ Fri Aug 29 22:58:56 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpHV1zTz/file36fe2a1baa028d/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 @ Fri Aug 29 22:58:56 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpHV1zTz/file36fe2a1baa028d/matched_reads.fastq.gz -> /tmp/RtmpHV1zTz/file36fe2a1baa028d/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 @ Fri Aug 29 22:58:56 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 @ Fri Aug 29 22:59:06 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpHV1zTz/file36fe2a1baa028d/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpHV1zTz/file36fe2a1baa028d/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/RtmpHV1zTz/file36fe2a1baa028d/matched_reads.fastq.gz -> /tmp/RtmpHV1zTz/file36fe2a1baa028d/realign2transcript.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 8 threads...

[bam_sort_core] merging from 0 files and 8 in-memory blocks...
Indexing bam files
-- Running step: transcript_quantification @ Fri Aug 29 22:59:06 2025 ----------
22:59:06 Fri Aug 29 2025 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Inputs:  ['/tmp/RtmpHV1zTz/file36fe2a41869f4f/sample1_realign2transcript.bam', '/tmp/RtmpHV1zTz/file36fe2a41869f4f/sample2_realign2transcript.bam', '/tmp/RtmpHV1zTz/file36fe2a41869f4f/sample3_realign2transcript.bam'] /tmp/RtmpHV1zTz/file36fe2a41869f4f/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/RtmpHV1zTz/file36fe2a27d12069/config_file_3604010.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 @ Fri Aug 29 22:59:07 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpHV1zTz/file36fe2a27d12069/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 @ Fri Aug 29 22:59:07 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpHV1zTz/file36fe2a27d12069/matched_reads.fastq.gz ->/tmp/RtmpHV1zTz/file36fe2a27d12069/align2genome.bam
-- Running step: isoform_identification @ Fri Aug 29 22:59:26 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 @ Fri Aug 29 22:59:35 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/RtmpHV1zTz/file36fe2a27d12069/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpHV1zTz/file36fe2a27d12069/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/RtmpHV1zTz/file36fe2a27d12069/matched_reads.fastq.gz ->/tmp/RtmpHV1zTz/file36fe2a27d12069/realign2transcript.bam
-- Running step: transcript_quantification @ Fri Aug 29 22:59:54 2025 ----------
22:59:54 Fri Aug 29 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/RtmpHV1zTz/file36fe2a519bd030/config_file_3604010.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 @ Fri Aug 29 22:59:55 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/RtmpHV1zTz/file36fe2a519bd030/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 @ Fri Aug 29 22:59:55 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpHV1zTz/file36fe2a519bd030/matched_reads.fastq.gz -> /tmp/RtmpHV1zTz/file36fe2a519bd030/align2genome.bam
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: isoform_identification @ Fri Aug 29 22:59:56 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 @ Fri Aug 29 22:59:56 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/RtmpHV1zTz/file36fe2a519bd030/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpHV1zTz/file36fe2a519bd030/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/RtmpHV1zTz/file36fe2a519bd030/matched_reads.fastq.gz -> /tmp/RtmpHV1zTz/file36fe2a519bd030/realign2transcript.bam
Sorting BAM files by 8 with CB threads...

[bam_sort_core] merging from 0 files and 8 in-memory blocks...
-- Running step: transcript_quantification @ Fri Aug 29 22:59:56 2025 ----------
2025-08-30T02:59:56.695442Z  INFO oarfish: setting user-provided filter parameters.
2025-08-30T02:59:56.695873Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpHV1zTz/file36fe2a519bd030/realign2transcript.bam, contains 10 reference sequences.
2025-08-30T02:59:56.695881Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-30T02:59:56.695884Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-30T02:59:56.695952Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-30T02:59:56.695960Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-08-30T02:59:56.706910Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpHV1zTz/file36fe2a5d91ffdf/config_file_3604010.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 @ Fri Aug 29 22:59:57 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/RtmpHV1zTz/file36fe2a5d91ffdf/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 @ Fri Aug 29 22:59:57 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpHV1zTz/file36fe2a5d91ffdf/matched_reads.fastq.gz ->/tmp/RtmpHV1zTz/file36fe2a5d91ffdf/align2genome.bam
-- Running step: isoform_identification @ Fri Aug 29 23:00:16 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri Aug 29 23:00:16 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/RtmpHV1zTz/file36fe2a5d91ffdf/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpHV1zTz/file36fe2a5d91ffdf/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/RtmpHV1zTz/file36fe2a5d91ffdf/matched_reads.fastq.gz ->/tmp/RtmpHV1zTz/file36fe2a5d91ffdf/realign2transcript.bam
-- Running step: transcript_quantification @ Fri Aug 29 23:00:35 2025 ----------
2025-08-30T03:00:35.353038Z  INFO oarfish: setting user-provided filter parameters.
2025-08-30T03:00:35.353548Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpHV1zTz/file36fe2a5d91ffdf/realign2transcript.bam, contains 10 reference sequences.
2025-08-30T03:00:35.353560Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-30T03:00:35.353564Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-30T03:00:35.353648Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-30T03:00:35.353655Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-08-30T03:00:35.364463Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpHV1zTz/file36fe2a137b7598/config_file_3604010.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 @ Fri Aug 29 23:00:36 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/RtmpHV1zTz/file36fe2a137b7598/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 @ Fri Aug 29 23:00:36 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpHV1zTz/file36fe2a137b7598/matched_reads.fastq.gz -> /tmp/RtmpHV1zTz/file36fe2a137b7598/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 @ Fri Aug 29 23:00:36 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri Aug 29 23:00:37 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/RtmpHV1zTz/file36fe2a137b7598/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpHV1zTz/file36fe2a137b7598/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/RtmpHV1zTz/file36fe2a137b7598/matched_reads.fastq.gz -> /tmp/RtmpHV1zTz/file36fe2a137b7598/realign2transcript.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 8 threads...

[bam_sort_core] merging from 0 files and 8 in-memory blocks...
Indexing bam files
-- Running step: transcript_quantification @ Fri Aug 29 23:00:37 2025 ----------
23:00:37 Fri Aug 29 2025 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpHV1zTz/file36fe2a131825f0/config_file_3604010.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 @ Fri Aug 29 23:00:38 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/RtmpHV1zTz/file36fe2a131825f0/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 @ Fri Aug 29 23:00:38 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpHV1zTz/file36fe2a131825f0/matched_reads.fastq.gz ->/tmp/RtmpHV1zTz/file36fe2a131825f0/align2genome.bam
-- Running step: isoform_identification @ Fri Aug 29 23:00:56 2025 -------------
	Counter({'counted_reads': 368, 'unmapped': 4})
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri Aug 29 23:00:57 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/RtmpHV1zTz/file36fe2a131825f0/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpHV1zTz/file36fe2a131825f0/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/RtmpHV1zTz/file36fe2a131825f0/matched_reads.fastq.gz ->/tmp/RtmpHV1zTz/file36fe2a131825f0/realign2transcript.bam
-- Running step: transcript_quantification @ Fri Aug 29 23:01:16 2025 ----------
23:01:16 Fri Aug 29 2025 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpHV1zTz/file36fe2a16355cf2/config_file_3604010.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Fri Aug 29 23:01:17 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/RtmpHV1zTz/file36fe2a16355cf2/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpHV1zTz/file36fe2a16355cf2/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpHV1zTz/file36fe2a16355cf2/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpHV1zTz/file36fe2a16355cf2/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/RtmpHV1zTz/file36fe2a16355cf2/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpHV1zTz/file36fe2a16355cf2/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/RtmpHV1zTz/file36fe2a16355cf2/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpHV1zTz/file36fe2a16355cf2/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/RtmpHV1zTz/file36fe2a16355cf2/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpHV1zTz/file36fe2a16355cf2/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 @ Fri Aug 29 23:01:18 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpHV1zTz/file36fe2a16355cf2/sampleA_matched_reads.fastq.gz -> /tmp/RtmpHV1zTz/file36fe2a16355cf2/sampleA_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpHV1zTz/file36fe2a16355cf2/sample1_matched_reads.fastq.gz -> /tmp/RtmpHV1zTz/file36fe2a16355cf2/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpHV1zTz/file36fe2a16355cf2/sample2_matched_reads.fastq.gz -> /tmp/RtmpHV1zTz/file36fe2a16355cf2/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpHV1zTz/file36fe2a16355cf2/sample3_matched_reads.fastq.gz -> /tmp/RtmpHV1zTz/file36fe2a16355cf2/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Fri Aug 29 23:01:20 2025 ----------------
23:01:20 Fri Aug 29 2025 quantify genes 
Using BAM(s): '/tmp/RtmpHV1zTz/file36fe2a16355cf2/sampleA_align2genome.bam',
'/tmp/RtmpHV1zTz/file36fe2a16355cf2/sample1_align2genome.bam',
'/tmp/RtmpHV1zTz/file36fe2a16355cf2/sample2_align2genome.bam', and
'/tmp/RtmpHV1zTz/file36fe2a16355cf2/sample3_align2genome.bam'
	Counter({'counted_reads': 368, 'unmapped': 4})
parsing /tmp/RtmpHV1zTz/file36fe2a16355cf2/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 20.51gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 415047.50Read/s]
parsing /tmp/RtmpHV1zTz/file36fe2a16355cf2/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 48.90gene_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, 1338664.62Read/s]
parsing /tmp/RtmpHV1zTz/file36fe2a16355cf2/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 48.48gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1294058.99Read/s]
parsing /tmp/RtmpHV1zTz/file36fe2a16355cf2/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 34.25gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 727722.95Read/s]
-- Running step: isoform_identification @ Fri Aug 29 23:01:22 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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  |                                                                            
  |==================                                                    |  25%
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Detected 6 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Fri Aug 29 23:01:45 2025 -------------------
Checking for fastq file(s) /tmp/RtmpHV1zTz/file36fe2a16355cf2/fastq, /tmp/RtmpHV1zTz/file36fe2a16355cf2/fastq/sample1.fq.gz, /tmp/RtmpHV1zTz/file36fe2a16355cf2/fastq/sample2.fq.gz, /tmp/RtmpHV1zTz/file36fe2a16355cf2/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpHV1zTz/file36fe2a16355cf2/sampleA_matched_reads.fastq.gz, /tmp/RtmpHV1zTz/file36fe2a16355cf2/sample1_matched_reads.fastq.gz, /tmp/RtmpHV1zTz/file36fe2a16355cf2/sample2_matched_reads.fastq.gz, /tmp/RtmpHV1zTz/file36fe2a16355cf2/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpHV1zTz/file36fe2a16355cf2/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpHV1zTz/file36fe2a16355cf2/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpHV1zTz/file36fe2a16355cf2/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpHV1zTz/file36fe2a16355cf2/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpHV1zTz/file36fe2a16355cf2/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpHV1zTz/file36fe2a16355cf2/sampleA_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpHV1zTz/file36fe2a16355cf2/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpHV1zTz/file36fe2a16355cf2/sample1_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpHV1zTz/file36fe2a16355cf2/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpHV1zTz/file36fe2a16355cf2/sample2_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpHV1zTz/file36fe2a16355cf2/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpHV1zTz/file36fe2a16355cf2/sample3_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

-- Running step: transcript_quantification @ Fri Aug 29 23:01:45 2025 ----------
2025-08-30T03:01:45.992453Z  INFO oarfish: setting user-provided filter parameters.
2025-08-30T03:01:45.993052Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpHV1zTz/file36fe2a16355cf2/sampleA_realign2transcript.bam, contains 5 reference sequences.
2025-08-30T03:01:45.993078Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-30T03:01:45.993082Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-30T03:01:45.993144Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-30T03:01:45.993149Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-08-30T03:01:45.998904Z  INFO oarfish::single_cell: Processed 100 cells.
2025-08-30T03:01:46.334328Z  INFO oarfish: setting user-provided filter parameters.
2025-08-30T03:01:46.334849Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpHV1zTz/file36fe2a16355cf2/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-08-30T03:01:46.334858Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-30T03:01:46.334861Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-30T03:01:46.334940Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-30T03:01:46.334946Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-08-30T03:01:46.647752Z  INFO oarfish: setting user-provided filter parameters.
2025-08-30T03:01:46.648401Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpHV1zTz/file36fe2a16355cf2/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-08-30T03:01:46.648424Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-30T03:01:46.648431Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-30T03:01:46.648539Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-30T03:01:46.648551Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-08-30T03:01:46.952540Z  INFO oarfish: setting user-provided filter parameters.
2025-08-30T03:01:46.952881Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpHV1zTz/file36fe2a16355cf2/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-08-30T03:01:46.952890Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-30T03:01:46.952893Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-30T03:01:46.952949Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-30T03:01:46.952954Z  INFO oarfish: parsed reference information for 5 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpHV1zTz/file36fe2a31800f3d/config_file_3604010.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Fri Aug 29 23:01:47 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/RtmpHV1zTz/file36fe2a31800f3d/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpHV1zTz/file36fe2a31800f3d/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpHV1zTz/file36fe2a31800f3d/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpHV1zTz/file36fe2a31800f3d/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/RtmpHV1zTz/file36fe2a31800f3d/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpHV1zTz/file36fe2a31800f3d/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/RtmpHV1zTz/file36fe2a31800f3d/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpHV1zTz/file36fe2a31800f3d/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/RtmpHV1zTz/file36fe2a31800f3d/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpHV1zTz/file36fe2a31800f3d/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 @ Fri Aug 29 23:01:48 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpHV1zTz/file36fe2a31800f3d/sampleA_matched_reads.fastq.gz ->/tmp/RtmpHV1zTz/file36fe2a31800f3d/sampleA_align2genome.bam
/tmp/RtmpHV1zTz/file36fe2a31800f3d/sample1_matched_reads.fastq.gz ->/tmp/RtmpHV1zTz/file36fe2a31800f3d/sample1_align2genome.bam
/tmp/RtmpHV1zTz/file36fe2a31800f3d/sample2_matched_reads.fastq.gz ->/tmp/RtmpHV1zTz/file36fe2a31800f3d/sample2_align2genome.bam
/tmp/RtmpHV1zTz/file36fe2a31800f3d/sample3_matched_reads.fastq.gz ->/tmp/RtmpHV1zTz/file36fe2a31800f3d/sample3_align2genome.bam
-- Running step: gene_quantification @ Fri Aug 29 23:02:08 2025 ----------------
23:02:08 Fri Aug 29 2025 quantify genes 
Using BAM(s): '/tmp/RtmpHV1zTz/file36fe2a31800f3d/sampleA_align2genome.bam',
'/tmp/RtmpHV1zTz/file36fe2a31800f3d/sample1_align2genome.bam',
'/tmp/RtmpHV1zTz/file36fe2a31800f3d/sample2_align2genome.bam', and
'/tmp/RtmpHV1zTz/file36fe2a31800f3d/sample3_align2genome.bam'
parsing /tmp/RtmpHV1zTz/file36fe2a31800f3d/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 15.99gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 356743.44Read/s]
parsing /tmp/RtmpHV1zTz/file36fe2a31800f3d/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 52.85gene_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, 1324963.36Read/s]
parsing /tmp/RtmpHV1zTz/file36fe2a31800f3d/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 48.07gene_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, 1284862.15Read/s]
parsing /tmp/RtmpHV1zTz/file36fe2a31800f3d/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 35.61gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 751398.07Read/s]
-- Running step: isoform_identification @ Fri Aug 29 23:02:09 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 @ Fri Aug 29 23:02:31 2025 -------------------
Checking for fastq file(s) /tmp/RtmpHV1zTz/file36fe2a31800f3d/fastq, /tmp/RtmpHV1zTz/file36fe2a31800f3d/fastq/sample1.fq.gz, /tmp/RtmpHV1zTz/file36fe2a31800f3d/fastq/sample2.fq.gz, /tmp/RtmpHV1zTz/file36fe2a31800f3d/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpHV1zTz/file36fe2a31800f3d/sampleA_matched_reads.fastq.gz, /tmp/RtmpHV1zTz/file36fe2a31800f3d/sample1_matched_reads.fastq.gz, /tmp/RtmpHV1zTz/file36fe2a31800f3d/sample2_matched_reads.fastq.gz, /tmp/RtmpHV1zTz/file36fe2a31800f3d/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpHV1zTz/file36fe2a31800f3d/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpHV1zTz/file36fe2a31800f3d/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpHV1zTz/file36fe2a31800f3d/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpHV1zTz/file36fe2a31800f3d/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpHV1zTz/file36fe2a31800f3d/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpHV1zTz/file36fe2a31800f3d/sampleA_realign2transcript.bam
/tmp/RtmpHV1zTz/file36fe2a31800f3d/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpHV1zTz/file36fe2a31800f3d/sample1_realign2transcript.bam
/tmp/RtmpHV1zTz/file36fe2a31800f3d/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpHV1zTz/file36fe2a31800f3d/sample2_realign2transcript.bam
/tmp/RtmpHV1zTz/file36fe2a31800f3d/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpHV1zTz/file36fe2a31800f3d/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Fri Aug 29 23:02:51 2025 ----------
2025-08-30T03:02:51.275506Z  INFO oarfish: setting user-provided filter parameters.
2025-08-30T03:02:51.275996Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpHV1zTz/file36fe2a31800f3d/sampleA_realign2transcript.bam, contains 5 reference sequences.
2025-08-30T03:02:51.276006Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-30T03:02:51.276010Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-30T03:02:51.276065Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-30T03:02:51.276071Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-08-30T03:02:51.281889Z  INFO oarfish::single_cell: Processed 100 cells.
2025-08-30T03:02:51.623644Z  INFO oarfish: setting user-provided filter parameters.
2025-08-30T03:02:51.624117Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpHV1zTz/file36fe2a31800f3d/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-08-30T03:02:51.624127Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-30T03:02:51.624131Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-30T03:02:51.624203Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-30T03:02:51.624211Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-08-30T03:02:51.960933Z  INFO oarfish: setting user-provided filter parameters.
2025-08-30T03:02:51.961446Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpHV1zTz/file36fe2a31800f3d/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-08-30T03:02:51.961458Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-30T03:02:51.961462Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-30T03:02:51.961519Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-30T03:02:51.961525Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-08-30T03:02:52.303444Z  INFO oarfish: setting user-provided filter parameters.
2025-08-30T03:02:52.303820Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpHV1zTz/file36fe2a31800f3d/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-08-30T03:02:52.303831Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-30T03:02:52.303834Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-30T03:02:52.303894Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-30T03:02:52.303914Z  INFO oarfish: parsed reference information for 5 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpHV1zTz/file36fe2a28b8b0c2/config_file_3604010.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Fri Aug 29 23:02:52 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 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpHV1zTz/file36fe2a28b8b0c2/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpHV1zTz/file36fe2a28b8b0c2/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpHV1zTz/file36fe2a28b8b0c2/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpHV1zTz/file36fe2a28b8b0c2/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/RtmpHV1zTz/file36fe2a28b8b0c2/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpHV1zTz/file36fe2a28b8b0c2/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/RtmpHV1zTz/file36fe2a28b8b0c2/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpHV1zTz/file36fe2a28b8b0c2/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/RtmpHV1zTz/file36fe2a28b8b0c2/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpHV1zTz/file36fe2a28b8b0c2/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 @ Fri Aug 29 23:02:53 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpHV1zTz/file36fe2a28b8b0c2/sampleA_matched_reads.fastq.gz -> /tmp/RtmpHV1zTz/file36fe2a28b8b0c2/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/RtmpHV1zTz/file36fe2a28b8b0c2/sample1_matched_reads.fastq.gz -> /tmp/RtmpHV1zTz/file36fe2a28b8b0c2/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/RtmpHV1zTz/file36fe2a28b8b0c2/sample2_matched_reads.fastq.gz -> /tmp/RtmpHV1zTz/file36fe2a28b8b0c2/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/RtmpHV1zTz/file36fe2a28b8b0c2/sample3_matched_reads.fastq.gz -> /tmp/RtmpHV1zTz/file36fe2a28b8b0c2/sample3_align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Fri Aug 29 23:02:55 2025 ----------------
23:02:55 Fri Aug 29 2025 quantify genes 
Using BAM(s): '/tmp/RtmpHV1zTz/file36fe2a28b8b0c2/sampleA_align2genome.bam',
'/tmp/RtmpHV1zTz/file36fe2a28b8b0c2/sample1_align2genome.bam',
'/tmp/RtmpHV1zTz/file36fe2a28b8b0c2/sample2_align2genome.bam', and
'/tmp/RtmpHV1zTz/file36fe2a28b8b0c2/sample3_align2genome.bam'
parsing /tmp/RtmpHV1zTz/file36fe2a28b8b0c2/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 21.51gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 379108.43Read/s]
parsing /tmp/RtmpHV1zTz/file36fe2a28b8b0c2/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 51.09gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1358259.07Read/s]
parsing /tmp/RtmpHV1zTz/file36fe2a28b8b0c2/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 51.24gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1165343.41Read/s]
parsing /tmp/RtmpHV1zTz/file36fe2a28b8b0c2/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 34.95gene_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, 763544.75Read/s]
-- Running step: isoform_identification @ Fri Aug 29 23:02:56 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

  |                                                                            
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  |                                                                            
  |==================                                                    |  25%
  |                                                                            
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Detected 6 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Fri Aug 29 23:03:20 2025 -------------------
Checking for fastq file(s) /tmp/RtmpHV1zTz/file36fe2a28b8b0c2/fastq, /tmp/RtmpHV1zTz/file36fe2a28b8b0c2/fastq/sample1.fq.gz, /tmp/RtmpHV1zTz/file36fe2a28b8b0c2/fastq/sample2.fq.gz, /tmp/RtmpHV1zTz/file36fe2a28b8b0c2/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpHV1zTz/file36fe2a28b8b0c2/sampleA_matched_reads.fastq.gz, /tmp/RtmpHV1zTz/file36fe2a28b8b0c2/sample1_matched_reads.fastq.gz, /tmp/RtmpHV1zTz/file36fe2a28b8b0c2/sample2_matched_reads.fastq.gz, /tmp/RtmpHV1zTz/file36fe2a28b8b0c2/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpHV1zTz/file36fe2a28b8b0c2/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpHV1zTz/file36fe2a28b8b0c2/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpHV1zTz/file36fe2a28b8b0c2/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpHV1zTz/file36fe2a28b8b0c2/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpHV1zTz/file36fe2a28b8b0c2/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpHV1zTz/file36fe2a28b8b0c2/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/RtmpHV1zTz/file36fe2a28b8b0c2/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpHV1zTz/file36fe2a28b8b0c2/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/RtmpHV1zTz/file36fe2a28b8b0c2/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpHV1zTz/file36fe2a28b8b0c2/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/RtmpHV1zTz/file36fe2a28b8b0c2/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpHV1zTz/file36fe2a28b8b0c2/sample3_realign2transcript.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Fri Aug 29 23:03:21 2025 ----------
23:03:21 Fri Aug 29 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpHV1zTz/file36fe2a28b8b0c2/sampleA_realign2transcript.bam...
parsing /tmp/RtmpHV1zTz/file36fe2a28b8b0c2/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpHV1zTz/file36fe2a28b8b0c2/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpHV1zTz/file36fe2a28b8b0c2/sample1_realign2transcript.bam...
parsing /tmp/RtmpHV1zTz/file36fe2a28b8b0c2/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpHV1zTz/file36fe2a28b8b0c2/sample1_realign2transcript.bamdone
parsing /tmp/RtmpHV1zTz/file36fe2a28b8b0c2/sample2_realign2transcript.bam...
parsing /tmp/RtmpHV1zTz/file36fe2a28b8b0c2/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpHV1zTz/file36fe2a28b8b0c2/sample2_realign2transcript.bamdone
parsing /tmp/RtmpHV1zTz/file36fe2a28b8b0c2/sample3_realign2transcript.bam...
parsing /tmp/RtmpHV1zTz/file36fe2a28b8b0c2/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpHV1zTz/file36fe2a28b8b0c2/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpHV1zTz/file36fe2a49da18ea/config_file_3604010.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Fri Aug 29 23:03:23 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/RtmpHV1zTz/file36fe2a49da18ea/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpHV1zTz/file36fe2a49da18ea/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpHV1zTz/file36fe2a49da18ea/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpHV1zTz/file36fe2a49da18ea/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/RtmpHV1zTz/file36fe2a49da18ea/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpHV1zTz/file36fe2a49da18ea/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/RtmpHV1zTz/file36fe2a49da18ea/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpHV1zTz/file36fe2a49da18ea/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/RtmpHV1zTz/file36fe2a49da18ea/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpHV1zTz/file36fe2a49da18ea/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 @ Fri Aug 29 23:03:24 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpHV1zTz/file36fe2a49da18ea/sampleA_matched_reads.fastq.gz ->/tmp/RtmpHV1zTz/file36fe2a49da18ea/sampleA_align2genome.bam
/tmp/RtmpHV1zTz/file36fe2a49da18ea/sample1_matched_reads.fastq.gz ->/tmp/RtmpHV1zTz/file36fe2a49da18ea/sample1_align2genome.bam
/tmp/RtmpHV1zTz/file36fe2a49da18ea/sample2_matched_reads.fastq.gz ->/tmp/RtmpHV1zTz/file36fe2a49da18ea/sample2_align2genome.bam
/tmp/RtmpHV1zTz/file36fe2a49da18ea/sample3_matched_reads.fastq.gz ->/tmp/RtmpHV1zTz/file36fe2a49da18ea/sample3_align2genome.bam
-- Running step: gene_quantification @ Fri Aug 29 23:03:44 2025 ----------------
23:03:44 Fri Aug 29 2025 quantify genes 
Using BAM(s): '/tmp/RtmpHV1zTz/file36fe2a49da18ea/sampleA_align2genome.bam',
'/tmp/RtmpHV1zTz/file36fe2a49da18ea/sample1_align2genome.bam',
'/tmp/RtmpHV1zTz/file36fe2a49da18ea/sample2_align2genome.bam', and
'/tmp/RtmpHV1zTz/file36fe2a49da18ea/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/RtmpHV1zTz/file36fe2a49da18ea/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 21.98gene_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, 362239.96Read/s]
parsing /tmp/RtmpHV1zTz/file36fe2a49da18ea/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 49.14gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1194005.92Read/s]
parsing /tmp/RtmpHV1zTz/file36fe2a49da18ea/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 48.12gene_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, 1148369.29Read/s]
parsing /tmp/RtmpHV1zTz/file36fe2a49da18ea/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 35.17gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 631976.86Read/s]
-- Running step: isoform_identification @ Fri Aug 29 23:03:45 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 6 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Fri Aug 29 23:04:07 2025 -------------------
Checking for fastq file(s) /tmp/RtmpHV1zTz/file36fe2a49da18ea/fastq, /tmp/RtmpHV1zTz/file36fe2a49da18ea/fastq/sample1.fq.gz, /tmp/RtmpHV1zTz/file36fe2a49da18ea/fastq/sample2.fq.gz, /tmp/RtmpHV1zTz/file36fe2a49da18ea/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpHV1zTz/file36fe2a49da18ea/sampleA_matched_reads.fastq.gz, /tmp/RtmpHV1zTz/file36fe2a49da18ea/sample1_matched_reads.fastq.gz, /tmp/RtmpHV1zTz/file36fe2a49da18ea/sample2_matched_reads.fastq.gz, /tmp/RtmpHV1zTz/file36fe2a49da18ea/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpHV1zTz/file36fe2a49da18ea/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpHV1zTz/file36fe2a49da18ea/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpHV1zTz/file36fe2a49da18ea/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpHV1zTz/file36fe2a49da18ea/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpHV1zTz/file36fe2a49da18ea/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpHV1zTz/file36fe2a49da18ea/sampleA_realign2transcript.bam
/tmp/RtmpHV1zTz/file36fe2a49da18ea/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpHV1zTz/file36fe2a49da18ea/sample1_realign2transcript.bam
/tmp/RtmpHV1zTz/file36fe2a49da18ea/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpHV1zTz/file36fe2a49da18ea/sample2_realign2transcript.bam
/tmp/RtmpHV1zTz/file36fe2a49da18ea/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpHV1zTz/file36fe2a49da18ea/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Fri Aug 29 23:04:27 2025 ----------
23:04:27 Fri Aug 29 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpHV1zTz/file36fe2a49da18ea/sampleA_realign2transcript.bam...
parsing /tmp/RtmpHV1zTz/file36fe2a49da18ea/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpHV1zTz/file36fe2a49da18ea/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpHV1zTz/file36fe2a49da18ea/sample1_realign2transcript.bam...
parsing /tmp/RtmpHV1zTz/file36fe2a49da18ea/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpHV1zTz/file36fe2a49da18ea/sample1_realign2transcript.bamdone
parsing /tmp/RtmpHV1zTz/file36fe2a49da18ea/sample2_realign2transcript.bam...
parsing /tmp/RtmpHV1zTz/file36fe2a49da18ea/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpHV1zTz/file36fe2a49da18ea/sample2_realign2transcript.bamdone
parsing /tmp/RtmpHV1zTz/file36fe2a49da18ea/sample3_realign2transcript.bam...
parsing /tmp/RtmpHV1zTz/file36fe2a49da18ea/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpHV1zTz/file36fe2a49da18ea/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpHV1zTz/file36fe2adefbe45/config_file_3604010.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Fri Aug 29 23:04:29 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/RtmpHV1zTz/file36fe2adefbe45/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpHV1zTz/file36fe2adefbe45/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpHV1zTz/file36fe2adefbe45/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpHV1zTz/file36fe2adefbe45/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/RtmpHV1zTz/file36fe2adefbe45/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpHV1zTz/file36fe2adefbe45/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/RtmpHV1zTz/file36fe2adefbe45/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpHV1zTz/file36fe2adefbe45/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/RtmpHV1zTz/file36fe2adefbe45/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpHV1zTz/file36fe2adefbe45/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 @ Fri Aug 29 23:04:29 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpHV1zTz/file36fe2adefbe45/sampleA_matched_reads.fastq.gz -> /tmp/RtmpHV1zTz/file36fe2adefbe45/sampleA_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpHV1zTz/file36fe2adefbe45/sample1_matched_reads.fastq.gz -> /tmp/RtmpHV1zTz/file36fe2adefbe45/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpHV1zTz/file36fe2adefbe45/sample2_matched_reads.fastq.gz -> /tmp/RtmpHV1zTz/file36fe2adefbe45/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpHV1zTz/file36fe2adefbe45/sample3_matched_reads.fastq.gz -> /tmp/RtmpHV1zTz/file36fe2adefbe45/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Fri Aug 29 23:04:31 2025 ----------------
23:04:31 Fri Aug 29 2025 quantify genes 
Using BAM(s): '/tmp/RtmpHV1zTz/file36fe2adefbe45/sampleA_align2genome.bam',
'/tmp/RtmpHV1zTz/file36fe2adefbe45/sample1_align2genome.bam',
'/tmp/RtmpHV1zTz/file36fe2adefbe45/sample2_align2genome.bam', and
'/tmp/RtmpHV1zTz/file36fe2adefbe45/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/RtmpHV1zTz/file36fe2adefbe45/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 22.37gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 440097.37Read/s]
parsing /tmp/RtmpHV1zTz/file36fe2adefbe45/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 47.20gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1281251.22Read/s]
parsing /tmp/RtmpHV1zTz/file36fe2adefbe45/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 51.39gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1259702.07Read/s]
parsing /tmp/RtmpHV1zTz/file36fe2adefbe45/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 35.86gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 773286.14Read/s]
-- Running step: isoform_identification @ Fri Aug 29 23:04:32 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri Aug 29 23:04:32 2025 -------------------
Checking for fastq file(s) /tmp/RtmpHV1zTz/file36fe2adefbe45/fastq, /tmp/RtmpHV1zTz/file36fe2adefbe45/fastq/sample1.fq.gz, /tmp/RtmpHV1zTz/file36fe2adefbe45/fastq/sample2.fq.gz, /tmp/RtmpHV1zTz/file36fe2adefbe45/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpHV1zTz/file36fe2adefbe45/sampleA_matched_reads.fastq.gz, /tmp/RtmpHV1zTz/file36fe2adefbe45/sample1_matched_reads.fastq.gz, /tmp/RtmpHV1zTz/file36fe2adefbe45/sample2_matched_reads.fastq.gz, /tmp/RtmpHV1zTz/file36fe2adefbe45/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpHV1zTz/file36fe2adefbe45/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpHV1zTz/file36fe2adefbe45/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpHV1zTz/file36fe2adefbe45/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpHV1zTz/file36fe2adefbe45/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpHV1zTz/file36fe2adefbe45/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpHV1zTz/file36fe2adefbe45/sampleA_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpHV1zTz/file36fe2adefbe45/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpHV1zTz/file36fe2adefbe45/sample1_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpHV1zTz/file36fe2adefbe45/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpHV1zTz/file36fe2adefbe45/sample2_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpHV1zTz/file36fe2adefbe45/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpHV1zTz/file36fe2adefbe45/sample3_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

-- Running step: transcript_quantification @ Fri Aug 29 23:04:34 2025 ----------
2025-08-30T03:04:34.630090Z  INFO oarfish: setting user-provided filter parameters.
2025-08-30T03:04:34.630550Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpHV1zTz/file36fe2adefbe45/sampleA_realign2transcript.bam, contains 14 reference sequences.
2025-08-30T03:04:34.630563Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-30T03:04:34.630566Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-30T03:04:34.630644Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-30T03:04:34.630651Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-08-30T03:04:34.642235Z  INFO oarfish::single_cell: Processed 100 cells.
2025-08-30T03:04:35.186580Z  INFO oarfish: setting user-provided filter parameters.
2025-08-30T03:04:35.187037Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpHV1zTz/file36fe2adefbe45/sample1_realign2transcript.bam, contains 14 reference sequences.
2025-08-30T03:04:35.187047Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-30T03:04:35.187050Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-30T03:04:35.187135Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-30T03:04:35.187144Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-08-30T03:04:35.740800Z  INFO oarfish: setting user-provided filter parameters.
2025-08-30T03:04:35.741331Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpHV1zTz/file36fe2adefbe45/sample2_realign2transcript.bam, contains 14 reference sequences.
2025-08-30T03:04:35.741342Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-30T03:04:35.741345Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-30T03:04:35.741429Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-30T03:04:35.741437Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-08-30T03:04:36.290949Z  INFO oarfish: setting user-provided filter parameters.
2025-08-30T03:04:36.291322Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpHV1zTz/file36fe2adefbe45/sample3_realign2transcript.bam, contains 14 reference sequences.
2025-08-30T03:04:36.291333Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-30T03:04:36.291336Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-30T03:04:36.291415Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-30T03:04:36.291422Z  INFO oarfish: parsed reference information for 14 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpHV1zTz/file36fe2a496d4912/config_file_3604010.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Fri Aug 29 23:04:37 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/RtmpHV1zTz/file36fe2a496d4912/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpHV1zTz/file36fe2a496d4912/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpHV1zTz/file36fe2a496d4912/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpHV1zTz/file36fe2a496d4912/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/RtmpHV1zTz/file36fe2a496d4912/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpHV1zTz/file36fe2a496d4912/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/RtmpHV1zTz/file36fe2a496d4912/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpHV1zTz/file36fe2a496d4912/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/RtmpHV1zTz/file36fe2a496d4912/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpHV1zTz/file36fe2a496d4912/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 @ Fri Aug 29 23:04:37 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpHV1zTz/file36fe2a496d4912/sampleA_matched_reads.fastq.gz ->/tmp/RtmpHV1zTz/file36fe2a496d4912/sampleA_align2genome.bam
/tmp/RtmpHV1zTz/file36fe2a496d4912/sample1_matched_reads.fastq.gz ->/tmp/RtmpHV1zTz/file36fe2a496d4912/sample1_align2genome.bam
/tmp/RtmpHV1zTz/file36fe2a496d4912/sample2_matched_reads.fastq.gz ->/tmp/RtmpHV1zTz/file36fe2a496d4912/sample2_align2genome.bam
/tmp/RtmpHV1zTz/file36fe2a496d4912/sample3_matched_reads.fastq.gz ->/tmp/RtmpHV1zTz/file36fe2a496d4912/sample3_align2genome.bam
-- Running step: gene_quantification @ Fri Aug 29 23:04:59 2025 ----------------
23:04:59 Fri Aug 29 2025 quantify genes 
Using BAM(s): '/tmp/RtmpHV1zTz/file36fe2a496d4912/sampleA_align2genome.bam',
'/tmp/RtmpHV1zTz/file36fe2a496d4912/sample1_align2genome.bam',
'/tmp/RtmpHV1zTz/file36fe2a496d4912/sample2_align2genome.bam', and
'/tmp/RtmpHV1zTz/file36fe2a496d4912/sample3_align2genome.bam'
parsing /tmp/RtmpHV1zTz/file36fe2a496d4912/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 21.77gene_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, 350987.78Read/s]
parsing /tmp/RtmpHV1zTz/file36fe2a496d4912/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 44.32gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1135313.99Read/s]
parsing /tmp/RtmpHV1zTz/file36fe2a496d4912/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 45.43gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1040357.18Read/s]
parsing /tmp/RtmpHV1zTz/file36fe2a496d4912/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 33.91gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 646152.33Read/s]
-- Running step: isoform_identification @ Fri Aug 29 23:05:00 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri Aug 29 23:05:00 2025 -------------------
Checking for fastq file(s) /tmp/RtmpHV1zTz/file36fe2a496d4912/fastq, /tmp/RtmpHV1zTz/file36fe2a496d4912/fastq/sample1.fq.gz, /tmp/RtmpHV1zTz/file36fe2a496d4912/fastq/sample2.fq.gz, /tmp/RtmpHV1zTz/file36fe2a496d4912/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpHV1zTz/file36fe2a496d4912/sampleA_matched_reads.fastq.gz, /tmp/RtmpHV1zTz/file36fe2a496d4912/sample1_matched_reads.fastq.gz, /tmp/RtmpHV1zTz/file36fe2a496d4912/sample2_matched_reads.fastq.gz, /tmp/RtmpHV1zTz/file36fe2a496d4912/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpHV1zTz/file36fe2a496d4912/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpHV1zTz/file36fe2a496d4912/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpHV1zTz/file36fe2a496d4912/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpHV1zTz/file36fe2a496d4912/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpHV1zTz/file36fe2a496d4912/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpHV1zTz/file36fe2a496d4912/sampleA_realign2transcript.bam
/tmp/RtmpHV1zTz/file36fe2a496d4912/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpHV1zTz/file36fe2a496d4912/sample1_realign2transcript.bam
/tmp/RtmpHV1zTz/file36fe2a496d4912/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpHV1zTz/file36fe2a496d4912/sample2_realign2transcript.bam
/tmp/RtmpHV1zTz/file36fe2a496d4912/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpHV1zTz/file36fe2a496d4912/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Fri Aug 29 23:05:20 2025 ----------
2025-08-30T03:05:20.954186Z  INFO oarfish: setting user-provided filter parameters.
2025-08-30T03:05:20.954738Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpHV1zTz/file36fe2a496d4912/sampleA_realign2transcript.bam, contains 14 reference sequences.
2025-08-30T03:05:20.954765Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-30T03:05:20.954769Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-30T03:05:20.954867Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-30T03:05:20.954875Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-08-30T03:05:20.967033Z  INFO oarfish::single_cell: Processed 100 cells.
2025-08-30T03:05:21.699979Z  INFO oarfish: setting user-provided filter parameters.
2025-08-30T03:05:21.700430Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpHV1zTz/file36fe2a496d4912/sample1_realign2transcript.bam, contains 14 reference sequences.
2025-08-30T03:05:21.700443Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-30T03:05:21.700447Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-30T03:05:21.700544Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-30T03:05:21.700552Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-08-30T03:05:22.450534Z  INFO oarfish: setting user-provided filter parameters.
2025-08-30T03:05:22.451006Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpHV1zTz/file36fe2a496d4912/sample2_realign2transcript.bam, contains 14 reference sequences.
2025-08-30T03:05:22.451015Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-30T03:05:22.451018Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-30T03:05:22.451102Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-30T03:05:22.451110Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-08-30T03:05:23.062976Z  INFO oarfish: setting user-provided filter parameters.
2025-08-30T03:05:23.063467Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpHV1zTz/file36fe2a496d4912/sample3_realign2transcript.bam, contains 14 reference sequences.
2025-08-30T03:05:23.063478Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-30T03:05:23.063482Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-30T03:05:23.063569Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-30T03:05:23.063578Z  INFO oarfish: parsed reference information for 14 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpHV1zTz/file36fe2a31aeba6/config_file_3604010.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Fri Aug 29 23:05:23 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/RtmpHV1zTz/file36fe2a31aeba6/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpHV1zTz/file36fe2a31aeba6/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpHV1zTz/file36fe2a31aeba6/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpHV1zTz/file36fe2a31aeba6/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/RtmpHV1zTz/file36fe2a31aeba6/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpHV1zTz/file36fe2a31aeba6/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/RtmpHV1zTz/file36fe2a31aeba6/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpHV1zTz/file36fe2a31aeba6/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/RtmpHV1zTz/file36fe2a31aeba6/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpHV1zTz/file36fe2a31aeba6/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 @ Fri Aug 29 23:05:24 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpHV1zTz/file36fe2a31aeba6/sampleA_matched_reads.fastq.gz -> /tmp/RtmpHV1zTz/file36fe2a31aeba6/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/RtmpHV1zTz/file36fe2a31aeba6/sample1_matched_reads.fastq.gz -> /tmp/RtmpHV1zTz/file36fe2a31aeba6/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/RtmpHV1zTz/file36fe2a31aeba6/sample2_matched_reads.fastq.gz -> /tmp/RtmpHV1zTz/file36fe2a31aeba6/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/RtmpHV1zTz/file36fe2a31aeba6/sample3_matched_reads.fastq.gz -> /tmp/RtmpHV1zTz/file36fe2a31aeba6/sample3_align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Fri Aug 29 23:05:26 2025 ----------------
23:05:26 Fri Aug 29 2025 quantify genes 
Using BAM(s): '/tmp/RtmpHV1zTz/file36fe2a31aeba6/sampleA_align2genome.bam',
'/tmp/RtmpHV1zTz/file36fe2a31aeba6/sample1_align2genome.bam',
'/tmp/RtmpHV1zTz/file36fe2a31aeba6/sample2_align2genome.bam', and
'/tmp/RtmpHV1zTz/file36fe2a31aeba6/sample3_align2genome.bam'
parsing /tmp/RtmpHV1zTz/file36fe2a31aeba6/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 21.69gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 348017.26Read/s]
parsing /tmp/RtmpHV1zTz/file36fe2a31aeba6/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  7.13gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  7.12gene_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, 1326136.33Read/s]
parsing /tmp/RtmpHV1zTz/file36fe2a31aeba6/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 50.04gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1149502.30Read/s]
parsing /tmp/RtmpHV1zTz/file36fe2a31aeba6/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 34.73gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 722757.10Read/s]
-- Running step: isoform_identification @ Fri Aug 29 23:05:27 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri Aug 29 23:05:27 2025 -------------------
Checking for fastq file(s) /tmp/RtmpHV1zTz/file36fe2a31aeba6/fastq, /tmp/RtmpHV1zTz/file36fe2a31aeba6/fastq/sample1.fq.gz, /tmp/RtmpHV1zTz/file36fe2a31aeba6/fastq/sample2.fq.gz, /tmp/RtmpHV1zTz/file36fe2a31aeba6/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpHV1zTz/file36fe2a31aeba6/sampleA_matched_reads.fastq.gz, /tmp/RtmpHV1zTz/file36fe2a31aeba6/sample1_matched_reads.fastq.gz, /tmp/RtmpHV1zTz/file36fe2a31aeba6/sample2_matched_reads.fastq.gz, /tmp/RtmpHV1zTz/file36fe2a31aeba6/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpHV1zTz/file36fe2a31aeba6/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpHV1zTz/file36fe2a31aeba6/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpHV1zTz/file36fe2a31aeba6/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpHV1zTz/file36fe2a31aeba6/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpHV1zTz/file36fe2a31aeba6/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpHV1zTz/file36fe2a31aeba6/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/RtmpHV1zTz/file36fe2a31aeba6/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpHV1zTz/file36fe2a31aeba6/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/RtmpHV1zTz/file36fe2a31aeba6/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpHV1zTz/file36fe2a31aeba6/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/RtmpHV1zTz/file36fe2a31aeba6/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpHV1zTz/file36fe2a31aeba6/sample3_realign2transcript.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Fri Aug 29 23:05:28 2025 ----------
23:05:28 Fri Aug 29 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpHV1zTz/file36fe2a31aeba6/sampleA_realign2transcript.bam...
parsing /tmp/RtmpHV1zTz/file36fe2a31aeba6/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpHV1zTz/file36fe2a31aeba6/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpHV1zTz/file36fe2a31aeba6/sample1_realign2transcript.bam...
parsing /tmp/RtmpHV1zTz/file36fe2a31aeba6/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpHV1zTz/file36fe2a31aeba6/sample1_realign2transcript.bamdone
parsing /tmp/RtmpHV1zTz/file36fe2a31aeba6/sample2_realign2transcript.bam...
parsing /tmp/RtmpHV1zTz/file36fe2a31aeba6/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpHV1zTz/file36fe2a31aeba6/sample2_realign2transcript.bamdone
parsing /tmp/RtmpHV1zTz/file36fe2a31aeba6/sample3_realign2transcript.bam...
parsing /tmp/RtmpHV1zTz/file36fe2a31aeba6/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpHV1zTz/file36fe2a31aeba6/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpHV1zTz/file36fe2a322dea2c/config_file_3604010.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Fri Aug 29 23:05:32 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/RtmpHV1zTz/file36fe2a322dea2c/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpHV1zTz/file36fe2a322dea2c/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpHV1zTz/file36fe2a322dea2c/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpHV1zTz/file36fe2a322dea2c/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/RtmpHV1zTz/file36fe2a322dea2c/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpHV1zTz/file36fe2a322dea2c/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/RtmpHV1zTz/file36fe2a322dea2c/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpHV1zTz/file36fe2a322dea2c/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/RtmpHV1zTz/file36fe2a322dea2c/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpHV1zTz/file36fe2a322dea2c/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 @ Fri Aug 29 23:05:32 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpHV1zTz/file36fe2a322dea2c/sampleA_matched_reads.fastq.gz ->/tmp/RtmpHV1zTz/file36fe2a322dea2c/sampleA_align2genome.bam
/tmp/RtmpHV1zTz/file36fe2a322dea2c/sample1_matched_reads.fastq.gz ->/tmp/RtmpHV1zTz/file36fe2a322dea2c/sample1_align2genome.bam
/tmp/RtmpHV1zTz/file36fe2a322dea2c/sample2_matched_reads.fastq.gz ->/tmp/RtmpHV1zTz/file36fe2a322dea2c/sample2_align2genome.bam
/tmp/RtmpHV1zTz/file36fe2a322dea2c/sample3_matched_reads.fastq.gz ->/tmp/RtmpHV1zTz/file36fe2a322dea2c/sample3_align2genome.bam
-- Running step: gene_quantification @ Fri Aug 29 23:05:53 2025 ----------------
23:05:53 Fri Aug 29 2025 quantify genes 
Using BAM(s): '/tmp/RtmpHV1zTz/file36fe2a322dea2c/sampleA_align2genome.bam',
'/tmp/RtmpHV1zTz/file36fe2a322dea2c/sample1_align2genome.bam',
'/tmp/RtmpHV1zTz/file36fe2a322dea2c/sample2_align2genome.bam', and
'/tmp/RtmpHV1zTz/file36fe2a322dea2c/sample3_align2genome.bam'
	Counter({'counted_reads': 358})
	Counter({'counted_reads': 91})
	Counter({'counted_reads': 95})
	Counter({'counted_reads': 176})
parsing /tmp/RtmpHV1zTz/file36fe2a322dea2c/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 16.29gene_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, 419011.39Read/s]
parsing /tmp/RtmpHV1zTz/file36fe2a322dea2c/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 51.48gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1280938.19Read/s]
parsing /tmp/RtmpHV1zTz/file36fe2a322dea2c/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 49.90gene_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, 1282975.65Read/s]
parsing /tmp/RtmpHV1zTz/file36fe2a322dea2c/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 35.86gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 728683.81Read/s]
-- Running step: isoform_identification @ Fri Aug 29 23:05:54 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri Aug 29 23:05:55 2025 -------------------
Checking for fastq file(s) /tmp/RtmpHV1zTz/file36fe2a322dea2c/fastq, /tmp/RtmpHV1zTz/file36fe2a322dea2c/fastq/sample1.fq.gz, /tmp/RtmpHV1zTz/file36fe2a322dea2c/fastq/sample2.fq.gz, /tmp/RtmpHV1zTz/file36fe2a322dea2c/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpHV1zTz/file36fe2a322dea2c/sampleA_matched_reads.fastq.gz, /tmp/RtmpHV1zTz/file36fe2a322dea2c/sample1_matched_reads.fastq.gz, /tmp/RtmpHV1zTz/file36fe2a322dea2c/sample2_matched_reads.fastq.gz, /tmp/RtmpHV1zTz/file36fe2a322dea2c/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpHV1zTz/file36fe2a322dea2c/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpHV1zTz/file36fe2a322dea2c/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpHV1zTz/file36fe2a322dea2c/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpHV1zTz/file36fe2a322dea2c/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpHV1zTz/file36fe2a322dea2c/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpHV1zTz/file36fe2a322dea2c/sampleA_realign2transcript.bam
/tmp/RtmpHV1zTz/file36fe2a322dea2c/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpHV1zTz/file36fe2a322dea2c/sample1_realign2transcript.bam
/tmp/RtmpHV1zTz/file36fe2a322dea2c/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpHV1zTz/file36fe2a322dea2c/sample2_realign2transcript.bam
/tmp/RtmpHV1zTz/file36fe2a322dea2c/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpHV1zTz/file36fe2a322dea2c/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Fri Aug 29 23:06:15 2025 ----------
23:06:15 Fri Aug 29 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpHV1zTz/file36fe2a322dea2c/sampleA_realign2transcript.bam...
parsing /tmp/RtmpHV1zTz/file36fe2a322dea2c/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpHV1zTz/file36fe2a322dea2c/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpHV1zTz/file36fe2a322dea2c/sample1_realign2transcript.bam...
parsing /tmp/RtmpHV1zTz/file36fe2a322dea2c/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpHV1zTz/file36fe2a322dea2c/sample1_realign2transcript.bamdone
parsing /tmp/RtmpHV1zTz/file36fe2a322dea2c/sample2_realign2transcript.bam...
parsing /tmp/RtmpHV1zTz/file36fe2a322dea2c/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpHV1zTz/file36fe2a322dea2c/sample2_realign2transcript.bamdone
parsing /tmp/RtmpHV1zTz/file36fe2a322dea2c/sample3_realign2transcript.bam...
parsing /tmp/RtmpHV1zTz/file36fe2a322dea2c/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpHV1zTz/file36fe2a322dea2c/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 
720.597  45.237 771.824 

Example timings

FLAMES.Rcheck/FLAMES-Ex.timings

nameusersystemelapsed
BulkPipeline3.3900.1643.403
MultiSampleSCPipeline10.123 0.71111.361
SingleCellPipeline2.9060.1291.881
add_gene_counts0.2640.0120.276
annotation_to_fasta0.1740.0010.176
blaze 4.41714.18410.590
bulk_long_pipeline 2.36611.047 2.573
combine_sce0.7080.0630.770
config-set0.1530.0210.174
config0.1440.0270.174
controllers-set0.3540.0410.396
controllers0.2230.0110.234
convolution_filter0.0000.0000.001
create_config0.0100.0020.012
create_sce_from_dir3.5332.1823.650
create_se_from_dir2.5210.1452.664
cutadapt0.1040.0130.117
example_pipeline0.3280.0060.335
experiment2.1710.1182.287
filter_annotation0.0490.0000.048
filter_coverage1.0090.0731.084
find_barcode0.2880.0330.327
find_bin0.0060.0020.007
find_variants21.997 1.77123.159
get_coverage1.0060.0701.078
index_genome0.1550.0180.171
mutation_positions1.4200.1581.578
plot_coverage2.1440.0242.169
plot_demultiplex1.9370.1602.123
plot_demultiplex_raw1.0500.0481.105
plot_durations2.2900.0982.387
plot_isoform_heatmap6.6550.2896.943
plot_isoform_reduced_dim22.751 0.70923.461
plot_isoforms2.9490.0072.956
resume_FLAMES2.2950.0712.362
run_FLAMES2.2100.0922.301
run_step1.0700.0331.106
sc_DTU_analysis7.0101.9736.813
sc_gene_entropy1.4880.1331.770
sc_genotype2.9030.5482.463
sc_impute_transcript0.5650.0090.574
sc_long_multisample_pipeline7.9635.2197.592
sc_long_pipeline3.0591.2272.519
sc_mutations2.6370.3032.381
sc_plot_genotype10.015 0.238 9.112
show-FLAMESPipeline0.2910.0060.297
steps-set0.4240.0070.431
steps0.1380.0100.148
weight_transcripts0.0240.0040.029