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Also update the docker version in the CWL during the CGC deploy.

Hello everyone!
I'm trying to run RNApeg.sh after building it locally on my ArchLinux server.

When I use:

docker run --mount type=bind,source=/home/rnapeg/resultados/trueseq,target=/results 2022cd54d364 -b /home/Análises/Teste1/star/all_samples_ensembl_chimericwithinbam/99*.bam -f /home/references/cicero/reference/Homo_sapiens/GRCh38_no_alt/FASTA/GRCh38_no_alt.fa -r /home/references/cicero/reference/Homo_sapiens/GRCh38_no_alt/mRNA/RefSeq/refFlat.txt -O /home/Análises/Teste1/RNAPEG/trueseq/sample_5

OR

docker run -v /home/Análises/Teste1/RNAPEG/trueseq:/results ghcr.io/stjude/rnapeg:latest RNApeg.sh -b /home/Análises/Teste1/star/all_samples_ensembl_chimericwithinbam/99*.bam -f /home/references/cicero/reference/Homo_sapiens/GRCh38_no_alt/FASTA/GRCh38_no_alt.fa -r /home/references/cicero/reference/Homo_sapiens/GRCh38_no_alt/mRNA/RefSeq/refFlat.txt

OR

singularity run --containall --bind /home/Análises/Teste1/RNAPEG/trueseq:/results docker://ghcr.io/stjude/rnapeg:latest RNApeg.sh -b /home/Análises/Teste1/star/all_samples_ensembl_chimericwithinbam/99*.bam -f /home/references/cicero/reference/Homo_sapiens/GRCh38_no_alt/FASTA/GRCh38_no_alt.fa -r /home/references/cicero/reference/Homo_sapiens/GRCh38_no_alt/mRNA/RefSeq/refFlat.txt

I get:

`ERROR: unrecognized parameter RNApeg.sh

`
When I run the same, but without RNApeg.sh, I get:

docker run --mount type=bind,source=/home/rnapeg/resultados/trueseq,target=/results 2022cd54d364 -b /home/Análises/Teste1/star/all_samples_ensembl_chimericwithinbam/99*.bam -f /home/references/cicero/reference/Homo_sapiens/GRCh38_no_alt/FASTA/GRCh38_no_alt.fa -r /home/references/cicero/reference/Homo_sapiens/GRCh38_no_alt/mRNA/RefSeq/refFlat.txt -O /

OR

singularity run --containall --bind /home/Análises/Teste1/RNAPEG/trueseq:/results docker://ghcr.io/stjude/rnapeg:latest -b /home/Análises/Teste1/star/all_samples_ensembl_chimericwithinbam/99*.bam -f /home/references/cicero/reference/Homo_sapiens/GRCh38_no_alt/FASTA/GRCh38_no_alt.fa -r /home/references/cicero/reference/Homo_sapiens/GRCh38_no_alt/mRNA/RefSeq/refFlat.txt

OR

docker run -v /home/Análises/Teste1/RNAPEG/trueseq:/results ghcr.io/stjude/rnapeg:latest -b /home/Análises/Teste1/star/all_samples_ensembl_chimericwithinbam/99*.bam -f /home/references/cicero/reference/Homo_sapiens/GRCh38_no_alt/FASTA/GRCh38_no_alt.fa -r /home/references/cicero/reference/Homo_sapiens/GRCh38_no_alt/mRNA/RefSeq/refFlat.txt

I get:

ERROR: Bamfile '/home/Análises/Teste1/star/all_samples_ensembl_chimericwithinbam/99_FRAS202421989-1a_1.fqAligned.sortedByCoord.out.bam' DNE

What should I do?

An external user (email below) reports a BAM compatibility issue with RNApeg. Their instrument-generated BAM maps to a variant of hg19, which is a bit different from the GRCh37-lite that RNApeg uses. With that said, hg19 and GRCh37-lite are compatible for the primary chromosome sequences (chr1-22, X, and Y, however not MT). RNApeg verifies compatibility between the input BAM and by default dies with an error if all the sequences don't have the same lengths. However there is an option "-ignore-incompatible" that in this situation will process compatible reference sequences and ignore any others, which is a good workaround as it rescues the vast majority of the alignment. IMO this should be added to the default command line in the Docker wrapper (RNApeg.sh).

contact email:

From: Steven Shideler <[email protected]>
Date: Wednesday, May 24, 2023 at 3:14 PM
To: Tian, Liqing <[email protected]>
Subject: Re: Illumina produce bam

To the Team at St. Jude,

Hi we are looking to use CICERO for our illumina IRPE assay analysis and our bam files are produced by the Illumina NextSeq 2000 instrument.

I’m writing to inquire as your instructions mention that if we are using a bam input file that wasn’t produced by aligning to GRCh37-lite to contact you.

Our bam files are produced by the DRAGEN server onboard the instrument using “hg19_no_alt_haps_no_decoys” as the reference.

What modifications are necessary to CICERO as I’m currently having trouble generating the junctions file with RNApeg using this Illumina output bam file.

Regards,
Steven Shideler, BSc., MSc., B.I.T.
Bioinformatician, Molecular Pathology
Alberta Precision Laboratories (APL)

Hi,
I am trying to integrate RNApeg into a Nextflow pipeline and I have been getting an error that I think is related to Nextflow not supporting executable containers. When running the RNApeg related process Nextflow attempts to run a bash command, but the RNApeg container expects only parameters to run inside the RNApeg.sh. Do you know if there is a work around for this? I can provide more details if needed. Thanks for your time.

Hello, I'm trying to use RNApeg to prepare data for Cicero and cannot figure out this error I'm getting. When I run the test data from the CICERO github it works, but when I run other data I get an error I can't figure out.

I'm testing with the bam files from https://www.stjude.org/research/labs/zhang-lab/cicero.html. My command is:
docker run -v /Users/shannonkeating/Documents/rna_variantskipping/cicero/test_data/RNApeg:/results -v /Applications/CICERO-master/reference:/reference -v /Users/shannonkeating/Documents/rna_variantskipping/cicero/test_data:/data ghcr.io/stjude/rnapeg:v2.7.7 -b /data/MV4_11_RNAseq_1.bam -f /reference/Homo_sapiens/GRCh37-lite/FASTA/GRCh37-lite.fa -r /reference/Homo_sapiens/GRCh37-lite/mRNA/Combined/all_refFlats.txt

This creates the initial MV4_11_RNAseq_1.bam.junctions.tab file but at the pass 2 step, I get this error:

pass 2: detect shifts... Uncaught exception from user code: error calling floating_junction_fix.pl -jf-shift MV4_11_RNAseq_1.bam.junctions.tab -now -force 1 -no-config -jf-db /reference/Homo_sapiens/GRCh37-lite/mRNA/Combined/all_refFlats.txt -fasta /reference/Homo_sapiens/GRCh37-lite/FASTA/GRCh37-lite.fa -write-both, exit 9 at /RNApeg/src/bin/junction_extraction_wrapper.pl line 338. main::run_cmd("floating_junction_fix.pl -jf-shift MV4_11_RNAseq_1.bam.junctions.tab -now -force 1 -no-config -jf-db /reference/Homo_sapiens/GRCh37-lite/mRNA/Combined/all_refFlats.txt -fasta /reference/Homo_sapiens/GRCh37-lite/FASTA/GRCh37-lite.fa -write-both", "MV4_11_RNAseq_1.bam.junctions.tab.shifted.tab") called at /RNApeg/src/bin/junction_extraction_wrapper.pl line 253 main::run_cmd("floating_junction_fix.pl -jf-shift MV4_11_RNAseq_1.bam.junctions.tab -now -force 1 -no-config -jf-db /reference/Homo_sapiens/GRCh37-lite/mRNA/Combined/all_refFlats.txt -fasta /reference/Homo_sapiens/GRCh37-lite/FASTA/GRCh37-lite.fa -write-both", [MV4_11__RNAseq_output.txt](https://github.com/stjude/RNApeg/files/12339210/MV4_11__RNAseq_output.txt) "MV4_11_RNAseq_1.bam.junctions.tab.shifted.tab") called at /RNApeg/src/bin/junction_extraction_wrapper.pl line 253

I've attached the full output as well.

I'm not sure if this is due to something with the GRCh37-lite reference genome because both the demo data from https://www.stjude.org/research/labs/zhang-lab/cicero.html and my own data are mapped to this reference, but the test data from https://github.com/stjude/CICERO are mapped to GRCh38_no_alt and that works properly. Any help trying to interpret this error would be greatly appreciated.

Building XML-LibXML-2.0208, required for Bio::Tools::CodonTable, fails in (host) Ubuntu 20.04 LTS:

Building and testing XML-LibXML-2.0208 ... ! Installing XML::LibXML failed. See /root/.cpanm/work/1685495744.1/build.log for details. Retry with --force to force install it.
FAIL

The /root/.cpanm/work/1685495744.1/build.log file shows:

[...]

#   Failed test 'exception thrown during parse'
#   at t/35huge_mode.t line 58.
#          got: ''
#     expected: anything else

#   Failed test 'exception refers to entity reference loop'
#   at t/35huge_mode.t line 60.
#                   ''
#     doesn't match '(?^si:entity.*loop)'
# Looks like you failed 2 tests of 5.
t/35huge_mode.t ....................................
Dubious, test returned 2 (wstat 512, 0x200)
Failed 2/5 subtests

[...]

Old post from 2013 referring an issue with the same tests failing

The error message suggests to add --force to the respective line in the Dockerfile, the old post also suggests skipping the test, and indeed substituting the line with,
RUN cpanm --force --no-wget Bio::Tools::CodonTable
Allows to complete the image build, which seems to run fine.