RNA-Star FeatureCount error for REF RNA-seq training

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1

Hi everyone,

I am a newbie in RNA-seq data and I am trying to learn RNA-seq analysis using Galaxyserve.

I have followed the instruction form Reference Based RNA-seq data analysis in TrainingGalaxyProject (Here) . However, I am stucked in “Featurecount” steps. The server noticed there is an error due to No paired-end reads were detected in paired-end read library.

Please help me with this issue!

The error detail can be found below

export FC_PATH=$(command -v featureCounts | sed ‘s@/bin/featureCounts$@@’) && featureCounts -a ‘/data/dnb11/galaxy_db/files/d/4/b/dataset_d4bf1575-4bc9-4a6c-ad6a-ed7dd2fa9449.dat’ -F “GTF” -o “output” -T ${GALAXY_SLOTS:-2} -s 0 -Q 10 -t ‘exon’ -g ‘gene_id’ --minOverlap 1 --fracOverlap 0 --fracOverlapFeature 0 -p -C --countReadPairs ‘/data/dnb11/galaxy_db/files/f/a/0/dataset_fa0df423-81d5-4894-a63b-1bf66b83719d.dat’ && grep -v “^#” “output” | sed -e ‘s|/data/dnb11/galaxy_db/files/f/a/0/dataset_fa0df423-81d5-4894-a63b-1bf66b83719d.dat|GSM461177|g’ > body.txt && cut -f 1,7 body.txt > ‘/data/jwd05e/main/083/574/83574365/outputs/dataset_f4907f59-c471-4727-98b1-a27cc9a0443c.dat’ && cut -f 1,6 body.txt > ‘/data/jwd05e/main/083/574/83574365/outputs/dataset_af149bc3-2501-49c3-8b72-cd15ca4874a5.dat’ && sed -e ‘s|/data/dnb11/galaxy_db/files/f/a/0/dataset_fa0df423-81d5-4894-a63b-1bf66b83719d.dat|GSM461177|g’ ‘output.summary’ > ‘/data/jwd05e/main/083/574/83574365/outputs/dataset_6df26791-4404-4122-a3da-fbc8d810b05e.dat’

========== _____ _ _ ____ _____ ______ _____
===== / | | | | _ | __ | | /\ | __ \
===== | ( | | | | |) | |) | | / \ | | | |
==== _ | | | | _ <| _ /| | / /\ \ | | | |
==== ____) | || | |) | | \ | |___ / ____ | || |
========== |/ _/|/|| _______// ____/
v2.1.1

//========================== featureCounts setting ===========================\
|| ||
|| Input files : 1 BAM file ||
|| ||
|| dataset_fa0df423-81d5-4894-a63b-1bf66b83719d.dat ||
|| ||
|| Output file : output ||
|| Summary : output.summary ||
|| Paired-end : yes ||
|| Count read pairs : yes ||
|| Annotation : dataset_d4bf1575-4bc9-4a6c-ad6a-ed7dd2fa9449 … ||
|| Dir for temp files : ./ ||
|| ||
|| Threads : 2 ||
|| Level : meta-feature level ||
|| Multimapping reads : not counted ||
|| Multi-overlapping reads : not counted ||
|| Min overlapping bases : 1 ||
|| ||
\============================================================================//

//================================= Running ==================================\
|| ||
|| Load annotation file dataset_d4bf1575-4bc9-4a6c-ad6a-ed7dd2fa9449.dat … ||
|| Features : 195484 ||
|| Meta-features : 23932 ||
|| Chromosomes/contigs : 29 ||
|| ||
|| Process BAM file dataset_fa0df423-81d5-4894-a63b-1bf66b83719d.dat… ||
ERROR: No paired-end reads were detected in paired-end read library : /data/dnb11/galaxy_db/files/f/a/0/dataset_fa0df423-81d5-4894-a63b-1bf66b83719d.dat

Thank you

2

Welcome @philongtran

Thanks for posting all the details, very helpful!

This error

Is referring to a conflict between your input BAM file here

And the parameter set on the form here

Your data was paired end, yes?

In short, you need to tell the tool what to expect in the input, then it runs a quick double check as it processes the input file. If the check fails, meaning the parameter and inputs are different, it knows how to report that back to you in the logs.

Go back to this view in the job details using the i-icon to see it formatted nicer. You can expand the input BAM dataset and click on the job details for that prior job from here – then keep going until you see the mapping job. Did it have forward and reverse reads input for mapping? That means the data is paired end (two reads per sample), instead of single end (one read, not two, per sample).

A nice full description of this concept is in this tutorial. → Hands-on: NGS data logistics / NGS data logistics / Introduction to Galaxy Analyses

Then use the rerun-icon on the failed dataset to bring back up the original tool form. On this you can toggle that parameter to better match your data content, and submit the job, to see if that is enough. :slight_smile:

Most problems with errors can be solved in a similar way: the job details, the job logs, then checking the inputs and parameters based on the error messages. You can search here at this forum to find prior Q&A like these → Search results for 'ERROR: No paired-end reads were detected in paired-end read library' - Galaxy Community Help, but you can also just search the web to start with. If you ever get stuck, come back here and we can try to help, too!

Hope this helps!