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Question: htseq-count error with strand data
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gravatar for Nmojica
16 months ago by
Nmojica • 20
Nmojica • 20 wrote:

Hi!

I am running htseq-count for a RNA-seq and I got this error:

Fatal error: Unknown error occured [bam_sort_core] merging from 28 files... Error occured when processing GFF file (line 1 of file /galaxy-repl/main/files/020/688/dataset_20688618.dat): Feature TRINITY_DN17_c0_g1_i1 at TRINITY_DN17_c0_g1_i1:[0,272)/. does not have strand information but you are running htseq-count in stranded mode. Use '--stranded=no'. [Exception type: ValueError, raised in count.py:57]

Apparently, my data is not from a strand-specific assay , but the data was obtained with Illumina Truseq Stranded mRNA library PrepKit. I have understood that for this protocol, the library type in TopHat should be fr-firststrand and in htseq-count I should select "reverse" in the strand option. However, I am getting this error. Should I use the unstranded mode instead? I am very new to RNA-Seq and I would highly appreciate your help.

rna-seq htseq-count • 637 views
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modified 16 months ago • written 16 months ago by Nmojica • 20
1
gravatar for Jennifer Hillman Jackson
16 months ago by
Jennifer Hillman Jackson ♦ 25k
United States
Jennifer Hillman Jackson ♦ 25k wrote:

Hello,

Yes, run in unstranded mode. The reference transcriptome strandedness is a different metric from the original fastq read strandedness.

This is an RNA tutorial that may help clarify with an example protocol: https://galaxyproject.github.io/training-material//topics/transcriptomics/tutorials/de-novo/tutorial.html

And more tutorials for RNA-seq are available here: https://galaxyproject.org/learn/

Thanks! Jen, Galaxy team
ADD COMMENT • link written 16 months ago by Jennifer Hillman Jackson ♦ 25k
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gravatar for Nmojica
16 months ago by
Nmojica • 20
Nmojica • 20 wrote:

Thanks! It finally worked.

ADD COMMENT • link written 16 months ago by Nmojica • 20

Great news, thanks for following up!

ADD REPLY • link written 16 months ago by Jennifer Hillman Jackson ♦ 25k
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