Question: htseq-count error with strand data
0
gravatar for Nmojica
4 months ago by
Nmojica10
Nmojica10 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 • 133 views
ADD COMMENTlink modified 4 months ago • written 4 months ago by Nmojica10
1
gravatar for Jennifer Hillman Jackson
4 months ago by
United States
Jennifer Hillman Jackson23k 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 COMMENTlink written 4 months ago by Jennifer Hillman Jackson23k
1
gravatar for Nmojica
4 months ago by
Nmojica10
Nmojica10 wrote:

Thanks! It finally worked.

ADD COMMENTlink written 4 months ago by Nmojica10

Great news, thanks for following up!

ADD REPLYlink written 4 months ago by Jennifer Hillman Jackson23k
Please log in to add an answer.

Help
Access

Use of this site constitutes acceptance of our User Agreement and Privacy Policy.
Powered by Biostar version 16.09
Traffic: 100 users visited in the last hour