DESeq work flow in Galaxy

Question

[kanwarjag]

I am trying to analyze RNA-seq data in DESeq in Galaxy and wonder if anyone has a detailed instructions or work flow how DEseq can be used after alignment in galaxy. Any pointer should be helpful. Since it is galaxy question I have also posted similar question on galxay but though this area may have better coverage. Thanks

Answer 1

[Devon Ryan]

The only real workflow would be to put the alignment files through either htseq-count or featureCounts, then putting the resulting files into DESeq. For featureCounts you'll need to select the correct output format (make sure to use the "DESeq2 IUC compatible ..." one). If you have a specific question about this then just ask.

As an aside, we literally did a training on this today here in Freiburg. You can find the training material online here.

Answer 2

[sapna01t]

Hi Devon

I am using htseq-count for making count tables to be used with deseq later, but i am getting error .. no exon found. tThe gff file of brassica juncea does not have feature exon.. what can I do to solve this problem?

[Devon Ryan]

Either that species lacks UTRs or it's unknown where they are. So try using CDS instead of exon. Note that I'm not sure how well htseq-count handles GFF files, so if that doesn't work then just post a comment here and I'll make a proper GTF file for you.

[sapna01t]

I am using HTseq on galaxy and it seems it is not accepting option CDS in feature type box. It will be a great help if you could make a proper GTF file for me. Thanks in advance :)

GFF file for brassica looks like this....

Seqname Source Feature Start End Score Strand Frame Group Scaffold02034 glean gene 5106 6434 . + 0 ID=BjuA000715; Scaffold02034 glean mRNA 5106 6434 . + 0 ID=BjuA000715;Parent=BjuA000715; Scaffold02034 glean CDS 5106 5174 0.99 + 0 ID=BjuA000715.cds;Parent=BjuA000715; Scaffold02034 glean CDS 5275 5425 0.97 + 0 ID=BjuA000715.cds;Parent=BjuA000715; Scaffold02034 glean CDS 6307 6434 0.61 + 2 ID=BjuA000715.cds;Parent=BjuA000715; Scaffold00650 glean gene 12497 13500 459.56 + . ID=BjuB006329; Scaffold00650 glean mRNA 12497 13500 459.56 + . ID=BjuB006329;Parent=BjuB006329;

[Devon Ryan]

Here you go. Make sure you explicitly label the file type as "gtf" when you upload it to Galaxy.

[sapna01t]

Thank you very much Sir ...