Question: htseq-count issues with Deseq2
0
gravatar for VY
18 months ago by
VY120
United States
VY120 wrote:

This is the first time im trying this but I keep running into issues. I'm running the htseq-count on my aligned BAM files to create count tables that I can then feed into Deseq2. I run the htseq-count using the default settings and the UCSC Main on Mouse: refGene (Genome) as the GFF file. The job finishes but there are no counts on any of the genes that I can see.

Just out of curiosity I fed the files into Deseq2 and the job crushed with this comment :

Fatal error: An undefined error occured, please check your intput carefully and contact your administrator. Creating a generic function for 'nchar' from package 'base' in package 'S4Vectors' all samples have 0 counts for all genes. check the counting script. all genes have equal values for all samples. will not be able to perform differential analysis estimating size factors Error in estimateSizeFactorsForMatrix(counts(object), locfunc = locfunc, : every gene contains at least one zero, cannot compute log geometric means Calls: DESeq ... estimateSizeFactors -> .local -> estimateSizeFactorsForMatrix

Any Help would be much appreciated Thanks :)

rna-seq htseq-count • 805 views
ADD COMMENTlink modified 18 months ago by Jennifer Hillman Jackson23k • written 18 months ago by VY120
1

Hello,

There may be a reference genome mismatch problem. This is how to check: https://wiki.galaxyproject.org/Support#Reference_genomes

If that is not the problem, please send in a bug report so that we can review. https://wiki.galaxyproject.org/Support#Reporting_tool_errors

Thanks for reporting the problem, Jen, Galaxy team

ADD REPLYlink written 18 months ago by Jennifer Hillman Jackson23k

Dear Jennifer thanks for the help,

The reads were mapped by the people doing the sequencing back to the mouse Grcm38.ERCC reference genome. What I did is import the GTF from the UCSC table browser Dec.2011(GRCm38/mm10) for the "Genes & Gene Predictions", shouldn't this be the correct reference?

Thanks

ADD REPLYlink written 18 months ago by VY120
0
gravatar for Jennifer Hillman Jackson
18 months ago by
United States
Jennifer Hillman Jackson23k wrote:

Hello,

Thanks for the extra information. I can confirm that these are different builds of the same base reference genome. Choices about how to go forward from here are in the link for reference genomes above with this case-specific help here in a prior post: Mouse mm10 genome

Hopefully this helps, Jen, Galaxy team

ADD COMMENTlink written 18 months ago by Jennifer Hillman Jackson23k
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