I have two questions related to how to use Deseq. 1) I have total three sample treatments. I am doing pairwise comparison between treatments. I am wondering what sample pool I should use to calcualte SizeFactors and Dispersion. Should I use only the two treatments data that I use in the subsequent nbinomTest step or use all my three sample treatments to calculate SizeFactors and Dispersion. 2) My sample is bacterial that includes chromosome and plasmids, should I study them separately or together ? More specifically, should I run your deseq pipeline independently on chromosome and plasmid or should I combine chromosome and plasmid data first then run your deseq pipeline. thanks, shihai

hi Shihai, We generally recommend users upgrade to DESeq2, which is faster and more sensitive. Additionally, it would make the pairwise comparisons a bit easier. You could use for example: dds <- DESeq(dds) results(dds, contrast=c("treatment","B","A")) results(dds, contrast=c("treatment","C","A")) results(dds, contrast=c("treatment","C","B")) Which would estimate size factors and dispersions using all samples in the first line. The following three lines build results tables contrasting all pairs of the three treatments. I would guess you can run them together, as genes from different chromosomes are combined in a standard analysis. But if I weren't sure, you could compute the size factors from the chromosomes and from the plasmids separately. If these are similar, then I would run them all together. If you have two vectors which identify the plasmid rows and the chromosome rows: estimateSizeFactorsFromMatrix( counts(x)[ plasmidRows, ] ) estimateSizeFactorsFromMatrix( counts(x)[ chromosomeRows, ] ) where x is the dataset, either CountDataSet or DESeqDataSet for DESeq2. Mike On Tue, Jun 10, 2014 at 3:14 PM, Feng, Shihai <sfeng at="" lanl.gov=""> wrote: > I have two questions related to how to use Deseq. > > 1) I have total three sample treatments. I am doing pairwise comparison between treatments. I am wondering what sample pool I should use to calcualte SizeFactors and Dispersion. Should I use only the two treatments data that I use in the subsequent nbinomTest step or use all my three sample treatments to calculate SizeFactors and Dispersion. > > 2) My sample is bacterial that includes chromosome and plasmids, should I study them separately or together ? More specifically, should I run your deseq pipeline independently on chromosome and plasmid or should I combine chromosome and plasmid data first then run your deseq pipeline. > > thanks, > shihai > _______________________________________________ > Bioconductor mailing list > Bioconductor at r-project.org > https://stat.ethz.ch/mailman/listinfo/bioconductor > Search the archives: http://news.gmane.org/gmane.science.biology.informatics.conductor