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Question: How to test or estimate differential TSS/promoter usage/expression based on RNA-seq data only
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gravatar for Steffen Heyne
5 months ago by
MPI-IE, Freiburg, Germany
Steffen Heyne10 wrote:

Hi,

there are many methods/workflows out now to get (differential) expression on the transcript-level. Like Salmon+Sleuth, Salmon+tximport+deseq2 or Tophat+StringTie+Ballgown.

However I just want to detect the most effected (highest foldchange or hightest absolute expression change) promoter/TSS per gene between two conditions. To get the corresponding (most effected Isoform(s) ) would be also nice!
I want to correlate these things later to chromatin marks and I'm not interested in things related to splicing etc. I have no cage data!

It seems that cuffdiff can do something like this: from http://www.nature.com/nprot/journal/v7/n3/full/nprot.2012.016.html (also see Figure 4):

"By grouping transcripts into biologically meaningful groups (such as transcripts that share the same transcription start site (TSS)), Cuffdiff identifies genes that are differentially regulated at the transcriptional or post-transcriptional level.   [...]

The software groups together isoforms of a gene that have the same TSS. These TSS groups represent isoforms that are all derived from the same pre-mRNA; accordingly, changes in abundance relative to one another reflect differential splicing of their common pre-mRNA. Cuffdiff also calculates the total expression level of a TSS group by adding up the expression levels of the isoforms within it. When a gene has multiple TSSs, Cuffdiff looks for changes in relative abundance between them, which reflect changes in TSS (and thus promoter) preference between conditions."

I would like to do something similar. What would be an appropriate workflow by not using cuffdiff?

I thought of:

1) collapsing all isoforms using the same TSS/Promoter into one isoform, then estimate counts +differences on these with eg. Salmon+Sleuth
2) collapsing with tximport not on gene level, but by same promoter/TSS, then use eg. DESeq
3) using DEXSeq, test for differences only on the first (TSS) exons per gene

Thanks for any suggestions!

ADD COMMENTlink modified 5 months ago • written 5 months ago by Steffen Heyne10
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