[BioC] DE analysis with reference transcriptome

[Michael Love]

hi Nicole,

Here was my response to a similar question:
https://stat.ethz.ch/pipermail/bioconductor/2014-May/059479.html,
basically saying the same as you quote from the RSEM authors. Briefly,
you can use DESeq2 on estimated counts, with the caveat that our
software is not taking into account the uncertainty of the estimation
of counts. I am not certain about the details of the RSEM/EBSeq
handoff, so I can't comment on that. You mention,

> > I've seen the
>> "Count-based differential expression analysis of RNA sequencing data using R
>> and Bioconductor" publication online and in it is mentioned that in the case
>> of no genome, a reference transcriptome can be built, reads aligned to it
>> and counted and then the standard pipeline for differential analysis used.

It seems reasonable to try this approach as well. Regardless, I would
recommend looking at the results of any pipeline by eye, and
cross-referencing with raw data (or raw-ish data, e.g. aligned reads
to your reference transcriptome in IGV).

Mike


>> The documentation for DESeq (and DESeq2), says to use raw counts, and
>> nothing (rounded) normalised or counts of covered base pairs. I had a look
>> at the RSEM and eXpress documentation and both seem to do some kind of
>> estimation due to the isoforms inherent in a transcriptome? On the RSEM
>> website it mentions that "popular di!
>
> ff!
>>
>>   erential expression (DE) analysis tools such as edgeR and DESeq do not
>> take variance due to read mapping uncertainty into consideration. Beacause
>> read mapping ambiguity is prevalent among isoforms and de novo assembled
>> transcripts, these tools are not ideal for DE detection in such conditions."
>> They suggest to use EBSeq, but I found max a handful of papers on google
>> scholar that actually used RSEM-EBSeq. I'm new to all this and it's getting
>> quite confusing. Could you please help? What would I have to do with my data
>> and/or my reference transcriptome to be able to use eg the RSEM - DESeq
>> (maybe DESeq2) pipeline? Is there a pipeline that you could recommend in my
>> situation?
>> Thank you so much for your time.
>> Kind Regards,
>> Nicole
>> University of the Sunshine Coast, Locked Bag 4, Maroochydore DC,
>> Queensland, 4558 Australia.
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