Question: Can I combine quants from SE and PE reads from replicates of same sample in Salmon Quasi-mapping mode?
gravatar for Sandip Darji
14 months ago by
USA/New York/Nathan Kline Institute for Psychiatric Research
Sandip Darji0 wrote:

Dear Dr. Patro, developers of Salmon and BioC community,

I have put great amount of effort to find out answer to this question on internet. Couldn't find it anywhere (bioconductor, biostars, seqanswers, github etc.).

I have 3 replicates for each sample and one of the replicates from each sample is single-end and other two are paired-end as SE and PE were processed at different facilities (I know I have to do batch correction in downstream analysis). Now I want to use transcript level abundance from quant.sf file which I derived for each replicates using Salmon's quasi-mapping pipeline (used appropriate flags for SE and PE reads). All these (SE & PE) reads are strand specific. 

My question is, can I use quant.sf directly from these replicates for downstream DE analysis using tximport or do SE or PE requires separate kind of processing before I can use them together as replicates for downstream analysis. I am planning to use limma-voom for my DE analysis.

Thank you so much for your time and apologies if the question was answered already.
Sandip Darji 

ADD COMMENTlink modified 14 months ago by Michael Love20k • written 14 months ago by Sandip Darji0
gravatar for Michael Love
14 months ago by
Michael Love20k
United States
Michael Love20k wrote:

As far as importing the quantifications, there is nothing special you would do to import SE or PE samples using tximport. When it comes to statistical analysis, you could include the library type as a variable in the design.

ADD COMMENTlink written 14 months ago by Michael Love20k

Thank you so much Dr. Love for your prompt response. 

I am including "batch" vector (batch 1 for SE reads and batch 2 for PE reads). So to confirm, you can treat quants from biological replicates as if they are normal replicates and it doesn't matter whether they are SE or PE. In other words, you can have biological replicates from SE and PE and treat them together as any other normal replicates. correct? I knew the fact, while dealing with raw counts (e.g. featurecounts or the counts from star-aligner), one can treat both SE and PE the same way as if they are all from same library (all from SE or all from PE) as long as they are biological replicates. Just wasn't sure the same is true while dealing with quants from salmon.

Thanks again,

Sandip Darji

ADD REPLYlink written 14 months ago by Sandip Darji0

Yes, that's what I meant

ADD REPLYlink written 14 months ago by Michael Love20k

Thanks, again.

ADD REPLYlink written 14 months ago by Sandip Darji0
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