Question: DESeq2: Tests of log2 differences
1
gravatar for fabian
2.4 years ago by
fabian20
fabian20 wrote:

Dear all

I have sequencing data reflecting the amount of nascent mRNA and I have from the same biological samples data reflecting the polyA fraction. both for two conditions (treated and unteated). I plan to do differential gene expression analysis on the nascent RNA-seq (to get an idea of differential transcription). In a next step I would like to test the polyA fraction sequencing data if they differ from the nascent RNAseq data, which could be indicative of differential decay/stability. To this end I do not want to test the hypothesis that the log2FC(polyA gene) != 0 but log2FC(polyA gene) != log2FC(nascent gene).

I did find the  lfcThreshold, altHypothesis options. But I can not provide a lfcThreshold for each gene individually. Furthermore I do not have an unequal altHypothesis.

I thought about doing it with contrasts but could not figure out how. If I would do something like '~ nascent + condition' I would fake additional replicas.

Any idea how to do this properly is highly welcomed. Thank you very much in advance.

ADD COMMENTlink modified 2.4 years ago • written 2.4 years ago by fabian20

Dear Michael,

thank you very much your helpful and prompt help. The suggested approach seems to fit indeed. I see only one potential problem.  Since I do have nascent RNA and polyA signal from the same biological replica, so I do have paired data. If I now go along your suggested line, I somehow pretend to have 3 replica x 2 condition x 2 assays = 12 biological replicas. But in fact I do only have 6 biological replicas. wouldn't that make the whole approach assailable since I underestimate the true biological variance?

Thank you again for your precious time and help.

 

ADD REPLYlink written 2.4 years ago by fabian20

You have 6 biological replicates, but you have 12 experiments. You can add a term for replicate, to control for within-replicate differences. And then you will only be performing inference on whether the nascent/polyA signal is different across condition, controlling for replicate. See this section in the vignette:

https://bioconductor.org/packages/release/bioc/vignettes/DESeq2/inst/doc/DESeq2.html#group-specific-condition-effects-individuals-nested-within-groups

ADD REPLYlink written 2.4 years ago by Michael Love25k

Those replies come almost as fast as if I would call you.

Thank you very much for your assistance.

ADD REPLYlink written 2.4 years ago by fabian20
Answer: DESeq2: Tests of log2 differences
0
gravatar for Michael Love
2.4 years ago by
Michael Love25k
United States
Michael Love25k wrote:

Does this answer? 

DESeq2 testing ratio of ratios (RIP-Seq, CLIP-Seq, ribosomal profiling)

 

 

ADD COMMENTlink written 2.4 years ago by Michael Love25k
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