[BioC] Expected number of DE genes?

[Steve Lianoglou]

Hi,

On Tue, Jul 15, 2014 at 5:54 PM, Jessica Perry Hekman
<hekman2 at illinois.edu> wrote:
> I'm getting only a few dozen differentially expressed genes when I analyze
> my RNA-Seq data with DESeq2 (79) and EdgeR (34) (even fewer when I use
> EBSeq). I had expected many more -- hundreds or even a thousand. If this is
> the real answer, I'm fine with it, but I'm concerned that I'm doing
> something wrong. What are the ranges of numbers of differentially expressed
> genes that one would expect from DESeq2 or EdgeR?

This is an impossible question to answer as this will, of course,
depend on your experiment.

There are many different reasons why you found so few DGEs. Maybe this
is reality, maybe this is a failed experiment (ie. some failure at the
bench), or maybe there is a mistake in your analysis.

We can only help you to answer the last point, but to do so we will
need to see the code you used and an explanation of your dataset (ie.
which samples are which condition).

Also: why do you expect to get upwards of a thousand differentially
expressed genes? Have you (or others) done this experiment before and
verified those results, or?

> More information:
>
> I'm in the midst of my first RNA-seq project (as many of you have probably
> surmised from my frequent postings to a variety of lists). My initial goal
> is to get a list of differentially expressed (DE) genes.
>
> I have 24 samples, 12 from each of 2 treatment groups.

That's an amount of replication that most of us would be envious of.
Assuming the experiment was done w/o problems, then you should be well
powered to detect true DE events.

> My species is fox (Vulpes vulpes), which aligns very nicely to dog (Canis
> familiaris).

Tricky.

> My current approach is to use the dog reference genome (to which my fox
> reads align at about 83%) + GTF with location of exons.

What does the enrichment of reads that align to exons vs. intergenic
reads look like?

Have you (or others) had success doing an analysis this way before w/
previous data/experiments?

> Can I feel confident about DESeq2 and EdgeR's calls?

For the most part, yes, as long as you have done the analysis properly.

-steve

-- 
Steve Lianoglou
Computational Biologist
Genentech