Correct for GC content bias RNASeq DE

Question

[debitboro]

Hi all,

Shall I need to correct for GC content bias when I'm trying to compare 2 tissue types coming from human in DE analysis ?, since (from what I understood) the GC content is gene-specific and is the same across the samples under comparison (same species).

Thanks in advance

Answer 1

[Rob]

Hi /u/debitboro,

One of the main technical issues with GC bias in RNA-seq data is that there can be sample-specific, fragment-level GC bias. That is, the probability of sampling a particular fragment (i.e, paired-end read) can depend upon the GC content of that fragment (see the excellent blog post by Mike Love here). These biases can differ even between the same tissue type in the same species. If you're comparing samples that were sequenced at the same time, in the same conditions, in the same lab, using the same protocol, etc., then you can reasonably expect the biases to be similar. Though bias will affect the actual accuracy of quantification, the fact that it is very similar between samples means that it will likely have only a small effect on differential analysis. However, if the samples were sequenced at different times, in slightly different conditions, at different labs, by different technicians, etc., then there could be technical differences that would affect downstream differential analysis. Again, I highly recommend you take a look at Mike's post and the "Alpine" paper he cites within that describes such biases in detail.

[debitboro]

thank you Rob, your response is helpful, great appreciation of the Mike Love's blog.