Is the gene-specific PCR efficiency a serious concern for intrasample comparisons in RNA-Seq?

Question

[FedeXandeR]

Or "Everything You Always Wanted to Know About RNA-Seq (But Were Afraid to Ask) Part 2"

In RNA-Seq, it is common practice to compare the abundance of transcripts within the same sample after some form of intrasample normalizations (e.g., TPM) that take into account both transcript length and sequencing depth (although only the former is strictly necessary as long as no other samples are considered). But how reliable are these quantifications really? In particular, I wonder:

1. How influential is the GC-bias? Is it important to correct for it?
2. How much do biases that may come from the PCR amplification step due to the gene-specific transcription efficiency of the polymerase matter? Can this be accounted for in some way?
3. Are there other biases that undermine intrasample comparisons?

Many thanks to everyone who would like to share their experience and opinions!

[ crossposted on Bioconductor: https://support.bioconductor.org/p/9152192/ ]

[benformatics]

Maybe check the literature for #1 and #3: https://www.nature.com/articles/srep25533

For #2 UMIs can account for this

Answer 1

[Trivas]

For 1, many aligners/counters e.g. STAR/Salmon have GC-bias correction built in to the software. Whether you choose to use it is up to you.

For 2, I would say this is more of a problem at the second-strand synthesis step than it would be at the PCR amplification step. At the library amp step, you are using adaptor sequences to amplify your library so your primers are all binding the same region. Your RNA (well cDNA at this point) should also be fragmented to minimize any kind of sequence bias on the DNA pol activity.

For 3, you might want to consider the input RNA integrity and how that might affect your fragment size/overall sequence quality.