RNA Seq - Combining & Normalization of 3-library samples from two different sequencing runs

Question

[Destiny]

Hi all,

I am having trouble finding any help for this dataset I am currently working with. It is RNA-seq data prepared using UMI's. Due to sampling differences with these type samples, they opted to create 3 libraries per sample and they were unsure they would have enough sequencing depth so a month later (before they received the first sequencing data) they decided to have the same pool re-run.

So each of the 38 samples has 3 libraries in two different runs. What would be the best practice for combining and normalizing the libraries for each sample from both runs?

Thank you!

Answer 1

[swbarnes2]

Rerunning the same library on the same kind of instrument with the same read length does not introduce technical artifacts. You should be able to just combine the files at the fastq stage, or any stage.

Note that 3 libraries of the same sample are technical replicates, not biological replicates; you should join them all together too.

(All of this is assuming there are no glaring technical faults with any library prep or sequencing run)

Answer 2

[Tash]

There are approaches out there to address batch effects, such as this: https://academic.oup.com/nargab/article/2/3/lqaa078/5909519

However, as all the libraries were treated the same, and run across both flow cells, I agree with swbarnes2 that you would be able to combine the files without any issues.