Technical replicates of same library samples

[Rosmano]

Hello,

I have a doubt that I would want to know if you can answer to.
I performed a sequencing run of 150 RNA-seq samples on a Novaseq S4 flow cell. However, we did not achieve the required depth (80 million) for 45 of those samples.

I was considering if I could perform a 2x100 sequencing run with the NextSeq-2000 P2 flow-cell for library samples for which I did not get the desired number of reads and concatenate the .fq files obtained from this test run with the .fq files from the previous Novaseq S4 sequencing run.

I've read a Maroni et al. 2008 paper saying that this was OK however the Illumina tech support says this is not when I asked him if it would be okay. Would love to know the experience from people around here.

[Rosmano]

Another question: would this impact the detection of variants in genes with lower expression compared to obtaining the 80 million reads in just one run?

[Rosmano]

https://www.nature.com/articles/s41467-019-09026-y

This paper highlights a potential issue of merging technical replicates. Since it generates a troubling number of false positive variants. Since this RNA-seq data will be used for variant calling, probably performing technical replicates is not the best approach.