Duplication rate as reported by fastp.

Question

[Anjan]

I have sequenced a set of RNA extracts using Illumina's stranded total RNA prep protocol. The QC tool, fastp, reports that the read duplication rate for these samples is in the 39 to 40% range. Is this an acceptable duplication rate to move forward with downstream analysis, specifically, identifying differentially expressed genes? I would expect some level of duplication given that we are sequencing multiple copies of transcribed DNA. Thank you for your insight. Anjan

Answer 1

[GenoMax]

While some sequence duplication is expected for RNAseq (there can be multiple copies of same transcript), any experimental manipulation (e.g. extra PCR cycles to increase the amount of sequenceable material) can cause issues with sequence duplication.

You may want to take a look at this blog post --> https://sequencing.qcfail.com/articles/libraries-can-contain-technical-duplication/

You should move forward with the analysis as long as there is no batch effect in your samples (i.e. done by different tech, days etc). If you start noticing issues then come back and diagnose.