Sources and effects of intronic reads in ribo-zero (ribosome depleted) RNA-seq data

Question

[xieshaojun0621]

Hi there,

Recently I did some analysis for ribosome depleted RNA_seq data. I also got the corresponding mRNA-seq data. When I loaded these two dataset into IGV. There are significantly more reads in intron compared to mRNA-seq data. In this case for ribosome depleted RNA_seq, I can still tell the exon structure based on the read coverage. Please see the snapshot I got from IGV. But I also had some data, I can't even tell the exon structure based on read coverage.

Here are my questions: 1) Could someone tell me the potential reason of these intronic reads? The reason I think of is introns that have been spliced out, degraded transcripts. 2) If there are intronic reads, does this mean there are reads come from degraded RNA fragments? If so, will this affect the downstream analysis, like DEG identification? 3) Is there anything that I should be very careful when doing analysis on such data?

Thanks, Shaojun

Answer 1

[WouterDeCoster]

PolyA enrichment as is used in mRNA sequencing selects not only for mRNAs but also for mature mRNAs. What you see intronic are mRNAs which haven't been spliced yet.

Obviously you cannot compare ribo-zero with polyA enrichment library preps, but comparing multiple ribo-zero libraries with each other should be fine.