Small RNA seq degradation

[RikyJKD]

Hi, in your opinion, for a small RNA seq experiment, the percentage of the reads that map on rRNA could be a "degradation index" of the library?
What is a good value for this percentage?
Thank you.

[pmiguel]

I don't think this will work for most small RNA library construction methodologies.
Small RNA library construction generally involves ligation of adapters directly to the 5' and 3' ends of RNA. This ligation requires that the RNA have a 5' phosphate and a 3' hydroxyl.

Typical chunks of RNA degraded by heat or random RNAses will not have suitable 5' or 3' ends for ligation. So degraded rRNA likely will show up in very low amounts in most cases. Unless you are end-repairing your RNA prior to ligation. Said end-repair probably requiring more than just T4-PNK treatment as 5' caps need treatment with tobacco acid pyrophosphatase and 2'-3' cyclic phosphates probably need special treatment as well.


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Phillip

[RikyJKD]

Thank you for the explanation. If my protocol is specific for miRNAs why 20% of my reads are rRNAs reads?

[pmiguel]

"Specific"? I didn't write that. I just explained why degraded rRNA is unlikely to comprise much of a miRNAseq library.

Total RNA is generally greater than 80% rRNA and not uncommonly >90% rRNA. 20% rRNA isn't much. Nothing to explain really.

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Phillip