any pipeline for small non coding RNA analysis from total RNA-seq

Question

[lkianmehr]

Please help me with these questions

1- Do I get reliable information of siRNA like long hairpin RNAs from total RNA-seq data ( with reads 75 bp)?

2- how about any small non-coding RNA like piRNA, miRNA, tRNA, tfRNA?

3 could you please suggest to me a pipeline for that

Thank you so much!

Answer 1

[ATpoint]

1- Do I get reliable information of siRNA like long hairpin RNAs from total RNA-seq data ( with reads 75 bp)?

No, probably not as most library prep and RNA extraction kits have a certain size range they capture, and smallRNAs are too short for it. That is why special smallRNA kits exist.

2- how about any small non-coding RNA like piRNA, miRNA, tRNA, tfRNA?

Same as above. You might get some spurious reads for some of them but this is probably not reliable. "Standard" RNA-seq is simply the wrong experiment for this.

Answer 2

[A. Domingues]

If I remember correctly most total RNA protocols will have two steps that will hinder the analysis you want to do:

1. size select the library before sequencing (>200 bp I think)
2. select rRNA and possibly other very abundant RNA species.

This means that small RNA analysis from total RNA is nearly impossible, but I would advise to first check how the library was prepared.

Answer 3

[nkabo]

I could suggest an answer to the third question. For pipelines to analyze small non coding RNAs, I would suggest you to check DolphinNext, they provide several pipelines for such analyses and it is very useful.

Link: https://dolphinnext.readthedocs.io/en/latest/dolphinNext/quick.html