miRNA proportion in mir-seq

[moriah]

Hi All,

I received an Illumina HiSeq output on human mirna-seq and after trimming the adapters and alignment to the genome I realized that only 30% of my reads align to miRNAs while other 70% are tRNA,rRNA and other small RNAs.

I was wondering whether this miRNA proportion is normal?

Thanks!

[Nicolas]

Hi,

Did you collapse identical sequences (you should not...)? How did you size-selected your RNA? Which tissues is it from (piRNA are abundant in the germline)?

[moriah]

No, I didn't collapse and it is from breast tissue (tumor and normal).

Size selection? What do you mean?
If you mean the library preparation from total RNA, I don't know.

Thanks!

[Nicolas]

The protocol for smallRNA-Seq is not the same as for "classical" RNA-Seq, with the aim of purifying the sample from long RNA and enriching for "smallRNA". At one point, we do run a gel and select for the appropriate size (that might be fly-specific, because there is a very abundant 30nt rRNA in Drosophila).

Other users' input would be useful here!

[moriah]

Yes, the protocol for small RNA library preparation was followed. The small RNA where isolated for sure and sequenced. But this low proportion of miRNA in my data is what I am not sure about, is it normal? and if not, where can be the problem?

Thank you