Ideal size of a library for miRNA ?

Question

[Aurelie MLB]

Hello,

I was wondering what is your experience about miRNA sequencing please? What would be the ideal size for a library?

I found this documentation from Exiqon (http://www.exiqon.com/ls/Documents/Scientific/Service-Customer-Information-NGS.pdf) basically saying that 5 M reads should be sufficient for a library if I understand well (it is based on this article: https://www.researchgate.net/profile/Raghu_Metpally/publication/235786794_Comparison_of_Analysis_Tools_for_miRNA_High_Throughput_Sequencing_Using_Nerve_Crush_as_a_Model/links/02e7e51ed5d1e63a72000000.pdf).

For safety, they seem to sequence up to 10M read though.

Do you think these numbers are sufficient for a sensitive differential expression analysis please?

Many thanks

Answer 1

[Chris Fields]

I found it completely depends on the expected yield of small RNA in your samples. I have had seq data from samples which had large RNA degradation issues, so we saw lots of rRNA in the preps and total miRNA levels < 5% and thus distinguishing noise from signal was more difficult. But also had samples where the yield was much higher (low background rRNA); I think these were exosomal preps. In both cases the reads were between 5-20M and still useful, so it's probably a safe bet to use 10M if your confident in your sample quality.

[Aurelie MLB]

Thank you!