Question

To check quality of miRNA-seq library

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9.0 years ago

unique379 ▴ 120

Dear all,

I need to have some view from your great experience in the field of NGS.

We have SOLiD Sequencer to sequence color-space. We have generated library following manufacture protocol and obtained miR-Seq libraries. In order to analyze these seq, first I have trimmed the adapter from 3' and then aligned with genome. Next, I checked quality of library, to do so, I have quantified the genomic co-ordinates of each biotype from Ensembl and plotted percentage in a pie chart (find attachment figure)for QC purpose. Results I found is very disappointing that my each library reached only with few miRNAs and other transcripts (such as protein_coding genes) presented in huge amount.

So I need to figure it out why and how it happened ??

Due to short miRNA length, is aligner was confused to map in miRNA gene? And can map to other part of genome such as protein_coding or non_coding RNA species?
Is this really contamination in my libraries? OR in other words my library preparation was not good?
What is the final destination of those reads which has been mapped to other transcripts?

Note: I have quantified the miRNAs from miRBase gff3 file and most of miRNAs were 0 or almost 0.

Please find attachment here: https://www.dropbox.com/s/cp1f90nwemmnnnr/pie.png?dl=0

RNA-Seq miRNA Gene-Expression NGS • 2.3k views

ADD COMMENT • link updated 2.3 years ago by Ram 44k • written 9.0 years ago by unique379 ▴ 120

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Please see my answer below. I am new to Biostars and finding my way around!

ADD REPLY • link updated 5.0 years ago by Ram 44k • written 8.9 years ago by robm9119 ▴ 180

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Yes we have selected size of libraries (35 bp long) and also checked the quality of RNA before making libraries. These all prerequisite steps were done by following manufacture protocol. And my colleague is pretty sure that quality was very good to pick up small RNA.

ADD REPLY • link updated 5.0 years ago by Ram 44k • written 8.9 years ago by unique379 ▴ 120

score 0 · Answer 1 · 2015-12-18

I am assuming that you did some some kind of size selection for RNA (for miRNA the size selected is usually <30 bp). Did you check the quality of your RNA before making libraries? If you see a lot of reads from longer transcripts, it's most likely that your RNA was degraded and you practically sequenced the degraded longer transcripts.