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-   -   3'End polyA artefact in illumina TruSeq Small RNA Seq (http://seqanswers.com/forums/showthread.php?t=24089)

proteomania 10-13-2012 12:00 PM

3'End polyA artefact in illumina TruSeq Small RNA Seq
 
Hi All,

I've been using Illumina TruSeq Kit to prepare Small RNA libraries. I found a small RNA population (non-miRNA) has about 1 to 8 extra As at their 3'end (mismatch to genome). I was wondering could this be the artefact of the TruSeq Kit or adaptor trimming?

Thanks.

pmiguel 10-16-2012 04:51 AM

Could be a poly-A tail. TruSeq small RNA prep involves direct ligation of 3' and 5' adapter to 3' and 5' ends of an RNA. So as long as an RNA strand is in the right size range and has a 5' phosphate and 3' hydroxyl group, it could be ligated to such that the fragment becomes an amplicon, capable of being sequenced.

Now the vast majority of degradation processes (enzymes, heat, divalent cations) are not going to leave ligatable ends. Hence the selectivity of the kit. But it is all a numbers game. So you would expect some % of the degraded RNAs to happen to end up in the ligatable state.

--
Phillip


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