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petey 11-15-2012 03:59 PM

Library prep woes – unusual bioanalyzer profile of Illumina RNA-seq library
3 Attachment(s)
Hi everyone,

I have been preparing RNA-seq libraries using the Illumina Tru-seq V2 kit. My first Batch of libraries turned out just like the picture in the Illumina manual using half the ligation template and a 15 cycle PCR (see attachment “Batch 1” – trace is from the MultiNA not Bioanalyzer). However, when I prepared a second batch of libraries I got almost no product using a 15 cycle PCR (see Batch 2_15 cycle). So I increased the PCR cycles to 21 and I get a bucket load of product; however, I appear to have 2 peaks now including a very sharp peak at ~250 (Batch 2_21 cycle). My first thought is that this must be a low abundance artefact and that the libraries are useless but I made quite a few (20+) that have this profile so I want to be sure they are useless before starting again. For Batch 1 and Batch 2 I used different RNA samples but the RNA used for each was from the same tissue (Arabidopsis leaves) prepared in the same way and had same Bioanalyzer quality (RIN 7-8); only difference was I prepared more samples in Batch 2, and used a brand new kit.

I presume something went wrong in the library prep, perhaps I lost the library in one of the AMpure steps or maybe it was a dynabead mRNA isolation issue? Has anyone seen this before? Does anyone have any ideas of the cause? Any thoughts on whether these libraries will be usable?

My plan now is to spike a small amount of the defective libraries into another experiment that we will be sequencing (to get a ~1 mil reads or so, 100bp se – does this sound reasonable?), to try to determine what I have amplified and see if we can diagnose the problem (I used the inline controls from illumina, so perhaps they will be useful). Does this sound like the right approach?



NextGenSeq 11-19-2012 10:00 AM

The libraries are okay except the 21 cycle library is over-amplified and beginning to dimerize. If you can see the library on a DNA 1000 chip you have enough to sequence.

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