Adaptor dimer contamination
 

Dear All,

We are facing some problems with adaptor dimers in Illumina RNA seq library prep.

There is a small amount of adaptor dimer (at ~150 bp) in a few of the samples. We tried a second bead clean up on half of the sample, but lost a significant amount of material. We talked with Illumina tech support, and this is a tiny enough amount of adaptor that they felt if was fine to go forward. There will be some adaptor only reads that the analyst can filter from the data, but it should be a small percentage.

I attach the profile of the sample most contaminated, do you think this wont influence the amplicfication of my library??

Many thanks,
Paolo

Ampure Size Selection to remove adapter dimer
 

Hi Paolo,

What bead to sample volume ratio did you use for your cleanup? If you use the wrong ratio, loss of library is possible. See slide 52: http://www.broadinstitute.org/files/...PrepSlides.pdf

A ratio of 0.7-0.8x is pretty effective at removing the adapter dimer band without resulting in sample loss.

I would personally do one more cleanup to completely remove the dimer band. Try it out on a non-precious sample first to test.

- Genohub

That's a lot of adapter dimer relative to your sample! Adapter dimer tends to hybridize to the flow cell much better than your actual library. I'd be worried about sequencing mainly adapter dimer if I loaded that onto our MiSeq.

In my experience, that amount of adapter dimer would cause some significant problems on a MiSeq, and you would definitely lose quite a number of reads on HiSeq as well.

dilute your adaptors, up to 10 folds dilution, you can try different ratio to get the best result.

Yes, this will definitely fail quality control. Nowadays the beads have improved a lot. You can use AmPure XP or NVIGEN MagVigen. But MagVigen fares better. https://www.nvigen.com/dna-clean-up/

Disclosure: I work at NVIGEN. In case you find problems using MagVigen. We will be happily work with you to troubleshoot or fine tune it for your needs.