I am attempting to construct a DNA library for paired end sequencing using the illumina y-shaped adapters. An issue we are currently having is that we run into a lot of adapter dimers when we add the sequencing adapters and then run PCR to amp up the library. We are adding the adapters to MmeI digested DNA which have a cut site of NN.
Other sequencing approaches don't have this issue as they can remove the 5' phosphate group of the adapters to prevent dimeric binding. With the Illumina adapters though, due to their non-complimentary y-shape, this would result in the loss of one half of the adapter pair as there would be no complementary sequence to hold it in place before nick translation can repair the DNA backbone.
I am wondering if anyone has designed their own adapters which do not follow this y-shape. Alternatively has anyone derived a method to overcome this issue of adapter dimers.
I've not used adapters provided by illumina before - I synthesised my own (PE adapters) (with help of the original illumina patent protocol) however New England Biolabs I believe sell illumina adapters (SE and PE) that are not Y shaped but that do the same job.
I've used both my own and NEB adapters and both have worked well. For SE sequencing I used the NEB adapters (ChIP-Seq)
Matt, I ran into a very similar problem as yours. I'm very curious if adding PNK solved the nick issue? And I assume you did another round of DNA ligation after the PNK treatment yes?
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