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Old 07-27-2017, 01:42 AM   #4
Jafar Jabbari
Location: Melbourne

Join Date: Jan 2013
Posts: 1,196

Originally Posted by GMDickson View Post

Yes, we are using PCR to produce the amplicons initially but would prefer to avoid further PCRs usually necessary to attach the sequncing adaptors/cleanup/enrich. I have indexes and a variable number of Ns added to the end of my PCR primers so that I can distinguish individual samples and try avoid problems associated with low complexity samples but I am not sure if it is feasible to add the necessary sequencing (for MiSeq) adaptors on by ligation or whether a portion needs to also be included in the initial PCR primers (similar to a protocol I found for preping samples for the Ion Torrent). I need to multiplex samples in a run and am dealing with very low quality DNA so the fragment is small.

Do you know of any protocols that might describe the addition of sequncing adaptors without a PCR step?
You can use any DNA library prep kit with forked adapters to prepare PCR-free library from amplicons. You would need to avoid size selection step and just do couple of clean ups after ligation to remove excess adapters.

Generally using non-template Ns and barcode at 5' end of PCR primers will result in bias resulting in quantitative and and sometimes qualitative differences deppending on the barcode.
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