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

Join Date: Jan 2013
Posts: 1,196
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Quote:
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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