Cycle number? Indexing/enrichment NEBNext

[lmw.bio]

I'm preparing dual-indexed libraries using NEBNext Ultra Library Prep for targeted resequencing on Illumina MiSeq and can't decide on the number of PCR cycles to use. I started the library preparation with 500ng of input DNA (custom amplicons). After the adapter ligation and bead clean-up I quantified my samples again (only quantified 9 random samples out of all 80) and found concentrations between 180 and 450 ng total DNA.

I'm looking for two pieces of advice. First, what is a good amount of DNA to use in the final enrichment? The protocol says to use it all of the adapter ligated DNA fragments, but I wonder if I could get away with only using a portion, just in case I need to repeat the enrichment step. And my second question is, how should one choose a good number of cycles for the enrichment step?

I have not spoken with NEB about these particular questions yet. However, when I contacted them previously about how much starting amplicon DNA to use, NEB tech support said, "For example if you are using 100ng of amplicons as starting material you may want to try about 4-5 cycles of PCR. I would expect that amplicons may need less cycles of PCR than our recommendations in the manual."

Given that I started with 500 ng of amplicon DNA and don't seem to have lost a great deal of DNA through the preparations so far I'm not sure how many cycles to use. Because my starting material was amplicons I would like to minimize as much additional bias as possible by limiting the number of enrichment cycles.

Thank you for any advice you may have.

[nucacidhunter]

I am not sure which of the following you are going to do:
1- You have amplified target regions by PCR using target specific primers and ligated them to Illumina adapters and you are going to pool them for sequencing directly
2- You are going to do some enrichment by sequence capture (like Agilent SureSelect) and use those adapted libraries as pool in hybridisation step of capture

[lmw.bio]

Option 1. I have amplified target regions by PCR using specific primers and lighted them to Illumina adapters. Before these can be pooled for sequencing is the final step (and the one in question), to do a PCR 'enrichment' of those adapter lighted fragments using dual indexed primers. The NEB protocol says to use between 4 and 12 cycles depending on input DNA but their recommendations are not made for amplicon inputs, only for fragmented DNA.

[nucacidhunter]

You can use 100 ng of each in PCR and do 5 cycles. Assuming 10% ligation efficiency you would expect around 300 ng yield. On the other hand 20 ul of 5 nM library is more than enough for qPCR and clustering. Quntifying yield after 5 cycles will indicated if and how many more cycles needed.

[lmw.bio]

thanks

Thanks nucacidhunter I'm going to go for it and will report back