I'm currently generating 16S amplicon libraries for Illumin sequencing using Phusion polymerase, but we have noticed a high level of recombination during the enrichment PCR (~30% of sequences have the incorrect barcode combinations).
I've since read that proof-reading polymerases can give a higher chance of chimera formation. From the literature it doesn't seem like anyone is particularly using any of the published recommendations for avoiding chimera formation (longer extension times, low template input and limited PCR cycles).
I'm wondering if there is a particular polymerase that people prefer for generating 16S amplicons which gives both high fidelity and low chimera formation?
Thanks,
Cath
I've since read that proof-reading polymerases can give a higher chance of chimera formation. From the literature it doesn't seem like anyone is particularly using any of the published recommendations for avoiding chimera formation (longer extension times, low template input and limited PCR cycles).
I'm wondering if there is a particular polymerase that people prefer for generating 16S amplicons which gives both high fidelity and low chimera formation?
Thanks,
Cath
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