PCR Jumping (Template Switching) in Amplicon sequencing

[eladSeq]

Hi,

We performed a full plate amplicon run on 454 FLX, on a gene library in which a single mutation was introduced to each molecule at a different position. We are observing a ~5X higher mutation rate in the 454 library reads compared to the mutation rate observed in Sanger sequencing of variants, and higher than could be explained due to sequencing errors.

We are thinking this may be due so-called "PCR Jumping" during the amplicon PCRs in which incomplete products can hybridize to other templates and end up with hybrid products with multiple mutations. Has anyone seen this in their amplicon runs, or have any thoughts about how to reduce this?

Thanks,
Elad

[essvee]

These are chimeras. You should look up chimera filtering software such as chimera slayer, perseus, or uchime. These can also be reduced by optimizing your PCR conditions.

[ajthomas]

Here are a couple of papers that you might find useful:

Judo, M. S., A. B. Wedel, et al. (1998). "Stimulation and suppression of PCR-mediated recombination." Nucleic Acids Res 26(7): 1819-25.

Kanagawa, T. (2003). "Bias and artifacts in multitemplate polymerase chain reactions (PCR)." J Biosci Bioeng 96(4): 317-23.