My lab has designed a nested PCR assay (~500bp) for a bacterial plasmid (pMUM) for use in environmental detection. The assay works well, but in an effort to replicate our findings we encountered an anomaly. After sequencing the replicates, we found that one sample (from a fish mucosal microbiome) yielded two distinct plasmid sequences in the two replicates. The chromatograms are "clean" meaning there is no evidence of heteroplasmy in the data. In my experience with simple PCR, this anomaly would cause suspicion, and I would interrogate whether the samples were mislabeled at some point. However, this is a nested PCR assay, and I wonder if the lack of evidence for heteroplasmy might instead be an artifact of multiple rounds of amplification. Is it possible that one plasmid type would be cleanly sequenced in the first replicate, and another type would be cleanly sequenced in the second replicate? Is nested PCR known to reduce sensitivity for heteroplasy and/or heterozygosity?
Usually, you need to resort to nested PCR when starting levels of template are very low: under these conditions you're already into stochastic partitioning territory (
Thank you, John. Indeed, we are likely dealing with VERY low template concentration. This makes me feel better about describing the anomaly in the methods note.
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