Our “GoFish” paper is published in PLOS ONE (Stoeckle MY, Mishu MD, Charlop-Powers Z. GoFish: a versatile nested PCR strategy for environmental DNA assays for marine vertebrates). Excerpt from the abstract:
“GoFish assays amplify a mitochondrial 12S rDNA segment with vertebrate metabarcoding primers, followed by nested PCR with M13-tailed, species-specific primers. Sanger sequencing confirms positives detected by gel electrophoresis.
Unlike quantitative PCR (qPCR), GoFish does not require tissues of target and related species for assay development and a basic thermal cycler is sufficient. Unlike Illumina metabarcoding, indexing and batching samples are unnecessary and advanced bioinformatics expertise is not needed. From water collection to Sanger sequencing results, the assay can be carried out in three days.
The main limitations to this approach, which employs metabarcoding primers, are the same as for metabarcoding, namely, inability to distinguish species with shared target sequences and inconsistent amplification of rarer eDNA.
This approach will be a useful addition to current eDNA methods when analyzing presence/absence of known species, when turnaround time is important, and in educational settings.”
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