Aquatic invertebrate metabarcoding with Oxford Nanopore sequencing

Metabarcoding is useful for the rapid identification of species in a mixed sample, for which short-read DNA sequencing platforms have been commonly used. We here use Oxford Nanopore MinION sequencing as an alternative to short-read sequencing platforms and obtain highly accurate consensus reads for bulk sample metabarcoding, despite the high base call error rates of MinION reads. We use rolling circle amplification (RCA) to obtain long and repeated DNA fragments and summarize these repeated fragments to obtain a consensus sequence for each species with our newly developed bioinformatics pipeline ASHURE. Our study demonstrates that Nanopore sequencing can be accurately used for bulk sample metabarcoding. We predict that highly accurate and potentially field-based DNA metabarcoding is possible within the next few years.