Metabarcoding of aquatic invertebrates with Nanopore sequencing with high accuracy. Metabarcoding of aquatic invertebrates with 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. The effectiveness of our pipeline is documented by analyzing reads from two MinION™ runs that represent different molecular workflows that generated data for 50 species in a mixed aquatic invertebrate sample. Overall, we achieved a consensus accuracy of up to 99.3%. 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.