A comprehensive DNA barcode reference library for the macroinvertebrates of Scottish seagrass beds using Oxford Nanopore Flongle Flowcells

DNA Barcoding using Sanger sequencing is a popular technique for identifying species on a per specimen basis. However for larger projects, sequencing individual voucher specimens can be time and resource intensive and moreover is associated with high levels of sequencing failure and contamination. Oxford Nanopore Sequencing Technology (ONT) has emerged as a scalable alternative, capable of generating hundreds of DNA barcodes simultaneously using the portable, benchtop MinION sequencing device. In this study we aim to compare and contrast the sequencing outcomes of Oxford Nanopore R10 Flongle flowcells verses Sanger sequencing for DNA barcoding and produce a DNA barcode reference library. We demonstrate that DNA barcodes generated using ONT outperform those produced by Sanger sequencing in terms of recovery and sequence quality with lower rates of contamination. We then produced DNA barcodes for 146 seagrass associated marine invertebrate OTUs collected from four seagrass beds in Scotland, targeting COI and 18S V4 regions. Using both markers, we show the number of recovered OTUs was higher than if each marker was used in isolation and make use of degenerate and group-specific primer pairs to improve recovery. Furthermore we demonstrate how mapping ONT reads to pre-existing DNA barcodes can be used to reduce ambiguous basecalls and improve recovery of sequences from contaminated specimens. Overall this study informs prospective users intending to carry out multimarker DNA barcode projects using Oxford Nanopore Sequencing. Furthermore, we generated the first DNA barcode reference library for seagrass beds in Scotland to support future biomonitoring of these priority habitats.