A Comprehensive <scp>DNA</scp> Barcode Reference Library for the Macroinvertebrates of Scottish Seagrass Beds Using Oxford Nanopore Flongle Flowcells

Oxford Nanopore Sequencing Technology (ONT) has emerged as a scalable method for generating DNA barcode reference libraries, capable of sequencing hundreds of DNA barcodes simultaneously using the portable, benchtop MinION sequencing device. In this study, we use ONT Flongle flowcells to produce DNA barcodes for 146 seagrass-associated marine invertebrate OTUs collected from four seagrass beds in Scotland, targeting COI and 18S V4 regions. We make use of degenerate and group-specific primer pairs to improve recovery and 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 barcoding 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.