Since the release of the MinION sequencer in 2014, it has been applied to great effect in the remotest and harshest of environments, and even in space. One of the most common applications of MinION is for nanopore-based DNA barcoding in situ for species identification and discovery, yet the existing sample capability is limited (n ≤ 10). Here, we assembled a portable sequencing setup comprising the BentoLab and MinION and developed a workflow capable of processing 32 samples simultaneously. We demonstrated this enhanced capability out at sea, where we collected samples and barcoded them onboard a dive vessel moored off Sisters' Islands Marine Park, Singapore. In under 9 h, we generated 105 MinION barcodes, of which 19 belonged to fresh metazoans processed immediately after collection. Our setup is thus viable and would greatly fortify existing portable DNA barcoding capabilities. We also tested the performance of the newly released R10.3 nanopore flow cell for DNA barcoding, and showed that the barcodes generated were ~99.9% accurate when compared to Illumina references. A total of 80% of the R10.3 nanopore barcodes also had zero base ambiguities, compared to 50-60% for R9.4.1, suggesting an improved homopolymer resolution and making the use of R10.3 highly recommended.
Keywords: Oxford Nanopore Technologies (ONT); cytochrome c oxidase subunit I (COI); marine biodiversity; metazoa; next-generation sequencing (NGS); portable sequencing.