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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.

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Background Sharks and rays are some of the most threatened marine taxa due to the high levels of bycatch and significant demand for meat and fin-related products in many Asian communities. At least 25% of shark and ray species are considered to be threatened with extinction. In particular, the density of reef sharks in the Pacific has declined to 3–10% of pre-human levels. Elasmobranchs are thought to be sparse in highly urbanised and turbid environments. Low visibility coupled with the highly elusive behaviour of sharks and rays pose a challenge to diversity estimation and biomonitoring efforts as sightings are limited to chance encounters or from carcasses ensnared in nets. Here we utilised an eDNA metabarcoding approach to enhance the precision of elasmobranch diversity estimates in urbanised marine environments. Results We applied eDNA metabarcoding on seawater samples to detect elasmobranch species in the hyper-urbanised waters off Singapore. Two genes—vertebrate 12S and elasmobranch COI—were targeted and amplicons subjected to Illumina high-throughput sequencing. With a total of 84 water samples collected from nine localities, we found 47 shark and ray molecular operational taxonomic units, of which 16 had species-level identities. When data were compared against historical collections and contemporary sightings, eDNA detected 14 locally known species as well as two potential new records. Conclusions Local elasmobranch richness uncovered by eDNA is greater than the seven species sighted over the last two decades, thereby reducing phantom diversity. Our findings demonstrate that eDNA metabarcoding is effective in detecting shark and ray species despite the challenges posed by the physical environment, granting a more consistent approach to monitor these highly elusive and threatened species.

24Autonomous Reef Monitoring Structure (ARMS) are standardised devices for sampling 25 biodiversity in complex marine benthic habitats such as coral reefs. When coupled with DNA 26 sequencing, these devices greatly expand our ability to document marine biodiversity. 27Unfortunately, the existing workflow for processing macrofaunal samples (>2-mm) in the 28 ARMS pipeline-which involves Sanger sequencing-is expensive, laborious, and thus 29 prohibitive for ARMS researchers. Here, we propose a faster, more cost-effective alternative 30 by demonstrating a successful application of the MinION-based barcoding approach on the 31 >2mm-size fraction of ARMS samples. All data were available within 3.5-4 h, and 32 sequencing costs relatively low at approximately US$3 per MinION barcode. We sequenced 33 the 313-bp fragment of the cytochrome c oxidase subunit I (COI) for 725 samples on both 34 MinION and Illumina platforms, and retrieved 507-584 overlapping barcodes. MinION 35 barcodes were highly accurate (~99.9%) when compared with Illumina reference barcodes. 36 Molecular operational taxonomic units inferred between MinION and Illumina barcodes were 37 consistently stable, and match ratios demonstrated highly congruent clustering patterns 38 (≥0.96). Our method would make ARMS more accessible to researchers, and greatly expedite 39 the processing of macrofaunal samples; it can also be easily applied to other small-to-40 moderate DNA barcoding projects (<10,000 specimens) for rapid species identification and 41 discovery. 42 43 44 45 46 47 48