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- amplicon sequencing,
- cytochrome c oxidase subunit I (COI),
- DNA barcoding,
- metazoa,
- nextgeneration sequencing,
- Oxford Nanopore Technologies,
- species estimation
Autonomous Reef Monitoring Structure (ARMS) are standardized devices for sampling biodiversity in complex marine benthic habitats such as coral reefs. When coupled with DNA sequencing, these devices greatly expand our ability to document marine biodiversity. Unfortunately, the existing workflow for processing macrofaunal samples (>2-mm) in the ARMS pipeline—which involves Sanger sequencing—is expensive, laborious, and thus prohibitive for ARMS researchers. Here, we propose a faster, more cost-effective alternative by demonstrating a successful application of the MinION-based barcoding approach on the >2 mm-size fraction of ARMS samples. All data were available within 3.5–4 h, and sequencing costs relatively low at approximately US$3 per MinION barcode. We sequenced the 313-bp fragment of the cytochrome c oxidase subunit I (COI) for 725 samples on both MinION and Illumina platforms, and retrieved 507–584 overlapping barcodes. MinION barcodes were highly accurate (∼99.9%) when compared with Illumina reference barcodes. Molecular operational taxonomic units inferred between MinION and Illumina barcodes were consistently stable, and match ratios demonstrated highly congruent clustering patterns (≥0.96). Our method would make ARMS more accessible to researchers, and greatly expedite the processing of macrofaunal samples; it can also be easily applied to other small-to-moderate DNA barcoding projects (<10,000 specimens) for rapid species identification and discovery.
Available at: http://works.bepress.com/andrew-bauman/20/
Funding for this research was provided by the Singapore Ministry of Education Academic Research Fund Tier 1 (R-154-000-A63-114), the National Research Foundation, Prime Minister’s Office, Singapore under its Marine Science R&D Programme (MSRDP-P03), and an AXA postdoctoral fellowship to AB (R-154-000-649-507).
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmars.2020.00448/full#supplementary-material
Basecalled fastq files, demultiplex sheets, Illumina reference file and consolidated MinION barcodes are available at Zenodo (https://doi.org/10.5281/zenodo.3816816). Basecalled fastq files can also be found on the NCBI Sequence Read Archive under BioProject PRJNA631514 (Accession Nos. SRX8336250 and SRX8336251).