MinION-in-ARMS: Nanopore Sequencing to Expedite Barcoding of Specimen-Rich Macrofaunal Samples From Autonomous Reef Monitoring Structures

Authors

Jia Jin Marc Chang, National University of Singapore
Yin Cheong Aden Ip, National University of Singapore
Andrew G. Bauman, National University of SingaporeFollow
Danwei Huang, National University of Singapore

ORCID

0000-0001-9260-2153

Document Type

Article

Publication Title

Frontiers In Marine Science

ISSN

2296-7745

Publication Date

6-2020

Keywords

amplicon sequencing, cytochrome c oxidase subunit I (COI), DNA barcoding, metazoa, nextgeneration sequencing, Oxford Nanopore Technologies, species estimation

Abstract

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.

DOI

10.3389/fmars.2020.00448

Volume

7

Issue

448

First Page

1

Last Page

13

Comments

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).

Creative Commons License

This work is licensed under a Creative Commons Attribution 4.0 International License.

NSUWorks Citation

Jia Jin Marc Chang, Yin Cheong Aden Ip, Andrew G. Bauman, and Danwei Huang. 2020. MinION-in-ARMS: Nanopore Sequencing to Expedite Barcoding of Specimen-Rich Macrofaunal Samples From Autonomous Reef Monitoring Structures .Frontiers In Marine Science , (448) : 1 -13. https://nsuworks.nova.edu/occ_facarticles/1292.