Data and scripts from: Comparison of biological communities associated with oyster aquaculture and oyster reefs using environmental DNA metabarcoding and Nanopore sequencing

We compare the biological communities associated with intertidal oyster reefs and aquaculture in estuarine eastern North Carolina, USA across seasons to examine the habitat value of small-scale oyster aquaculture sites aquaculture gear using broad-spectrum metabarcoding of environmental DNA (eDNA) samples. We combine Nanopore sequencing and aquatic eDNA analyses to non-invasively record biological communities at broad taxonomic scales.

Water samples were collected in bleach-sterilized 1L Nalgene bottles and kept on ice until filtration on 0.45 um MCE filters, then stored at -80C until DNA extraction. Environmental data was collected using a handheld Thermo Scientific™ Elite PCTS probe.

DNA was extracted from 1/2 of each filter using the Qiagen Blood & Tissue kit.

PCR was performed in triplicate on each sample and blank using three primer sets tagged with Oxford Nanopore primer adapters. Three gene regions commonly used in metabarcoding studies were used to capture the widest breadth of organisms. Targeted gene regions included: a 313 bp Cytochrome C Oxidase (COI) region (mICOIintF/HCO2198 (Leray et al. 2013)), a 336 bp 18S V4 rRNA region (1F/400R (Pochon et al. 2013)), and the 163 bp 12S rRNA region (MiFish-UF MiFish-UR (Miya et al. 2015)). Indexing PCR was performed using the Oxford Nanopore PCR Barcoding Expansion

Libraries were sequenced on the Oxford Nanopore GridION using the LSK-110 kit. We used MinKNOW v.21.05.20 and Guppy v.5.0.13 natively on the GridION to basecall with high-accurary basecalling and demultiplex the sequences. Guppy parameters: trim_barcodes ‘on’ and require_barcodes_both_ends and detect_mid_strand_barcodes ‘off’. Barcodes were filtered to retain a minimum barcode score of 60 and Q-score of 9. Scripts to generate consensus sequences can be found on GitHub: https://github.com/lauragivens/ONTbroadsp_eDNA