Microbial DNA extraction from seawater filters using the E.Z.N.A.® Water DNA Kit and full-length 16S rRNA gene sequencing on Oxford Nanopore MinION v1

Abstract

This protocol describes the collection and processing of seawater samples for microbial community analysis using membrane filtration, DNA extraction with the E.Z.N.A. Water DNA Kit, and full‑length 16S rRNA gene sequencing on Oxford Nanopore Technologies platforms. Seawater (5 L per sample) is pre‑filtered through a 200 µm mesh to remove large particles and organisms and then filtered onto 0.2 µm polycarbonate membranes to capture bacterioplankton. Filters are stored frozen prior to extraction, then cut and processed following the manufacturer’s instructions for the E.Z.N.A. Water DNA Kit to obtain high‑quality environmental DNA suitable for amplicon library preparation. DNA yields are quantified with Qubit, with optional clean‑up and concentration for low‑yield samples, and full‑length 16S libraries are generated using the ONT 16S Barcoding Kit (SQK‑16S024) and a high‑fidelity polymerase. Barcoded libraries are bead‑purified, normalized, pooled, and sequenced on MinION flow cells (R9.4.1) to obtain long‑read 16S rRNA gene data for downstream analysis of marine bacterial community composition.