Full 18S metabarcoding of environmental samples of various substrates with Nanopore sequencing. v2

• DOI: 10.17504/protocols.io.3byl4qe48vo5/v2
• Publication Date: 2025-01-17T15:10:11Z
• Authors: Quentin Blandenier; Nicholas Gibson; Alexander K. Tice; Robert E Jones; Erin P. Jones; Richard E. Baird; Brendan A. Zurweller; Matthew W. Brown
• Keywords: Nanopore; Environmental DNA

We describe here a flexible protocol for eDNA metabarcoding with Oxford Nanopore's MinIon MK1C platform from sampling to sequencing. The first section summarizes some key steps of and sample preservation both aquatic terrestrial environments. second one describes the DNA extraction DNeasy PowerSoil Pro Kit Qiagen different types samples (i.e., soil, liquid dead plant materials). Our amplification bead purification protocols are characterized in third section. Finally, library prep Native amplicon Barcoding 96 V14 (SQK-NBD114.96) sequencing R10.4.1 flow cells (FLO-MIN114) MinION Mk1C device presented last sections. This has been optimized protists (microbial eukaryotes) 18S marker, but should be easily adjustable other organisms, by modifying sections, or markers, even longer ones such as full ribosomal operon, if need be. goal is teach/train researchers fields expertise on obtaining nanopore sequences environmental guiding them protocol, focusing steps, informing expected results based our 3 runs performed so far. A comprehensive bioinformatic pipeline treat data produced, well methodological article discussing this method best ways use it preparation.