ARISE DNA Barcoding using Oxford Nanopore Technologies v1

Abstract

This protocol is designed as a relatively low-cost, high-quality, easily-upscalable high-throughput DNA barcoding workflow. By sequencing 24 plates of samples (94 samples per plate, and two controls) on a single flow cell, the whole workflow for amplification and sequencing can be brought back to ca. € 2,20 (1-step PCR) to € 3,40 (2-step PCR) per sample. Processing more plates in a run would be possible, but doesn't significantly lower the per-sample costs. This protocol is the standard for DNA Barcoding within the ARISE program at Naturalis Biodiversity Center. For more information about ARISE, visit https://www.arise-biodiversity.nl/. The protocol largely follows the standard ONT protocol for ligation sequencing (Ligation Sequencing DNA V14), with a custom PCR setup.

Authors 6

1. Kevin Beentjes Aachen Naturalis Biodiversity Center

   Naturalis Biodiversity Center

   Affiliation as printed

   Naturalis Biodiversity Center

2. Nafiesa Ibrahim Aachen Naturalis Biodiversity Center

   Naturalis Biodiversity Center

   Affiliation as printed

   Naturalis Biodiversity Center

3. Tara van der Plas Aachen Naturalis Biodiversity Center

   Naturalis Biodiversity Center

   Affiliation as printed

   Naturalis Biodiversity Center

4. Cas Lekkerkerker Aachen Naturalis Biodiversity Center

   Naturalis Biodiversity Center

   Affiliation as printed

   Naturalis Biodiversity Center

5. Thirza van de Wetering Aachen Naturalis Biodiversity Center

   Naturalis Biodiversity Center

   Affiliation as printed

   Naturalis Biodiversity Center

6. Emma Wubben Aachen Naturalis Biodiversity Center

   Naturalis Biodiversity Center

   Affiliation as printed

   Naturalis Biodiversity Center