MinION barcodes: biodiversity discovery and identification by everyone, for everyone

Abstract

DNA barcodes are a useful tool for discovering, understanding, and monitoring biodiversity which are critical at a time when biodiversity loss is a major problem for many countries. However, widespread adoption of barcodes requires cost-effective and simple barcoding methods. We here present a workflow that satisfies these conditions. It was developed via “innovation through subtraction” and thus requires minimal lab equipment, can be learned within days, reduces the barcode sequencing cost to <10 cents, and allows fast turnaround from specimen to sequence by using the real-time sequencer MinION. We first describe cost-effective and rapid procedures for obtaining tagged amplicons. We then demonstrate how a portable MinION device can be used for real-time sequencing of tagged amplicons in many settings (field stations, biodiversity labs, citizen science labs, schools). Small projects can use the flow cell dongle (“Flongle”) while large projects can rely on MinION flow cells that can be stopped and re-used after collecting sufficient data for a given project. We also provide amplicon coverage recommendations that are based on several runs of MinION flow cells (R10.3) which suggest that each run can generate >10,000 barcodes. Next, we present a novel software, ONTbarcoder, which overcomes the bioinformatics challenges posed by the sequencing errors of MinION reads. This software is compatible with Windows10, Macintosh, and Linux, has a graphical user interface (GUI), and can generate thousands of barcodes on a standard laptop within hours based on two input files (FASTQ, demultiplexing file). We document that MinION barcodes are virtually identical to Sanger and Illumina barcodes for the same specimens (>99.99%). Lastly, we demonstrate how rapidly MinION data have improved by comparing the performance of sequential flow cell generations. We overall assert that barcoding with MinION is the way forward for government agencies, universities, museums, and schools because it combines low consumable and capital cost with scalability. Biodiversity loss is threatening the planet and the use of MinION barcodes will help with enabling an army of researchers and citizen scientists, which is necessary for effective biodiversity discovery and monitoring.