Barcode 100K Specimens: In a Single Nanopore Run

It is a global priority to better manage the biosphere, but action needs to be informed by monitoring shifts in the abundance and distribution of species across the domains of life. The acquisition of such information is currently constrained by the limited knowledge of biodiversity. Among the 20 million or more species of eukaryotes, just a tenth have scientific names. DNA barcoding can speed the registration of unknown animal species, the most diverse kingdom of eukaryotes, as the BIN system automates their recognition. However, inexpensive analytical protocols are critical as the census of all animal species will require processing a billion or more specimens. Barcoding involves DNA extraction followed by PCR and sequencing with the last step dominating costs until 2017. By recovering barcodes from highly multiplexed samples, the Sequel platforms from Pacific BioSciences slashed costs by 90%, but these instruments are only deployed in core facilities because of their expense. Sequencers from Oxford Nanopore Technologies provide an escape from high capital and service costs, but their low sequence fidelity has, until now, kept analytical cost above Sequel. However, the improved performance of its latest flow cells (R10.4.1) might erase this differential. This study demonstrates that a regular MinION flow cell can characterize an amplicon pool derived from 100,000 specimens while a Flongle flow cell can process one derived from several thousand. At $0.01 per specimen, DNA sequencing is now the least expensive step in the barcode workflow. By coupling simplified protocols for DNA extraction with ultra-low volume PCRs, it will be possible to move from specimen to DNA barcode for $0.10, a price point that will enable the census of all species within two decades.