Real-time DNA barcoding in a rainforest using nanopore sequencing: opportunities for rapid biodiversity assessments and local capacity building

Abstract

BackgroundAdvancements in portable scientific instruments provide promising avenues to expedite field work in order to understand the diverse array of organisms that inhabit our planet. Here, we tested the feasibility for in situ molecular analyses of endemic fauna using a portable laboratory fitting within a single backpack in one of the world's most imperiled biodiversity hotspots, the Ecuadorian Chocó rainforest. We used portable equipment, including the MinION nanopore sequencer (Oxford Nanopore Technologies) and the miniPCR (miniPCR), to perform DNA extraction, polymerase chain reaction amplification, and real-time DNA barcoding of reptile specimens in the field.FindingsWe demonstrate that nanopore sequencing can be implemented in a remote tropical forest to quickly and accurately identify species using DNA barcoding, as we generated consensus sequences for species resolution with an accuracy of >99% in less than 24 hours after collecting specimens. The flexibility of our mobile laboratory further allowed us to generate sequence information at the Universidad Tecnológica Indoamérica in Quito for rare, endangered, and undescribed species. This includes the recently rediscovered Jambato toad, which was thought to be extinct for 28 years. Sequences generated on the MinION required as few as 30 reads to achieve high accuracy relative to Sanger sequencing, and with further multiplexing of samples, nanopore sequencing can become a cost-effective approach for rapid and portable DNA barcoding.ConclusionsOverall, we establish how mobile laboratories and nanopore sequencing can help to accelerate species identification in remote areas to aid in conservation efforts and be applied to research facilities in developing countries. This opens up possibilities for biodiversity studies by promoting local research capacity building, teaching nonspecialists and students about the environment, tackling wildlife crime, and promoting conservation via research-focused ecotourism.

A sample-to-report solution for taxonomic identification of cultured bacteria in the clinical setting based on nanopore sequencing

“…Therefore, we excluded reads that were significantly shorter than the mode of the read length distribution (by 20 bases) from the analysis with the corresponding command line parameter in LORCAN. With these parameters being set, accurate consensus sequences (≥ 99% identity to sanger sequences produced from the same DNA) were reliably produced with as few as 100 size-filtered reads per sample confirming previous findings [42].…”

Categorization of species as native or nonnative using DNA sequence signatures without a complete reference library

“…New affordable sequencing technologies, coupled with decreases in traditional sequencing costs and in the time required to process specimens (see, for example, Pomerantz et al. ) have made it possible to sequence DNA from entire communities of individuals (Borisenko et al. , Kress et al.…”

“…The genomics revolution is transforming the studies of conservation biology, ecology, and evolution (Hudson 2008, Allendorf et al 2010. New affordable sequencing technologies, coupled with decreases in traditional sequencing costs and in the time required to process specimens (see, for example, Pomerantz et al 2018) have made it possible to sequence DNA from entire communities of individuals (Borisenko et al 2008, Kress et al 2009, Cristescu 2014 and allow for the production and publication of genomic information from non-model organisms from across the tree of life (Ekblom and Galindo 2011). One approach for collecting and analyzing sequence data that has become widely utilized is known as DNA sequence barcoding (also known as "DNA barcoding"; Hebert et al 2003, Savolainen et al 2005, Ratnasingham and Hebert 2007.…”

Portable sequencer in the fight against infectious disease

“…The portable sequencer, MinION, can be transported to countries or locations where performing sequencing is difficult or transferring samples to other countries or locations is forbidden. For example, MinION has been taken to a remote rainforest of Tanzania [17], Ecuador [18], the Canadian high Arctic [19], and even the International Space Station [20]. MinION helped the surveillance and sequencing of Zika virus in the 2016 Brazil outbreak [21].…”