DNA Barcoding of Nematodes Using the MinION

Abstract

Many nematode species are parasitic and threaten the health of plants and animals, including humans, on a global scale. Advances in DNA sequencing techniques have allowed for the rapid and accurate identification of many organisms including nematodes. However, the steps taken from sample collection in the field to molecular analysis and identification can take many days and depend on access to both immovable equipment and a specialized laboratory. Here, we present a protocol to genetically identify nematodes using 18S SSU rRNA sequencing using the MinION, a portable third generation sequencer, and proof that it is possible to perform all the molecular preparations on a fully portable molecular biology lab-the Bentolab. We show that both parasitic and free-living nematode species (Anisakis simplex, Panagrellus redivivus, Turbatrix aceti, and Caenorhabditis elegans) can be identified with a 96-100% accuracy compared to Sanger sequencing, requiring only 10-15 min of sequencing. This protocol is an essential first step toward genetically identifying nematodes in the field from complex natural environments (such as feces, soil, or marine sediments). This increased accessibility could in turn improve global information of nematode presence and distribution, aiding near-real-time global biomonitoring.

Takeaways from Mobile DNA Barcoding with BentoLab and MinION

“…We obtained 105 MinION barcodes, of which 19 were from samples obtained in the field. To our knowledge, the 31 samples and 19 DNA barcodes generated here represent one of the highest throughputs from published studies to date [ 20 , 21 , 31 , 32 ], with the entire sample-to-sequence workflow completed in under 9 h. In the following, we discuss our experiences with portable sequencing on the BentoLab and MinION in the form of three takeaways learnt from the entire process.…”

“…One of the strengths of second- and third-generation sequencers is the ability to reduce sequencing costs via sample multiplexing onto a flow cell [ 15 ]; the greater the number of samples, the lower the resultant cost of each barcode. MinION barcodes can cost as low as ~USD 0.35 each when multiplexing ~3000 samples per MinION flow cell [ 35 ], though such a volume is unlikely to be achieved in a field setting [ 32 , 53 ]. Users thus need to bear in mind that there are financial trade-offs with the lower throughput for portable sequencing.…”

“…Nanopore sequencing has also featured prominently in the monitoring of disease outbreaks such as Ebola [ 24 , 25 , 26 ], and more recently in the detection of SARS-CoV-2 [ 27 , 28 , 29 ]. However, portable DNA barcoding for biodiversity identification and discovery remains limited in application and is restricted to fairly small sample sizes ( n ≤ 10) [ 20 , 21 , 30 , 31 , 32 ]. We thus sought to assemble a mobile sequencing workflow that would enhance the capacity for in situ DNA barcoding.…”

“…We chose the BentoLab over other portable laboratory devices like the miniPCR TM (8-wells) as our proposed workflow was reliant on having a higher thermocycling capacity (see Methods). The BentoLab–MinION combination for portable sequencing is not new [ 32 , 34 ]. One study explored the rapid characterization of single-nucleotide polymorphisms for forensics [ 34 ], while another demonstrated the feasibility of in situ DNA barcoding of nematodes [ 32 ].…”

“…The BentoLab–MinION combination for portable sequencing is not new [ 32 , 34 ]. One study explored the rapid characterization of single-nucleotide polymorphisms for forensics [ 34 ], while another demonstrated the feasibility of in situ DNA barcoding of nematodes [ 32 ]. Here, our proposed pipeline is designed to comfortably handle larger sample sizes and more diverse fauna through the use of degenerate metazoan primers.…”