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.
Highlights
Nematodes are one of the most abundant groups of metazoan organisms (Seesao et al, 2017)
The use of the MinION opens up exciting possibilities for nextgeneration biomonitoring
Extrapolating our results to potential application in a field setting, our results suggest that barcoding with the MinION can generate enough reads for reliable identification within 15 min, assuming good DNA quality and depending on the number of samples that are multiplexed
Summary
Nematodes are one of the most abundant groups of metazoan organisms (Seesao et al, 2017). It is estimated that less than 4% of nematode species are currently known to science, with global species richness estimated between 106 and 108 (Lambshead, 2004). Many of these species are parasites that threaten the health of plants and animals, including humans. Distinguishable morphological characters are scarce in nematodes, making identification difficult, time-consuming and often unsuccessful to genus or species level (Decraemer and Baujard, 1998; Lawton et al, 1998; Karanastasi et al, 2001; Lambshead, 2004; Hope and Aryuthaka, 2009).
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