![]() ![]() Massive multiplexing of DNA barcode markers generates a great reduction of per sample sequencing costs and labor time compared to Sanger sequencing ( Schuster, 2008 Shokralla et al., 2015). ![]() The latter is defined as single-molecule real-time sequencing ( Van Dijk et al., 2014). The majority of these studies used Sanger sequencing but currently there are many sequencing technologies that have become more accessible and affordable for a wide array of applications ( Kircher and Kelso, 2010 Van Dijk et al., 2014 Goodwin et al., 2016) such as high-throughput sequencing (HTS) and third generation sequencing (TGS). There have been increased efforts in recent years to resolve the genetic taxonomy of nematodes and barcode nematode species using markers including the 18S small subunit ribosomal RNA gene (18S SSU rRNA), the 28S large subunit ribosomal RNA gene (28S LSU rRNA), the cytochrome oxidase I gene (COI) and the internal transcribed spacer (ITS) regions of the ribosomal RNA locus ( Blaxter et al., 1998 Bhadury et al., 2006a, b Hunt et al., 2016 O’Neil et al., 2017 Seesao et al., 2017 Pafčo et al., 2018). As a result, genetic identification is becoming increasingly important in nematology. For example, easily 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). Morphological identification is commonly used to identify nematode species, but also has significant drawbacks. For example, the World Health Organization estimates that worldwide infections with soil-transmitted nematodes cause a human annual disease burden of 3.8 million years lost to disabilities (YLD), a disease burden in the same range as HIV/AIDS (4 million YLD) and twice as high as malaria (1.7 million YLD) 1. Many of these species are parasites that threaten the health of plants and animals, including humans. It is estimated that less than 4% of nematode species are currently known to science, with global species richness estimated between 10 6 and 10 8 ( Lambshead, 2004). Nematodes are one of the most abundant groups of metazoan organisms ( Seesao et al., 2017). This increased accessibility could in turn improve global information of nematode presence and distribution, aiding near-real-time global biomonitoring. 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). 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. 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. 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. ![]() Advances in DNA sequencing techniques have allowed for the rapid and accurate identification of many organisms including nematodes. Many nematode species are parasitic and threaten the health of plants and animals, including humans, on a global scale. 2School of Biological and Environmental Sciences, Liverpool John Moores University, Liverpool, United Kingdom.1Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, Amsterdam, Netherlands. ![]()
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