Data_Sheet_2_DNA Barcoding of Nematodes Using the MinION.FASTA

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.

诸多线虫物种具有寄生性，在全球范围内威胁包括人类在内的动植物健康。DNA测序技术的进步，使得包括线虫在内的多种生物得以被快速、精准地鉴定。然而，从野外样本采集到分子分析与鉴定的整套流程往往耗时数日，且依赖固定式实验设备与专业实验室资源。本研究提出一套基于18S小亚基核糖体RNA（18S SSU rRNA）测序的线虫遗传鉴定方案，所用设备为便携式第三代测序仪MinION；同时验证了可在全便携分子生物学实验室Bentolab中完成全部分子制备流程。实验结果显示，相较于桑格（Sanger）测序，本方案可对多种寄生及自由生活线虫物种（包括简单异尖线虫（Anisakis simplex）、潘氏线虫（Panagrellus redivivus）、醋线虫（Turbatrix aceti）以及秀丽隐杆线虫（Caenorhabditis elegans））实现96%~100%的鉴定准确率，且测序时长仅需10~15分钟。该方案是实现从复杂自然环境（如粪便、土壤或海洋沉积物）中野外就地遗传鉴定线虫的关键第一步。这种检测便利性的提升，可进一步完善全球线虫分布与存在情况的相关数据，助力近实时全球生物监测。