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

Advancements 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 <em>in situ</em> molecular analyses of endemic fauna using a portable laboratory fitting within a single backpack, in one of the worlds most imperiled biodiversity hotspots: the Ecuadorian Chocó rainforest. We utilized portable equipment, including the MinION nanopore sequencer (Oxford Nanopore Technologies) and the miniPCR (miniPCR), to perform DNA extraction, PCR amplification and real-time DNA barcoding of reptile specimens in the field.<br>We 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 &gt;99% in less than 24 hours after collecting specimens. The flexibility of our mobile laboratory further allowed us to generate sequence information at Universidad Tecnológica Indoamérica in Quito for rare, endangered, and undescribed species. This includes the recently re-discovered Jambato toad, which was thought to be extinct for 28 years. Sequences generated on the MinION required as little 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.<br>Overall, 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 research by promoting local research capacity building, teaching laymen and students about the environment, tackling wildlife crime or by promoting conservation via research focused eco-tourism.

便携式科研仪器的革新为加速野外考察工作、深入探究栖息于地球的各类生物提供了极具前景的路径。本研究于全球受威胁最严重的生物多样性热点区域之一——厄瓜多尔乔科雨林中，利用可装入单个背包的便携式实验室，开展了针对特有动物的原位（in situ）分子分析可行性验证。我们采用了包括MinION纳米孔测序仪（Oxford Nanopore Technologies）与miniPCR扩增仪（miniPCR）在内的便携式设备，在野外完成了爬行动物标本的DNA提取、PCR扩增与实时DNA条形码鉴定。

研究结果表明，纳米孔测序可应用于偏远热带森林，通过DNA条形码技术快速且精准地完成物种鉴定：我们在采集标本后24小时内，即可获得用于物种分辨率判定的一致性序列，其准确率高于99%。我们搭建的移动实验室还具备灵活性，可在基多的因多美洲科技大学（Universidad Tecnológica Indoamérica）为珍稀、濒危及未描述物种生成序列信息，其中包括被认为已灭绝28年之久、近期重新被发现的詹巴托蟾蜍。

相较于桑格测序（Sanger sequencing），MinION测序仅需最少30条读长即可获得高精度序列；若进一步实现样本多重化，纳米孔测序将成为一种兼具成本效益的快速便携式DNA条形码鉴定方案。

综上，本研究明确了移动实验室与纳米孔测序如何助力偏远地区的物种鉴定加速工作，以辅助保育行动，并可应用于发展中国家的科研机构。该研究通过推动本地科研能力建设、面向普通民众与学生开展环境教育、打击野生动植物犯罪，或是依托聚焦科研的生态旅游促进保育工作，为生物多样性研究开辟了全新可能。