Data_Sheet_1_The Application of Nanopore Sequencing Technology to the Study of Dinoflagellates: A Proof of Concept Study for Rapid Sequence-Based Discrimination of Potentially Harmful Algae.pdf

Harmful algal blooms (HABs) are a naturally occurring global phenomena that have the potential to impact fisheries, leisure and ecosystems, as well as posing a significant hazard to animal and human health. There is significant interest in the development and application of methodologies to study all aspects of the causative organisms and toxins associated with these events. This paper reports the first application of nanopore sequencing technology for the detection of eukaryotic harmful algal bloom organisms. The MinION sequencing platform from Oxford Nanopore technologies provides long read sequencing capabilities in a compact, low cost, and portable format. In this study we used the MinION to sequence long-range PCR amplicons from multiple dinoflagellate species with a focus on the genus Alexandrium. Primers applicable to a wide range of dinoflagellates were selected, meaning that although the study was primarily focused on Alexandrium the applicability to three additional genera of toxic algae, namely; Gonyaulax, Prorocentrum, and Lingulodinium was also demonstrated. The amplicon generated here spanned approximately 3 kb of the rDNA cassette, including most of the 18S, the complete ITS1, 5.8S, ITS2 and regions D1 and D2 of the 28S. The inclusion of barcode genes as well as highly conserved regions resulted in identification of organisms to the species level. The analysis of reference cultures resulted in over 99% of all sequences being attributed to the correct species with an average identity above 95% from a reference list of over 200 species (see Supplementary Material 1). The use of mock community analysis within environmental samples highlighted that complex matrices did not prevent the ability to distinguish between phylogenetically similar species. Successful identification of causative organisms in environmental samples during natural toxic events further highlighted the potential of the assay. This study proves the suitability of nanopore sequencing technology for taxonomic identification of harmful algal bloom organisms and acquisition of data relevant to the World Health Organisations “one health” approach to marine monitoring.

有害藻华（Harmful Algal Blooms，HABs）是一种自然发生的全球性现象，其潜在影响范围涵盖渔业、休闲活动以及生态系统，同时亦对动物和人类健康构成重大威胁。对开发与应用旨在研究这些事件中致病原生物及其毒素的各方面方法的研究兴趣日益浓厚。本文报告了纳米孔测序技术在检测真核有害藻华生物中的应用首例。牛津纳米孔技术公司的MinION测序平台以紧凑、低成本和便携的格式提供了长读长测序能力。在本研究中，我们使用MinION对多种金藻目物种的广范围PCR扩增子进行测序，重点关注亚历山大属。选用的引物适用于广泛范围的藻类，这意味着尽管研究主要聚焦于亚历山大属，但对三种额外的有毒藻类属，即；金藻属（Gonyaulax）、原甲藻属（Prorocentrum）和舌形藻属（Lingulodinium）的应用性也得到了验证。生成的扩增子覆盖了约3 kb的rDNA盒，包括大部分的18S、完整的ITS1、5.8S、ITS2以及28S的D1和D2区域。包括条形码基因以及高度保守区域在内的扩增子，实现了物种水平的生物体鉴定。对参考菌株的分析导致超过99%的序列被归因于正确的物种，平均同源性高于95%，参照超过200种物种的列表（见补充材料1）。在环境样本中进行的模拟群落分析突显了复杂基质并不能阻止区分亲缘关系相似的物种的能力。在自然中毒事件期间，环境样本中成功鉴定致病原生物进一步凸显了该检测方法的潜力。本研究证明了纳米孔测序技术在有害藻华生物分类鉴定以及获取与世界卫生组织“一个健康”海洋监测方法相关的数据方面的适用性。