Oncorhynchus kisutch genetic stock ID SNP baseline

AbstractGenetic stock identification (GSI) using genotypes produced from genotyping-by-sequencing of SNP loci has become the gold standard for stock identification in Pacific salmon, which are found in mixed stocks in the ocean. Sequencing platforms currently applied require large batch sizes and multi-day processing in specialized facilities to perform genotyping by the thousands. However, recent advances in third-generation single-molecule sequencing platforms, like the Oxford Nanopore minION, provide base calling on portable, pocket-sized sequencers and promise real-time, in-field stock identification on variable batch sizes. Here we evaluate utility and comparability to established GSI platforms of at-sea stock identification of coho salmon Oncorhynchus kisutch based on targeted SNP amplicon sequencing on the minION platform during a high-sea winter expedition to the Gulf of Alaska. As long read sequencers are not optimized for short amplicons, we concatenate amplicons to increase coverage and throughput. Nanopore sequencing at-sea yielded stock assignment for 50 of the 80 assessed individuals. Nanopore-based SNP calls agreed with Ion Torrent based genotypes in 83.25%, but assignment of individuals to stock of origin only agreed in 61.5% of individuals highlighting inherent challenges of Nanopore sequencing, such as resolution of homopolymer tracts and indels. However, poor representation of assayed coho salmon in the queried baseline dataset contributed to poor assignment confidence on both platforms. Future improvements will focus on lowering turnaround time, accuracy, throughput, and cost, as well as augmentation of the existing baselines. If successfully implemented, Nanopore sequencing will provide an alternative method to the large-scale laboratory approach by providing mobile small batch genotyping to diverse stakeholders., MethodsThis SNP baseline used for genetic stock identification of coho salmon on the Oxford Nanopore minION is a combination of previously published baseline data from the Fisheries and Oceans Canada Molecular Genetics Laboratory at the Pacific Biological Station by Beacham et al. (https://datadryad.org/stash/dataset/doi:10.5061/dryad.msbcc2fvk; https://datadryad.org/stash/dataset/doi:10.5061/dryad.3g1r4v3; https://datadryad.org/stash/dataset/doi:10.5061/dryad.4md17)., Usage notesAn explanation of the SNP loci and alles can be found in Appendix Table A2 of Beacham, Terry D., et al. \"Population and individual identification of coho salmon in British Columbia through parentage-based tagging and genetic stock identification: an alternative to coded-wire tags.\" Canadian Journal of Fisheries and Aquatic Sciences 74.9 (2017): 1391-1410 (https://doi.org/10.1139/cjfas-2016-0452). The database is foramted for use in rubias (https://github.com/eriqande/rubias).## 摘要 基于单核苷酸多态性（single nucleotide polymorphism, SNP）位点的基于测序的基因分型（genotyping-by-sequencing）获得的基因型开展的遗传种群鉴定（genetic stock identification, GSI），已成为海洋混合种群太平洋鲑鱼（Pacific salmon）种群鉴定的金标准。当前主流测序平台需采用大批次样本量，并需在专业设施中完成多日处理，方可实现数千样本的基因分型。然而，以牛津纳米孔MinION（Oxford Nanopore minION）为代表的第三代单分子测序平台近年取得重要进展，可在便携的口袋式测序仪上完成碱基识别，有望实现可变批次样本的实时野外种群鉴定。本研究依托阿拉斯加湾公海冬季科考航行，利用MinION平台开展靶向SNP扩增子测序，评估其用于银大麻哈鱼（Oncorhynchus kisutch, coho salmon）野外种群鉴定的实用性，并与现有成熟GSI平台进行可比性分析。鉴于长读长测序仪未针对短扩增子优化，我们通过拼接扩增子以提升测序覆盖度与通量。本次公海纳米孔测序完成了80个待测个体中的50个的种群归属分配。基于纳米孔测序的SNP分型结果与Ion Torrent平台的基因型一致性达83.25%，但仅61.5%的个体的种群起源分配结果与之一致，这凸显了纳米孔测序固有的技术挑战，例如均聚物区域与插入缺失（insertions-deletions, indels）的分辨难题。不过，两个平台的种群分配置信度均偏低，其原因均为查询基准数据集对受试银大麻哈鱼的代表性不足。未来改进将聚焦于缩短周转时间、提升准确性与通量、降低成本，以及扩充现有基准数据集。若能成功落地，纳米孔测序可提供一种替代大规模实验室方案的可行方法，为各类利益相关者提供移动式小批量基因分型服务。 ## 方法 本研究用于牛津纳米孔MinION平台银大麻哈鱼遗传种群鉴定的SNP基准数据集，整合了Beacham等人于加拿大渔业与海洋部太平洋生物站分子遗传学实验室发布的既往基准数据（https://datadryad.org/stash/dataset/doi:10.5061/dryad.msbcc2fvk；https://datadryad.org/stash/dataset/doi:10.5061/dryad.3g1r4v3；https://datadryad.org/stash/dataset/doi:10.5061/dryad.4md17）。 ## 使用说明 关于SNP位点与等位基因的详细说明，可参见Beacham, Terry D.等人发表于《加拿大渔业与水产科学期刊》（*Canadian Journal of Fisheries and Aquatic Sciences*）的论文："Population and individual identification of coho salmon in British Columbia through parentage-based tagging and genetic stock identification: an alternative to coded-wire tags"，74卷第9期(2017): 1391-1410（https://doi.org/10.1139/cjfas-2016-0452）的附录表A2。本数据库格式适配rubias工具使用（https://github.com/eriqande/rubias）。