A highly flexible and repeatable genotyping method for aquaculture studies based on target amplicon sequencing using next-generation sequencing technology
A highly flexible and repeatable genotyping method for aquaculture studies based on target amplicon sequencing using next-generation sequencing technology
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DOI：
10.1038/s41598-019-43336-x
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发表时间：
2019-05-06
影响因子：
4.6
通讯作者：
Kikuchi, Kiyoshi
中科院分区：
文献类型：
--
作者：
Sato, Mana;Hosoya, Sho;Kikuchi, Kiyoshi
由于高通量测序技术的进步，使用全基因组单核苷酸多态性（SNP）的研究在遗传学和基因组学中已经变得司空见惯。由于所需的SNP和样品的数量根据每个研究目标而变化，因此已经深入研究了在SNP/样品数量方面具有高灵活性和高重复性的基因分型技术。例如，超高多重扩增子测序，离子AmpliSeq，已被用作主要用于诊断目的的高通量基因分型方法。在此，我们设计了一个针对红鳍东方鲀（Takifugu rubripes）3,187个全基因组SNP的自定义面板，并将其应用于养殖河豚的基因分型，以测试其在水产养殖研究中的可行性。我们在Illumina MiSeq测序仪上对由不同个体池组成的两个文库（每个n= 326）进行测序。因此，超过99%的目标区域（3，178个SNP）被扩增，并且在过滤步骤之后有2，655个SNP可用。在重复运行之间的每个SNP的平均覆盖深度中观察到强相关性（r= 0.993）。利用这些基因型数据进行遗传分析，成功地发现了已知的群体结构和河豚的性别决定位点。这些结果表明该方法具有上级的重复性和灵活性，适合包括分子育种在内的遗传研究，例如标记辅助和基因组选择。
Studies using genome-wide single nucleotide polymorphisms (SNPs) have become commonplace in genetics and genomics, due to advances in high-throughput sequencing technologies. Since the numbers of required SNPs and samples vary depending on each research goal, genotyping technologies with high flexibility in the number of SNPs/samples and high repeatability have been intensively investigated. For example, the ultrahigh-multiplexed amplicon sequencing, Ion AmpliSeq, has been used as a high-throughput genotyping method mainly for diagnostic purposes. Here, we designed a custom panel targeting 3,187 genome-wide SNPs of fugu, Takifugu rubripes, and applied it for genotyping farmed fugu to test its feasibility in aquaculture studies. We sequenced two libraries consisting of different pools of individuals (n= 326 each) on the Illumina MiSeq sequencer. Consequently, over 99% target regions (3,178 SNPs) were amplified and 2,655 SNPs were available after filtering steps. Strong correlation was observed in the mean depth of coverage of each SNP between duplicate runs (r= 0.993). Genetic analysis using these genotype data successfully detected the known population structure and the sex determining locus of fugu. These results show the method is superior in repeatability and flexibility, and suits genetic studies including molecular breeding, such as marker assisted and genomic selection.