Table4.xlsx

Next-generation sequencing (NGS) technologies have enabled genome re-sequencing for exploring genome-wide polymorphisms among individuals, as well as targeted re-sequencing for the rapid and simultaneous detection of polymorphisms in genes associated with various biological functions. Therefore, a simple and robust method for targeted re-sequencing should facilitate genotyping in a wide range of biological fields. In this study, we developed a simple, custom, targeted re-sequencing method, designated “multiplex PCR targeted amplicon sequencing (MTA-seq),” and applied it to the genotyping of the model grass Brachypodium distachyon. To assess the practical usability of MTA-seq, we applied it to the genotyping of genome-wide single-nucleotide polymorphisms (SNPs) identified in natural accessions (Bd1-1, Bd3-1, Bd21-3, Bd30-1, Koz-1, Koz-3, and Koz-4) by comparing the re-sequencing data with that of reference accession Bd21. Examination of SNP-genotyping accuracy in 443 amplicons from eight parental accessions and an F1 progeny derived by crossing of Bd21 and Bd3-1 revealed that ~95% of the SNPs were correctly called. The assessment suggested that the method provided an efficient framework for accurate and robust SNP genotyping. The method described here enables easy design of custom target SNP-marker panels in various organisms, facilitating a wide range of high-throughput genetic applications, such as genetic mapping, population analysis, molecular breeding, and genomic diagnostics.

下一代测序（Next-generation sequencing，NGS）技术已实现全基因组重测序，可用于探究个体间全基因组范围的多态性；同时也可开展靶向重测序，以快速同步检测与多种生物学功能相关的基因中的多态性。因此，一种简便且稳健的靶向重测序方法，将有助于推动众多生物学领域中的基因分型工作。本研究中，我们开发了一种简便的定制化靶向重测序方法，命名为"多重PCR靶向扩增子测序（multiplex PCR targeted amplicon sequencing，MTA-seq）"，并将其应用于模式草本植物二穗短柄草（Brachypodium distachyon）的基因分型。为评估MTA-seq的实际应用潜力，我们将其用于全基因组单核苷酸多态性（single-nucleotide polymorphisms，SNPs）的基因分型：这些SNPs是通过将自然株系（Bd1-1、Bd3-1、Bd21-3、Bd30-1、Koz-1、Koz-3及Koz-4）的重测序数据与参考株系Bd21的重测序数据比对后鉴定得到的。我们对8个亲本株系以及由Bd21与Bd3-1杂交获得的F1后代的共443个扩增子进行了SNP基因分型准确性检测，结果显示约95%的SNPs可被准确分型。本次评估表明，该方法为精准且稳健的SNP基因分型提供了高效的研究框架。本研究所述方法可轻松实现不同生物中定制化靶向SNP标记组合的设计，助力遗传作图、群体分析、分子育种及基因组诊断等多种高通量遗传学应用的开展。