Peng Ze, Fan Wen, Wang Liping, Paudel Dev, Leventini Dante, Tillman Barry L, Wang Jianping
Agronomy Department, University of Florida, Gainesville, FL, 32610, USA.
Genetics Institute, Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL, 32610, USA.
Mol Genet Genomics. 2017 Oct;292(5):955-965. doi: 10.1007/s00438-017-1327-z. Epub 2017 May 10.
Enabled by the next generation sequencing, target enrichment sequencing (TES) is a powerful method to enrich genomic regions of interest and to identify sequence variations. The objective of this study was to explore the feasibility of probe design from transcript sequences for TES application in calling sequence variants in peanut, an important allotetraploid crop with a large genome size. In this study, we applied an in-solution hybridization method to enrich DNA sequences of seven peanut genotypes. Our results showed that it is feasible to apply TES with probes designed from transcript sequences in polyploid peanut. Using a set of 31,123 probes, a total of 5131 and 7521 genes were targeted in peanut A and B genomes, respectively. For each genotype used in this study, the probe target capture regions were efficiently covered with high depth. The average on-target rate of sequencing reads was 42.47%, with a significant amount of off-target reads coming from genomic regions homologous to target regions. In this study, when given predefined genomic regions of interest and the same amount of sequencing data, TES provided the highest coverage of target regions when compared to whole genome sequencing, RNA sequencing, and genotyping by sequencing. Single nucleotide polymorphism (SNP) calling and subsequent validation revealed a high validation rate (85.71%) of homozygous SNPs, providing valuable markers for peanut genotyping. This study demonstrated the success of applying TES for SNP identification in peanut, which shall provide valuable suggestions for TES application in other non-model species without a genome reference available.
在新一代测序技术的支持下，靶向富集测序（TES）是一种富集感兴趣的基因组区域并识别序列变异的强大方法。本研究的目的是探索从转录本序列设计探针用于TES在花生（一种基因组庞大的重要异源四倍体作物）中检测序列变异的可行性。在本研究中，我们采用溶液内杂交方法富集了7个花生基因型的DNA序列。我们的结果表明，在多倍体花生中应用基于转录本序列设计的探针进行TES是可行的。使用一组31123个探针，分别在花生A和B基因组中靶向了总共5131个和7521个基因。对于本研究中使用的每个基因型，探针靶向捕获区域均被高效且深度覆盖。测序 reads 的平均靶向率为42.47%，大量的脱靶 reads 来自与靶区域同源的基因组区域。在本研究中，当给定预定义的感兴趣基因组区域和相同数量的测序数据时，与全基因组测序、RNA测序和测序基因分型相比，TES提供了最高的靶区域覆盖率。单核苷酸多态性（SNP）检测及后续验证显示纯合SNP的验证率很高（85.71%）为花生基因分型提供了有价值的标记。本研究证明了应用TES在花生中进行SNP鉴定的成功，这将为TES在其他没有可用基因组参考的非模式物种中的应用提供有价值的建议。
