Département de phytologie, Université Laval, Québec, QC, Canada.
Institut de biologie intégrative et des systèmes, Université Laval, Québec, QC, Canada.
BMC Genomics. 2019 Aug 6;20(1):634. doi: 10.1186/s12864-019-5998-1.
The effective use of mutant populations for reverse genetic screens relies on the population-wide characterization of the induced mutations. Genome- and population-wide characterization of the mutations found in fast neutron populations has been hindered, however, by the wide range of mutations generated and the lack of affordable technologies to detect DNA sequence changes. In this study, we therefore aimed to test whether genotyping-by-sequencing (GBS) technology could be used to characterize copy number variation (CNV) induced by fast neutrons in a soybean mutant population.
We called CNVs from GBS data in 79 soybean mutants and assessed the sensitivity and precision of this approach by validating our results against array comparative genomic hybridization (aCGH) data for 19 of these mutants as well as targeted PCR and ddPCR assays for a representative subset of the smallest events detected by GBS. Our GBS pipeline detected 55 of the 96 events found by aCGH, with approximate detection thresholds of 60 kb, 500 kb and 1 Mb for homozygous deletions, hemizygous deletions and duplications, respectively. Among the whole set of 79 mutants, the GBS data revealed 105 homozygous deletions, 32 hemizygous deletions and 19 duplications. This included several extremely large events, exhibiting maximum sizes of ~ 11.2 Mb for a homozygous deletion, ~ 11.6 Mb for a hemizygous deletion, and ~ 50 Mb for a duplication.
This study provides a proof of concept that GBS can be used as an affordable high-throughput method for assessing CNVs in fast neutron mutants. The modularity of this GBS approach allows combining as many different libraries or sequencing runs as is necessary for reaching the goals of a particular study. This method should enable the low-cost genome-wide characterization of hundreds to thousands of individuals in fast neutron mutant populations or any population with large genomic deletions and duplications.
反向遗传学筛选中有效利用突变体群体，依赖于对诱导突变的群体水平的特征描述。然而，由于产生的突变范围广泛，以及缺乏经济实惠的技术来检测 DNA 序列变化，因此限制了对快速中子群体中发现的突变进行全基因组和群体水平的特征描述。在这项研究中，我们旨在测试高通量测序（GBS）技术是否可用于鉴定快速中子诱导的大豆突变体群体中的拷贝数变异（CNV）。
我们从 79 个大豆突变体的 GBS 数据中鉴定出了 CNV，并通过对其中 19 个突变体的 GBS 数据与基于芯片的比较基因组杂交（aCGH）数据、针对 GBS 检测到的最小事件的代表性子集的靶向 PCR 和 ddPCR 检测的结果进行验证，评估了该方法的灵敏度和精确度。我们的 GBS 流程检测到了 aCGH 发现的 96 个事件中的 55 个，对于纯合缺失、杂合缺失和重复事件，近似检测阈值分别为 60kb、500kb 和 1Mb。在 79 个突变体的整个数据集，GBS 数据揭示了 105 个纯合缺失、32 个杂合缺失和 19 个重复事件。这包括几个非常大的事件，最大缺失约为 11.2Mb，最大缺失约为 11.6Mb，最大重复约为 50Mb。
本研究提供了一个概念验证，证明 GBS 可以作为一种经济实惠的高通量方法，用于评估快速中子突变体中的 CNV。这种 GBS 方法的模块化允许结合尽可能多的不同文库或测序运行，以达到特定研究的目标。这种方法应该能够在快速中子突变体群体或具有大量基因组缺失和重复的任何群体中，对数百到数千个个体进行低成本的全基因组特征描述。
