High-throughput genotyping enables the large-scale analysis of genetic diversity in population genomics and genome-wide association studies that combine the genotypic and phenotypic characterization of large collections of accessions. Sequencing-based approaches for genotyping are progressively replacing traditional genotyping methods because of the lower ascertainment bias. However, genome-wide genotyping based on sequencing becomes expensive in species with large genomes and a high proportion of repetitive DNA. Here we describe the use of CRISPR-Cas9 technology to deplete repetitive elements in the 3.76-Gb genome of lentil (Lens culinaris), 84% consisting of repeats, thus concentrating the sequencing data on coding and regulatory regions (single-copy regions). We designed a custom set of 566,766 gRNAs targeting 2.9 Gbp of repeats and excluding repetitive regions overlapping annotated genes and putative regulatory elements based on ATAC-seq data. The novel depletion method removed ∼40% of reads mapping to repeats, increasing those mapping to single-copy regions by ∼2.6-fold. When analyzing 25 million fragments, this repeat-to-single-copy shift in the sequencing data increased the number of genotyped bases of ∼10-fold compared to nondepleted libraries. In the same condition, we were also able to identify ∼12-fold more genetic variants in the single-copy regions and increased the genotyping accuracy by rescuing thousands of heterozygous variants that otherwise would be missed because of low coverage. The method performed similarly regardless of the multiplexing level, type of library or genotypes, including different cultivars and a closely related species (L. orientalis). Our results showed that CRISPR-Cas9-driven repeat depletion focuses sequencing data on single-copy regions, thus improving high-density and genome-wide genotyping in large and repetitive genomes.

中文翻译：

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基于 CRISPR-Cas9 的重复去除用于复杂植物基因组的高通量基因分型

高通量基因分型能够对群体基因组学和全基因组关联研究中的遗传多样性进行大规模分析，结合大量种质的基因型和表型特征。由于确定偏差较低，基于测序的基因分型方法正在逐步取代传统的基因分型方法。然而，对于基因组较大且重复 DNA 比例较高的物种，基于测序的全基因组基因分型变得昂贵。在这里，我们描述了使用 CRISPR-Cas9 技术来消除扁豆 ( Lens culinaris) 3.76-Gb 基因组中的重复元件），84% 由重复组成，因此将测序数据集中在编码和调控区域（单拷贝区域）。我们设计了一组定制的 566,766 个 gRNA，目标是 2.9 Gbp 的重复序列，并根据 ATAC-seq 数据排除重叠注释基因和推定调控元件的重复区域。这种新颖的去除方法去除了~40%的映射到重复的读段，将映射到单拷贝区域的读段增加了~2.6倍。当分析 2500 万个片段时，测序数据中的重复到单拷贝的转变使基因分型碱基的数量比未耗尽的文库增加了约 10 倍。在同样的条件下，我们还能够在单拷贝区域中识别出约 12 倍的遗传变异，并通过挽救数千个杂合变异来提高基因分型的准确性，否则这些变异会因覆盖率低而被遗漏。无论多重水平、文库类型或基因型如何，该方法的表现都相似，包括不同的品种和密切相关的物种。东方乳杆菌）。我们的结果表明，CRISPR-Cas9 驱动的重复消除将测序数据集中在单拷贝区域，从而改善大型重复基因组中的高密度和全基因组基因分型。