CRISPR/Cas9-mediated enrichment coupled to nanopore sequencing provides a valuable tool for the precise reconstruction of large genomic target regions

Complete and accurate identification of genetic variants associated with specific phenotypes can be challenging when there is a high level of genomic divergence between individuals in a study and the corresponding reference genome. However, CRISPR/Cas9-mediated enrichment can be combined with nanopore sequencing to facilitate the reconstruction of such genomic regions by targeted de novo genome assembly. We used this approach to reconstruct a 250-kbp target region on chromosome 5 of the common bean genome (Phaseolus vulgaris) associated with the shattering phenotype. Comparing a non-shattering cultivar (Midas) with the reference genome revealed many single-nucleotide variants and structural variants in this region. We cut five 50-kbp tiled sub-regions of Midas genomic DNA using Cas9, followed by sequencing on a MinION device and de novo assembly, generating a single contig spanning the whole 250-kbp region. This assembly increased the number of Illumina reads mapping to genes in the region, improving their genotypability for downstream analysis. The Cas9 tiling approach for target enrichment and sequencing is a valuable alternative to whole-genome sequencing for the assembly of ultra-long regions of interest, improving the accuracy of downstream genotype-phenotype association analysis.

当研究个体与对应参考基因组间存在高度基因组分化时，完整且精准地鉴定与特定表型相关的遗传变异往往颇具挑战。不过，CRISPR/Cas9介导的富集技术可与纳米孔测序结合，通过靶向从头基因组组装助力这类基因组区域的重建。我们依托该方法，在普通菜豆（Phaseolus vulgaris）第5号染色体上重建了一个与裂荚表型相关的250千碱基对（kbp）目标区域。对比非裂荚品种Midas与参考基因组后发现，该区域内存在大量单核苷酸变异与结构变异。我们利用Cas9切割Midas基因组DNA的五个50千碱基对平铺式子区域，随后在MinION测序仪上完成测序并开展从头组装，最终得到一条覆盖整个250千碱基对区域的重叠群（contig）。该组装提升了比对至该区域基因的Illumina读段数量，优化了下游分析中对这些基因进行基因分型的可行性。相较于全基因组测序，这种用于目标区域富集与测序的Cas9平铺式方法，是组装超长目标区域的优质替代方案，可有效提升下游基因型-表型关联分析的准确性。