A substantial fraction of the human genome is difficult to interrogate with short-read DNA sequencing technologies due to paralogy, complex haplotype structures, or tandem repeats. Long-read sequencing technologies, such as Oxford Nanopore's MinION, enable direct measurement of complex loci without introducing many of the biases inherent to short-read methods, though they suffer from relatively lower throughput. This limitation has motivated recent efforts to develop amplification-free strategies to target and enrich loci of interest for subsequent sequencing with long reads. Here, we present CaBagE, a novel method for target enrichment that is efficient and useful for sequencing large, structurally complex targets. The CaBagE method leverages the stable binding of Cas9 to its DNA target to protect desired fragments from digestion with exonuclease. Enriched DNA fragments are then sequenced with Oxford Nanopore's MinION long-read sequencing technology. Enrichment with CaBagE resulted in up to 416X coverage of target loci when tested on five genomic targets ranging from 4-20kb in length using healthy donor DNA. Four cancer gene targets were enriched in a single reaction and multiplexed on a single MinION flow cell. We further demonstrate the utility of CaBagE in two ALS patients with C9orf72 short tandem repeat expansions to produce genotype estimates commensurate with genotypes derived from repeat-primed PCR for each individual. With CaBagE there is a physical enrichment of on-target DNA in a given sample prior to sequencing. This feature allows adaptability across sequencing platforms and potential use as an enrichment strategy for applications beyond sequencing. CaBagE is a rapid enrichment method that can illuminate regions of the 'hidden genome' underlying human disease.

中文翻译：

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CaBagE：基于Cas9的背景消除策略，用于有针对性的长期阅读DNA测序

由于寄生虫，复杂的单倍型结构或串联重复，人类基因组的很大一部分很难用短读DNA测序技术进行询问。牛津纳米孔（Oxford Nanopore）的MinION等长读测序技术可直接测量复杂基因座，而不会引入短读方法固有的许多偏见，尽管它们的吞吐量相对较低。这种局限性激发了最近的努力，以开发无扩增策略来靶向和富集感兴趣的基因座，以供后续长读测序。在这里，我们介绍了CaBagE，这是一种新型的富集靶标的方法，可用于对大型，结构复杂的靶标进行测序。CaBagE方法利用Cas9与其DNA靶标的稳定结合来保护所需片段免遭核酸外切酶消化。然后用牛津纳米孔的MinION长读测序技术对富集的DNA片段进行测序。使用健康供体DNA在5个长度为4-20kb的基因组靶标上进行测试时，CaBagE的富集可导致高达416X的靶基因座覆盖率。在单个反应中富集了四个癌症基因靶标，并在单个MinION流通池中进行了多重处理。我们进一步证明了CaBagE在两名C9orf72短串联重复扩增的ALS患者中的效用，以产生与每个人从重复启动PCR产生的基因型相对应的基因型估计。使用CaBagE进行测序之前，给定样品中的目标DNA会发生物理富集。此功能可实现跨测序平台的适应性，并有可能用作测序以外应用程序的丰富策略。CaBagE是一种快速富集方法，可以照亮人类疾病的“隐藏基因组”区域。