CaBagE: A Cas9-based Background Elimination strategy for targeted, long-read DNA sequencing.
CaBagE: A Cas9-based Background Elimination strategy for targeted, long-read DNA sequencing.
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DOI：
10.1371/journal.pone.0241253
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发表时间：
2021
期刊：
影响因子：
3.7
通讯作者：
Quinlan AR
中科院分区：
文献类型：
--
作者：
Wallace AD;Sasani TA;Swanier J;Gates BL;Greenland J;Pedersen BS;Varley KE;Quinlan AR
由于同源关系、复杂的单倍型结构或串联重复，很大一部分人类基因组很难用短读DNA测序技术进行查询。长读测序技术，如牛津纳米孔的minion，能够直接测量复杂的基因座，而不会引入许多短读方法固有的偏差，尽管它们的吞吐量相对较低。这一限制促使最近努力开发无扩增策略，以靶向和丰富感兴趣的基因座，以便随后进行长阅读测序。在这里，我们介绍了CABAGE，这是一种目标丰富的方法，对大型、结构复杂的目标进行排序是有效和有用的。Cabage方法利用Cas9与其DNA靶标的稳定结合来保护所需片段不被核酸外切酶消化。然后用牛津纳米孔的Minion长读测序技术对浓缩的DNA片段进行测序。当使用健康供体DNA对长度从4-20kb的五个基因组目标进行测试时，用Cabage进行浓缩导致目标基因座的中位数覆盖率为116倍(范围为39-416)。四个肿瘤基因靶点在一次反应中被浓缩，并在单个Minion Flow细胞上多重。我们进一步证明了CABAGE在两个带有C9orf72短串联重复序列扩增的ALS患者中的实用性，以产生与重复启动的聚合酶链式反应所产生的每个个体的基因类型相称的基因估计。使用Cabage，在测序之前，可以在给定的样本中对靶标DNA进行物理浓缩。这一功能允许跨测序平台的适应性，并可能用作测序以外的应用的丰富策略。CABAGE是一种快速浓缩方法，可以揭示隐藏在人类疾病背后的“基因组”区域。
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 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 a median of 116X coverage (range 39–416) 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.
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