Robust long-read native DNA sequencing using the ONT CsgG Nanopore system.
Robust long-read native DNA sequencing using the ONT CsgG Nanopore system.
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DOI：
10.12688/wellcomeopenres.11246.3
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发表时间：
2017-04-06
影响因子：
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通讯作者：
Hussain S
中科院分区：
文献类型：
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作者：
Carter JM;Hussain S
背景：在DNA测序过程中获得长读长的能力具有几个潜在的重要实际应用。已经报道了使用目前可从Oxford Nanopore Technologies（ONT）商购获得的Nanopore测序方法的特别长的读取长度。然而，早期的报道已经证明了组合的通量和序列准确性的水平仅是有限的。最近，ONT发布了一种新的CsgG孔测序系统以及具有改进潜力的250 b/s易位化学。研究方法：我们在ONT微型“MinION”装置上使用这些组件，并对从近单倍体癌细胞系HAP 1获得的天然基因组DNA进行测序。我们的数据分析是利用最近描述的为纳米孔/长读序测序输出量身定制的计算工具进行的，在这里我们提出了我们的关键发现。结果：从单次测序运行中，我们获得了约240，000个高质量的映射读数，共包含约23亿个碱基。获得了9.6kb的平均读长和~ 17 kb的N50，而序列与参考序列的平均同一性为85%。值得注意的是，我们获得了线粒体基因组的~ 68 X覆盖率，并且能够实现测序的mtDNA读数的平均一致性为99.8%。结论：随着改进的测序化学已经发布和更高通量的仪器在管道中，这个早期的研究表明，基于ONT CsgG的测序可能是一个有用的选择，用于与复杂基因组相关的潜在的实际长读段应用。
Background: The ability to obtain long read lengths during DNA sequencing has several potentially important practical applications. Especially long read lengths have been reported using the Nanopore sequencing method, currently commercially available from Oxford Nanopore Technologies (ONT). However, early reports have demonstrated only limited levels of combined throughput and sequence accuracy. Recently, ONT released a new CsgG pore sequencing system as well as a 250b/s translocation chemistry with potential for improvements. Methods: We made use of such components on ONTs miniature ‘MinION’ device and sequenced native genomic DNA obtained from the near haploid cancer cell line HAP1. Analysis of our data was performed utilising recently described computational tools tailored for nanopore/long-read sequencing outputs, and here we present our key findings. Results: From a single sequencing run, we obtained ~240,000 high-quality mapped reads, comprising a total of ~2.3 billion bases. A mean read length of 9.6kb and an N50 of ~17kb was achieved, while sequences mapped to reference with a mean identity of 85%. Notably, we obtained ~68X coverage of the mitochondrial genome and were able to achieve a mean consensus identity of 99.8% for sequenced mtDNA reads. Conclusions: With improved sequencing chemistries already released and higher-throughput instruments in the pipeline, this early study suggests that ONT CsgG-based sequencing may be a useful option for potential practical long-read applications with relevance to complex genomes.