The use of Oxford Nanopore native barcoding for complete genome assembly.
The use of Oxford Nanopore native barcoding for complete genome assembly.
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DOI：
10.1093/gigascience/gix001
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发表时间：
2017-03-01
期刊：
影响因子：
9.2
通讯作者：
Feil EJ
中科院分区：
文献类型：
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作者：
Bayliss SC;Hunt VL;Yokoyama M;Thorpe HA;Feil EJ
牛津纳米孔技术公司的MINION(TM)是一种移动式DNA测序仪，可以在很短的周转时间内产生长阅读序列。在这里，我们报告了使用牛津纳米孔天然条形码进行单个重叠群基因组组装的第一个演示，当应用于12个样本的多路文库并与现有的Illumina短读数据相结合时。考虑到现有短读数据的可用性，这为从一次Minion(TM)测序运行中关闭多个细菌基因组铺平了道路。我们使用的菌株MHO_001代表了重要的社区获得的耐甲氧西林金黄色葡萄球菌谱系USA300。利用现有的短读取序列和来自多路数据的条形码长读取序列的杂交组装，我们完成了金黄色葡萄球菌USA300株MHO_001的基因组。长读数据仅占平均Minion(TM)运行(∼7x基因组覆盖率)的5%至10%，但使用标准工具，这足以完成金黄色葡萄球菌MHO_001株的环状染色体(2.86Mb)和两个完整的质粒(27kb和3kb)。与USA300参考基因组USA300_FPR3757相比，发现了微小的差异，包括可移动遗传元件的易位、丢失和获得。在这里，我们演示了使用本地条形码进行多路复用的minion(TM)读取，可以与短读取数据结合使用，以完整地完成细菌基因组。从一次Minion(TM)运行中完成多个基因组的能力将影响我们对辅助基因组内容、质粒多样性和基因组重排的理解。
The Oxford Nanopore Technologies MinION(TM) is a mobile DNA sequencer that can produce long read sequences with a short turn-around time. Here we report the first demonstration of single contig genome assembly using Oxford Nanopore native barcoding when applied to a multiplexed library of 12 samples and combined with existing Illumina short read data. This paves the way for the closure of multiple bacterial genomes from a single MinION(TM) sequencing run, given the availability of existing short read data. The strain we used, MHO_001, represents the important community-acquired methicillin-resistant Staphylococcus aureus lineage USA300. Using a hybrid assembly of existing short read and barcoded long read sequences from multiplexed data, we completed a genome of the S. aureus USA300 strain MHO_001. The long read data represented only ∼5% to 10% of an average MinION(TM) run (∼7x genomic coverage), but, using standard tools, this was sufficient to complete the circular chromosome of S. aureus strain MHO_001 (2.86 Mb) and two complete plasmids (27 Kb and 3 Kb). Minor differences were noted when compared to USA300 reference genome, USA300_FPR3757, including the translocation, loss, and gain of mobile genetic elements. Here we demonstrate that MinION(TM) reads, multiplexed using native barcoding, can be used in combination with short read data to fully complete a bacterial genome. The ability to complete multiple genomes, for which short read data is already available, from a single MinION(TM) run is set to impact our understanding of accessory genome content, plasmid diversity, and genome rearrangements.