Evaluation of strategies for the assembly of diverse bacterial genomes using MinION long-read sequencing
Evaluation of strategies for the assembly of diverse bacterial genomes using MinION long-read sequencing
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DOI：
10.1186/s12864-018-5381-7
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发表时间：
2019-01-09
期刊：
影响因子：
4.4
通讯作者：
Klassen, Jonathan L.
中科院分区：
文献类型：
--
作者：
Goldstein, Sarah;Beka, Lidia;Klassen, Jonathan L.
背景短读测序技术使微生物基因组测序变得便宜和容易。然而，关闭基因组通常是昂贵的，并且从重复的和/或具有极端%GC含量的基因组组装短读段仍然具有挑战性。长时间阅读，单分子测序技术，如牛津纳米孔MinION有可能克服这些困难，虽然利用其潜力的最佳方法仍然很差evaluated.ResultsWe测序九个细菌基因组跨越了广泛的GC内容使用Illumina MiSeq和牛津纳米孔MinION测序技术，以确定每种方法的优点，无论是单独和组合。仅使用MiSeq读段的组装高度准确，但缺乏邻接性，通过将MinION读段添加到这些组装中可以部分克服这一缺陷。通过使用MinION read进行初始组装来生成甚至更多连续的基因组组装，但是这些组装更容易出错并且需要进一步抛光。当Illumina文库有偏差时，这一点尤其明显，我们的菌株具有高和低GC含量。增加基因组的连续性大大提高了注释的插入序列和次级代谢物生物合成基因簇，可能是因为长的读取可以消除这些高度重复的，但生物学上重要的基因组regions.ConclusionsGenome组装使用短的读取的挑战，重复序列和极端GC内容。我们的研究结果表明，这些困难可以在很大程度上克服使用单分子，长读序测序技术，如牛津纳米孔MinION。使用MinION读数进行组装，然后用Illumina读数进行抛光，以足够的准确度生成最连续的基因组，以能够准确注释重要但难以测序的基因组特征，例如插入序列和次级代谢物生物合成基因簇。因此，Oxford Nanopore和Illumina测序的结合可以经济有效地推进微生物进化和基因组驱动的药物发现的研究。
BackgroundShort-read sequencing technologies have made microbial genome sequencing cheap and accessible. However, closing genomes is often costly and assembling short reads from genomes that are repetitive and/or have extreme %GC content remains challenging. Long-read, single-molecule sequencing technologies such as the Oxford Nanopore MinION have the potential to overcome these difficulties, although the best approach for harnessing their potential remains poorly evaluated.ResultsWe sequenced nine bacterial genomes spanning a wide range of GC contents using Illumina MiSeq and Oxford Nanopore MinION sequencing technologies to determine the advantages of each approach, both individually and combined. Assemblies using only MiSeq reads were highly accurate but lacked contiguity, a deficiency that was partially overcome by adding MinION reads to these assemblies. Even more contiguous genome assemblies were generated by using MinION reads for initial assembly, but these assemblies were more error-prone and required further polishing. This was especially pronounced when Illumina libraries were biased, as was the case for our strains with both high and low GC content. Increased genome contiguity dramatically improved the annotation of insertion sequences and secondary metabolite biosynthetic gene clusters, likely because long-reads can disambiguate these highly repetitive but biologically important genomic regions.ConclusionsGenome assembly using short-reads is challenged by repetitive sequences and extreme GC contents. Our results indicate that these difficulties can be largely overcome by using single-molecule, long-read sequencing technologies such as the Oxford Nanopore MinION. Using MinION reads for assembly followed by polishing with Illumina reads generated the most contiguous genomes with sufficient accuracy to enable the accurate annotation of important but difficult to sequence genomic features such as insertion sequences and secondary metabolite biosynthetic gene clusters. The combination of Oxford Nanopore and Illumina sequencing can therefore cost-effectively advance studies of microbial evolution and genome-driven drug discovery.