Development of a Dual-Index Sequencing Strategy and Curation Pipeline for Analyzing Amplicon Sequence Data on the MiSeq Illumina Sequencing Platform
Development of a Dual-Index Sequencing Strategy and Curation Pipeline for Analyzing Amplicon Sequence Data on the MiSeq Illumina Sequencing Platform
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DOI：
10.1128/aem.01043-13
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发表时间：
2013-09-01
影响因子：
4.4
通讯作者：
Schloss, Patrick D.
中科院分区：
文献类型：
--
作者：
Kozich, James J.;Westcott, Sarah L.;Schloss, Patrick D.
测序技术的快速发展改变了微生物生态学的实验景观。在过去的10年里，该领域已经从使用克隆文库对每次研究的数百个16S rRNA基因片段进行测序发展到使用来自454和Illumina的下一代测序技术对每次研究的数百万个片段进行测序。随着这些技术的进步，评估这些平台的优势，弱点和整体适用性对于微生物群落的询问至关重要。在这里，我们提出了一种改进的方法，使用Illumina的MiSeq平台对16S rRNA基因内的可变区进行测序，该平台目前能够产生配对的250个核苷酸读数。我们评估了16S rRNA基因的三个长度不同的重叠区域（即，V34、V4和V45）。通过滴定应用于流动池的16S rRNA基因扩增子的浓度，并使用基于质量评分的方法来校正用于构建重叠群的读数之间的差异，我们能够将错误率降低多达两个数量级。最后，我们重新处理了来自先前研究的样品，以证明大量样品可以与鸟枪宏基因组并行地进行多重和测序。这些分析表明，我们的方法可以提供至少与454平台产生的数据一样好的数据，同时以一小部分成本提供相当高的测序覆盖率。
Rapid advances in sequencing technology have changed the experimental landscape of microbial ecology. In the last 10 years, the field has moved from sequencing hundreds of 16S rRNA gene fragments per study using clone libraries to the sequencing of millions of fragments per study using next-generation sequencing technologies from 454 and Illumina. As these technologies advance, it is critical to assess the strengths, weaknesses, and overall suitability of these platforms for the interrogation of microbial communities. Here, we present an improved method for sequencing variable regions within the 16S rRNA gene using Illumina's MiSeq platform, which is currently capable of producing paired 250-nucleotide reads. We evaluated three overlapping regions of the 16S rRNA gene that vary in length (i.e., V34, V4, and V45) by resequencing a mock community and natural samples from human feces, mouse feces, and soil. By titrating the concentration of 16S rRNA gene amplicons applied to the flow cell and using a quality score-based approach to correct discrepancies between reads used to construct contigs, we were able to reduce error rates by as much as two orders of magnitude. Finally, we reprocessed samples from a previous study to demonstrate that large numbers of samples could be multiplexed and sequenced in parallel with shotgun metagenomes. These analyses demonstrate that our approach can provide data that are at least as good as that generated by the 454 platform while providing considerably higher sequencing coverage for a fraction of the cost.