Genome Institute of Singapore, Genome #02-01, Singapore, Singapore.
PLoS One. 2013 Apr 8;8(4):e60811. doi: 10.1371/journal.pone.0060811. Print 2013.
The high throughput and cost-effectiveness afforded by short-read sequencing technologies, in principle, enable researchers to perform 16S rRNA profiling of complex microbial communities at unprecedented depth and resolution. Existing Illumina sequencing protocols are, however, limited by the fraction of the 16S rRNA gene that is interrogated and therefore limit the resolution and quality of the profiling. To address this, we present the design of a novel protocol for shotgun Illumina sequencing of the bacterial 16S rRNA gene, optimized to amplify more than 90% of sequences in the Greengenes database and with the ability to distinguish nearly twice as many species-level OTUs compared to existing protocols. Using several in silico and experimental datasets, we demonstrate that despite the presence of multiple variable and conserved regions, the resulting shotgun sequences can be used to accurately quantify the constituents of complex microbial communities. The reconstruction of a significant fraction of the 16S rRNA gene also enabled high precision (>90%) in species-level identification thereby opening up potential application of this approach for clinical microbial characterization.
高通量和低成本效益的短读测序技术，从原则上讲，使研究人员能够以前所未有的深度和分辨率对复杂微生物群落进行 16S rRNA 分析。然而，现有的 Illumina 测序方案受到所检测的 16S rRNA 基因部分的限制，因此限制了分析的分辨率和质量。为了解决这个问题，我们提出了一种新的用于细菌 16S rRNA 基因的 shotgun Illumina 测序的方案设计，该方案经过优化，可扩增 Greengenes 数据库中超过 90%的序列，并且与现有方案相比，能够区分近两倍数量的种水平 OTUs。使用多个计算机模拟和实验数据集，我们证明，尽管存在多个可变和保守区域，但产生的 shotgun 序列可用于准确量化复杂微生物群落的组成成分。16S rRNA 基因的重建也实现了高精确度（>90%）的种水平鉴定，从而为该方法在临床微生物特征分析中的潜在应用开辟了道路。
