Comparison of two next-generation sequencing technologies for resolving highly complex microbiota composition using tandem variable 16S rRNA gene regions.
Comparison of two next-generation sequencing technologies for resolving highly complex microbiota composition using tandem variable 16S rRNA gene regions.
复制标题
DOI：
10.1093/nar/gkq873
复制
发表时间：
2010-12
影响因子：
14.9
通讯作者：
O'Toole PW
中科院分区：
文献类型：
--
作者：
Claesson MJ;Wang Q;O'Sullivan O;Greene-Diniz R;Cole JR;Ross RP;O'Toole PW
高通量分子技术可以高分辨率地分析微生物群落，即使在复杂的环境中，如肠道微生物群。下一代测序技术的最新改进允许更精细的分辨率。我们比较了较长（454钛）序列读数与较短但数量更多的配对末端读数（Illumina）的系统发育分析。对于这两种方法，我们针对从人类粪便样本中提取的微生物DNA中的16 S rRNA基因可变区的六个串联组合，以研究其局限性和潜力。计算机模拟评估预测，V3/V4和V4/V5区域将为两种技术提供最高的分类准确度。然而，V3/V4区域的实验测序显示与其他区域相比存在显著的扩增偏倚，强调了实验验证引物对的必要性。454和Illumina技术的最新发展与之前的版本相比提供了更高的分辨率，并且显示出彼此的相对一致性。然而，大多数Illumina读段由于其较短的长度和超过60 nt的较高错误率而不能被分类到属水平。尽管如此，随着质量的提高和阅读时间的延长，Illumina的覆盖范围更大，有望对人类肠道等高度多样化和复杂的环境提供无与伦比的见解。
High-throughput molecular technologies can profile microbial communities at high resolution even in complex environments like the intestinal microbiota. Recent improvements in next-generation sequencing technologies allow for even finer resolution. We compared phylogenetic profiling of both longer (454 Titanium) sequence reads with shorter, but more numerous, paired-end reads (Illumina). For both approaches, we targeted six tandem combinations of 16S rRNA gene variable regions, in microbial DNA extracted from a human faecal sample, in order to investigate their limitations and potentials. In silico evaluations predicted that the V3/V4 and V4/V5 regions would provide the highest classification accuracies for both technologies. However, experimental sequencing of the V3/V4 region revealed significant amplification bias compared to the other regions, emphasising the necessity for experimental validation of primer pairs. The latest developments of 454 and Illumina technologies offered higher resolution compared to their previous versions, and showed relative consistency with each other. However, the majority of the Illumina reads could not be classified down to genus level due to their shorter length and higher error rates beyond 60 nt. Nonetheless, with improved quality and longer reads, the far greater coverage of Illumina promises unparalleled insights into highly diverse and complex environments such as the human gut.