Department of Microbiology, University College Cork, Cork, Ireland.
Nucleic Acids Res. 2010 Dec;38(22):e200. doi: 10.1093/nar/gkq873. Epub 2010 Sep 29.
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.
高通量分子技术可以在复杂的环境中（如肠道微生物群）以高分辨率对微生物群落进行分析。新一代测序技术的最新进展允许更精细的分辨率。我们比较了较长（454 钛）序列读数和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，我们都针对从人类粪便样本中提取的微生物 DNA 中的六个 16S rRNA 基因可变区的串联组合进行了靶向，以研究它们的局限性和潜力。计算机模拟评估预测，V3/V4 和 V4/V5 区将为这两种技术提供最高的分类准确性。然而，与其他区域相比，V3/V4 区域的实验测序显示出显著的扩增偏差，这强调了对引物对进行实验验证的必要性。454 和 Illumina 技术的最新发展与它们的前几代相比提供了更高的分辨率，并彼此相对一致。然而，由于长度较短且超过 60nt 时错误率较高，大多数 Illumina 读数无法分类到属水平。尽管如此，Illumina 的高质量和长读长保证了无与伦比的覆盖范围，有望深入了解高度多样化和复杂的环境，如人类肠道。
