School of Biotechnology and Biomolecular Sciences and Centre for Marine Bio-Innovation, University of New South Wales, Sydney, New South Wales, Australia.
PLoS One. 2012;7(6):e39948. doi: 10.1371/journal.pone.0039948. Epub 2012 Jun 27.
Direct sequencing of environmental DNA (metagenomics) has a great potential for describing the 16S rRNA gene diversity of microbial communities. However current approaches using this 16S rRNA gene information to describe community diversity suffer from low taxonomic resolution or chimera problems. Here we describe a new strategy that involves stringent assembly and data filtering to reconstruct full-length 16S rRNA genes from metagenomicpyrosequencing data. Simulations showed that reconstructed 16S rRNA genes provided a true picture of the community diversity, had minimal rates of chimera formation and gave taxonomic resolution down to genus level. The strategy was furthermore compared to PCR-based methods to determine the microbial diversity in two marine sponges. This showed that about 30% of the abundant phylotypes reconstructed from metagenomic data failed to be amplified by PCR. Our approach is readily applicable to existing metagenomic datasets and is expected to lead to the discovery of new microbial phylotypes.
直接对环境 DNA（宏基因组学）进行测序在描述微生物群落的 16S rRNA 基因多样性方面具有很大的潜力。然而，目前使用这些 16S rRNA 基因信息来描述群落多样性的方法存在分类分辨率低或嵌合体问题。在这里，我们描述了一种新的策略，该策略涉及严格的组装和数据过滤，以从宏基因组焦磷酸测序数据中重建全长 16S rRNA 基因。模拟表明，重建的 16S rRNA 基因真实地反映了群落多样性，嵌合体形成的比率最小，并能达到属级别的分类分辨率。该策略还与基于 PCR 的方法进行了比较，以确定两种海洋海绵中的微生物多样性。结果表明，从宏基因组数据重建的丰富的生物型中，约有 30%无法通过 PCR 扩增。我们的方法易于应用于现有的宏基因组数据集，预计将发现新的微生物生物型。
