Targeted recovery of novel phylogenetic diversity from next-generation sequence data.
Targeted recovery of novel phylogenetic diversity from next-generation sequence data.
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DOI：
10.1038/ismej.2012.50
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发表时间：
2012-11
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--
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下一代测序技术使人们认识到了所谓的“稀有生物圈”。这些微生物操作分类单位(OTU)由低相对丰度定义，可能特别适合于维持低种群数量。我们假设，挖掘低丰度的下一代16S核糖体RNA(RRNA)基因数据将导致发现新的系统发育多样性，反映出以前的采样工作尚未发现的微生物。在这里，我们通过结合分子和生物信息学方法来检验这一假设，以在稀有生物圈OTUS中有针对性地检索系统发育新颖性。我们结合了BLASTN网络分析、系统发育和有针对性的引物设计，从独特的潜在细菌谱系中扩增出16S rRNA基因序列，这些细菌谱系包括来自北极冻土带土壤样本的数百万序列数据集的稀有生物圈的一部分。七个恢复的系统发育谱系中的三个代表了极其不同的分类实体，证明了这里开发的方案的可行性。这些不同的靶序列对应于(A)BRC1候选门中以前未知的谱系，(B)早期分歧和目前公认的单一种蓝藻Gloebacter的姊妹群，一个包含多个半形性特征的属和(C)在线粒体内的高度分化的谱系。与来自额外土壤的12个下一代数据集的比较表明，这些新的16S rRNA基因持续低丰度分布。结果表明，这种序列分析和检索流水线适用于探索未被充分代表的系统发育新颖性和恢复可能代表细菌进化的重要步骤的分类群。
Next-generation sequencing technologies have led to recognition of a so-called ‘rare biosphere'. These microbial operational taxonomic units (OTUs) are defined by low relative abundance and may be specifically adapted to maintaining low population sizes. We hypothesized that mining of low-abundance next-generation 16S ribosomal RNA (rRNA) gene data would lead to the discovery of novel phylogenetic diversity, reflecting microorganisms not yet discovered by previous sampling efforts. Here, we test this hypothesis by combining molecular and bioinformatic approaches for targeted retrieval of phylogenetic novelty within rare biosphere OTUs. We combined BLASTN network analysis, phylogenetics and targeted primer design to amplify 16S rRNA gene sequences from unique potential bacterial lineages, comprising part of the rare biosphere from a multi-million sequence data set from an Arctic tundra soil sample. Demonstrating the feasibility of the protocol developed here, three of seven recovered phylogenetic lineages represented extremely divergent taxonomic entities. These divergent target sequences correspond to (a) a previously unknown lineage within the BRC1 candidate phylum, (b) a sister group to the early diverging and currently recognized monospecific Cyanobacteria Gloeobacter, a genus containing multiple plesiomorphic traits and (c) a highly divergent lineage phylogenetically resolved within mitochondria. A comparison to twelve next-generation data sets from additional soils suggested persistent low-abundance distributions of these novel 16S rRNA genes. The results demonstrate this sequence analysis and retrieval pipeline as applicable for exploring underrepresented phylogenetic novelty and recovering taxa that may represent significant steps in bacterial evolution.
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影响因子：
4.4
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