PhylOTU: a high-throughput procedure quantifies microbial community diversity and resolves novel taxa from metagenomic data.
PhylOTU: a high-throughput procedure quantifies microbial community diversity and resolves novel taxa from metagenomic data.
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DOI：
10.1371/journal.pcbi.1001061
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发表时间：
2011-01-20
影响因子：
4.3
通讯作者：
Pollard KS
中科院分区：
文献类型：
--
作者：
Sharpton TJ;Riesenfeld SJ;Kembel SW;Ladau J;O'Dwyer JP;Green JL;Eisen JA;Pollard KS
微生物多样性的典型特征是将核糖体RNA （SSU-rRNA）序列聚类为操作分类单位（otu）。通过PCR对环境SSU-rRNA标记进行靶向测序，由于在引物和扩增上的偏差，可能无法检测到OTUs。散弹枪测序环境DNA的分析，被称为宏基因组学，避免了扩增偏差，但产生了片段，非重叠的序列读取，无法通过现有的otu查找方法聚类。为了规避这些限制，我们开发了PhylOTU，这是一个通过使用系统发育原理和概率序列谱从宏基因组SSU-rRNA序列数据中识别otu的计算工作流程。使用模拟宏基因组数据，我们量化了PhylOTU簇读取otu的准确性。比较PCR和霰弹枪测序的SSU-rRNA标记，发现PCR文库在每个测序残基中鉴定出更多的otu，而宏基因组文库恢复了更大的otu分类多样性。此外，我们还在宏基因组文库中发现了新的种、属和科，包括PCR序列分析缺失的门的OTUs。综上所述，这些结果表明PhylOTU能够表征目前基于pcr的多样性调查中隐藏的部分生物圈。微生物构成了地球上大部分的生物多样性。由于绝大多数微生物不容易在实验室中培养，研究人员经常依靠基于pcr的基因组序列研究来表征微生物多样性。这些分析极大地扩展了我们对生物多样性的理解，但由于方法上的偏差，基于pcr的方法可能只揭示了部分微生物生物圈。环境DNA的鸟枪测序，被称为宏基因组学，避免了与基因组序列的靶向扩增相关的偏见，并可以提供对传统调查所隐藏的多样性的见解。然而，霰弹枪序列数据的碎片性、非重叠性使得现有工具难以分析。在这里，我们提出了PhylOTU，一种新的计算方法，可以从宏基因组数据中准确表征微生物多样性。我们处理了来自全球开放海洋的超过1000万个宏基因组序列，以鉴定新的细菌分类群，并揭示了来自相同样品的基于pcr的序列调查所忽略的微生物的存在。这些结果表明，为了充分表征微生物多样性，需要一个新的生物信息学工具箱来分析霰弹枪宏基因组数据。
Microbial diversity is typically characterized by clustering ribosomal RNA (SSU-rRNA) sequences into operational taxonomic units (OTUs). Targeted sequencing of environmental SSU-rRNA markers via PCR may fail to detect OTUs due to biases in priming and amplification. Analysis of shotgun sequenced environmental DNA, known as metagenomics, avoids amplification bias but generates fragmentary, non-overlapping sequence reads that cannot be clustered by existing OTU-finding methods. To circumvent these limitations, we developed PhylOTU, a computational workflow that identifies OTUs from metagenomic SSU-rRNA sequence data through the use of phylogenetic principles and probabilistic sequence profiles. Using simulated metagenomic data, we quantified the accuracy with which PhylOTU clusters reads into OTUs. Comparisons of PCR and shotgun sequenced SSU-rRNA markers derived from the global open ocean revealed that while PCR libraries identify more OTUs per sequenced residue, metagenomic libraries recover a greater taxonomic diversity of OTUs. In addition, we discover novel species, genera and families in the metagenomic libraries, including OTUs from phyla missed by analysis of PCR sequences. Taken together, these results suggest that PhylOTU enables characterization of part of the biosphere currently hidden from PCR-based surveys of diversity? Microorganisms comprise the majority of the biodiversity on the planet. Because the overwhelming majority of microbes are not readily cultured in the laboratory, researchers often rely on PCR-based investigations of genomic sequence to characterize microbial diversity. These analyses have dramatically expanded our understanding of biodiversity, but due to methodological biases PCR-based approaches may only reveal part of the microbial biosphere. Shotgun sequencing of environmental DNA, known as metagenomics, avoids the biases associated with targeted amplification of genomic sequence and can provide insight into the diversity hidden from traditional investigations. However, the fragmentary, non-overlapping nature of shotgun sequence data makes it intractable to analyze with existing tools. Here, we present PhylOTU, a novel computational method that enables accurate characterization of microbial diversity from metagenomic data. We process over 10 million metagenomic sequences obtained from the global open ocean to identify novel Bacterial taxa and reveal the presence of microorganisms overlooked by investigation of PCR-based sequences from the same samples. These results suggest that to fully characterize microbial biodiversity requires a novel bioinformatics toolbox for analysis of shotgun metagenomic data.
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64.8
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