Generation of Multimillion-Sequence 16S rRNA Gene Libraries from Complex Microbial Communities by Assembling Paired-End Illumina Reads
Generation of Multimillion-Sequence 16S rRNA Gene Libraries from Complex Microbial Communities by Assembling Paired-End Illumina Reads
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DOI：
10.1128/aem.02772-10
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发表时间：
2011-06-01
影响因子：
4.4
通讯作者：
Neufeld, Josh D.
中科院分区：
文献类型：
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作者：
Bartram, Andrea K.;Lynch, Michael D. J.;Neufeld, Josh D.
微生物群落拥有无与伦比的分类多样性。尽管16S rRNA基因测序技术已经出现，但对环境及宿主相关样本进行充分的特征描述对微生物学家来说仍然是一个挑战。为了增加对不同细菌群落采样的深度，我们开发了一种利用Illumina基因组分析仪对16S rRNA基因（跨越V3区域；约200个核苷酸）的数百万对末端读段进行测序和组装的方法。为了确认可重复性，并确定一个适合数据分析的计算流程，我们对已知培养细菌分离株的DNA确定混合物（>100万个组装后序列）和北极苔原土壤样本（>600万个组装后序列）都制备了双份的序列文库。Illumina 16S rRNA基因文库相较于所有现有的新一代测序方法（例如，454焦磷酸测序），其序列数量有了大幅增加，而125个碱基的对末端读段的组装通过为每个16S rRNA基因序列纳入一个初始质量控制步骤，提供了一种方法学上的优势。这种方法结合了带索引的引物，能够在单个流动池泳道中对多个微生物群落进行特征描述，可以很容易地进行修改以针对其他可变区域或基因，并且展示了对相对丰度较低的生物的DNA进行前所未有的经济获取方式。
Microbial communities host unparalleled taxonomic diversity. Adequate characterization of environmental and host-associated samples remains a challenge for microbiologists, despite the advent of 16S rRNA gene sequencing. In order to increase the depth of sampling for diverse bacterial communities, we developed a method for sequencing and assembling millions of paired-end reads from the 16S rRNA gene (spanning the V3 region; similar to 200 nucleotides) by using an Illumina genome analyzer. To confirm reproducibility and to identify a suitable computational pipeline for data analysis, sequence libraries were prepared in duplicate for both a defined mixture of DNAs from known cultured bacterial isolates (>1 million postassembly sequences) and an Arctic tundra soil sample (>6 million postassembly sequences). The Illumina 16S rRNA gene libraries represent a substantial increase in number of sequences over all extant next-generation sequencing approaches (e. g., 454 pyrosequencing), while the assembly of paired-end 125-base reads offers a methodological advantage by incorporating an initial quality control step for each 16S rRNA gene sequence. This method incorporates indexed primers to enable the characterization of multiple microbial communities in a single flow cell lane, may be modified readily to target other variable regions or genes, and demonstrates unprecedented and economical access to DNAs from organisms that exist at low relative abundances.