Youssef Noha, Sheik Cody S, Krumholz Lee R, Najar Fares Z, Roe Bruce A, Elshahed Mostafa S
Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, OK 74074, USA.
Appl Environ Microbiol. 2009 Aug;75(16):5227-36. doi: 10.1128/AEM.00592-09. Epub 2009 Jun 26.
Pyrosequencing-based 16S rRNA gene surveys are increasingly utilized to study highly diverse bacterial communities, with special emphasis on utilizing the large number of sequences obtained (tens to hundreds of thousands) for species richness estimation. However, it is not yet clear how the number of operational taxonomic units (OTUs) and, hence, species richness estimates determined using shorter fragments at different taxonomic cutoffs correlates with the number of OTUs assigned using longer, nearly complete 16S rRNA gene fragments. We constructed a 16S rRNA clone library from an undisturbed tallgrass prairie soil (1,132 clones) and used it to compare species richness estimates obtained using eight pyrosequencing candidate fragments (99 to 361 bp in length) and the nearly full-length fragment. Fragments encompassing the V1 and V2 (V1+V2) region and the V6 region (generated using primer pairs 8F-338R and 967F-1046R) overestimated species richness; fragments encompassing the V3, V7, and V7+V8 hypervariable regions (generated using primer pairs 338F-530R, 1046F-1220R, and 1046F-1392R) underestimated species richness; and fragments encompassing the V4, V5+V6, and V6+V7 regions (generated using primer pairs 530F-805R, 805F-1046R, and 967F-1220R) provided estimates comparable to those obtained with the nearly full-length fragment. These patterns were observed regardless of the alignment method utilized or the parameter used to gauge comparative levels of species richness (number of OTUs observed, slope of scatter plots of pairwise distance values for short and nearly complete fragments, and nonparametric and parametric species richness estimates). Similar results were obtained when analyzing three other datasets derived from soil, adult Zebrafish gut, and basaltic formations in the East Pacific Rise. Regression analysis indicated that these observed discrepancies in species richness estimates within various regions could readily be explained by the proportions of hypervariable, variable, and conserved base pairs within an examined fragment.
基于焦磷酸测序的16S rRNA基因调查越来越多地用于研究高度多样化的细菌群落，特别强调利用获得的大量序列（数万到数十万）来估计物种丰富度。然而，目前尚不清楚在不同分类阈值下使用较短片段确定的操作分类单元（OTU）数量以及物种丰富度估计值与使用较长的、近乎完整的16S rRNA基因片段指定的OTU数量之间如何关联。我们从一个未受干扰的高草草原土壤构建了一个16S rRNA克隆文库（1132个克隆），并用它来比较使用八个焦磷酸测序候选片段（长度为99至361 bp）和近乎全长片段获得的物种丰富度估计值。包含V1和V2（V1 + V2）区域以及V6区域（使用引物对8F - 338R和967F - 1046R生成）的片段高估了物种丰富度；包含V3、V7和V7 + V8高变区域（使用引物对338F - 530R、1046F - 1220R和1046F - 1392R生成）的片段低估了物种丰富度；而包含V4、V5 + V6和V6 + V7区域（使用引物对530F - 805R、805F - 1046R和967F - 1220R生成）的片段提供的估计值与使用近乎全长片段获得的估计值相当。无论使用何种比对方法或用于衡量物种丰富度比较水平的参数（观察到的OTU数量、短片段和近乎完整片段的成对距离值散点图的斜率以及非参数和参数物种丰富度估计值），都观察到了这些模式。在分析另外三个来自土壤、成年斑马鱼肠道和东太平洋海隆玄武岩地层的数据集时也获得了类似的结果。回归分析表明，在各个区域内观察到的物种丰富度估计值差异可以很容易地由所检查片段内高变、可变和保守碱基对的比例来解释。
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