Division of Periodontology, College of Dentistry, The Ohio State University, Columbus, Ohio, United States of America.
PLoS One. 2011;6(6):e20956. doi: 10.1371/journal.pone.0020956. Epub 2011 Jun 29.
Pyrosequencing of 16S rRNA genes allows for in-depth characterization of complex microbial communities. Although it is known that primer selection can influence the profile of a community generated by sequencing, the extent and severity of this bias on deep-sequencing methodologies is not well elucidated. We tested the hypothesis that the hypervariable region targeted for sequencing and primer degeneracy play important roles in influencing the composition of 16S pyrotag communities. Subgingival plaque from deep sites of current smokers with chronic periodontitis was analyzed using Sanger sequencing and pyrosequencing using 4 primer pairs. Greater numbers of species were detected by pyrosequencing than by Sanger sequencing. Rare taxa constituted nearly 6% of each pyrotag community and less than 1% of the Sanger sequencing community. However, the different target regions selected for pyrosequencing did not demonstrate a significant difference in the number of rare and abundant taxa detected. The genera Prevotella, Fusobacterium, Streptococcus, Granulicatella, Bacteroides, Porphyromonas and Treponema were abundant when the V1-V3 region was targeted, while Streptococcus, Treponema, Prevotella, Eubacterium, Porphyromonas, Campylobacter and Enterococcus predominated in the community generated by V4-V6 primers, and the most numerous genera in the V7-V9 community were Veillonella, Streptococcus, Eubacterium, Enterococcus, Treponema, Catonella and Selenomonas. Targeting the V4-V6 region failed to detect the genus Fusobacterium, while the taxa Selenomonas, TM7 and Mycoplasma were not detected by the V7-V9 primer pairs. The communities generated by degenerate and non-degenerate primers did not demonstrate significant differences. Averaging the community fingerprints generated by V1-V3 and V7-V9 primers provided results similar to Sanger sequencing, while allowing a significantly greater depth of coverage than is possible with Sanger sequencing. It is therefore important to use primers targeted to these two regions of the 16S rRNA gene in all deep-sequencing efforts to obtain representational characterization of complex microbial communities.
16S rRNA 基因焦磷酸测序可深入分析复杂微生物群落。尽管人们知道引物选择会影响测序产生的群落特征，但这种偏倚对高通量测序方法的程度和严重程度尚不清楚。我们检验了这样一个假设，即测序的靶向高变区和引物简并性在影响 16S pyrotag 群落组成方面起着重要作用。使用 Sanger 测序和 4 对引物的焦磷酸测序分析了慢性牙周炎深牙周袋的龈下菌斑。焦磷酸测序比 Sanger 测序检测到更多的物种。每个 pyrotag 群落的稀有分类单元约占 6%，而 Sanger 测序群落不到 1%。然而，用于焦磷酸测序的不同靶区在检测到的稀有和丰富分类单元数量上没有显著差异。当靶向 V1-V3 区时，普雷沃氏菌属、梭杆菌属、链球菌属、颗粒链菌属、拟杆菌属、卟啉单胞菌属和密螺旋体属等属是丰富的，而当靶向 V4-V6 引物时，链球菌属、密螺旋体属、普雷沃氏菌属、真杆菌属、卟啉单胞菌属、弯曲菌属和肠球菌属占主导地位，V7-V9 区数量最多的属是韦荣球菌属、链球菌属、真杆菌属、肠球菌属、密螺旋体属、卡他莫拉菌属和唾液乳杆菌属。靶向 V4-V6 区未能检测到梭杆菌属，而 Selenomonas、TM7 和支原体属则不能被 V7-V9 引物对检测到。简并和非简并引物产生的群落没有显著差异。对 V1-V3 和 V7-V9 引物生成的群落指纹图谱进行平均处理，结果与 Sanger 测序相似，但比 Sanger 测序具有更大的覆盖深度。因此，在所有高通量测序工作中，使用靶向 16S rRNA 基因这两个区域的引物对于获得复杂微生物群落的代表性特征非常重要。
