Target region selection is a critical determinant of community fingerprints generated by 16S pyrosequencing.
Target region selection is a critical determinant of community fingerprints generated by 16S pyrosequencing.
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DOI：
10.1371/journal.pone.0020956
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发表时间：
2011
期刊：
影响因子：
3.7
通讯作者：
Camerlengo T
中科院分区：
文献类型：
--
作者：
Kumar PS;Brooker MR;Dowd SE;Camerlengo T
16S rRNA 基因的焦磷酸测序可以深入表征复杂的微生物群落。尽管众所周知，引物选择可以影响测序生成的群落概况，但这种对深度测序方法的偏差的程度和严重性尚未得到很好的阐明。我们测试了以下假设：用于测序和引物简并的高变区在影响 16S 焦标签群落的组成方面发挥着重要作用。使用桑格测序和焦磷酸测序（使用 4 个引物对）分析患有慢性牙周炎的当前吸烟者深层的龈下菌斑。焦磷酸测序检测到的物种数量比桑格测序检测到的物种数量更多。稀有类群占每个热标签群落的近 6%，占桑格测序群落的不到 1%。然而，选择用于焦磷酸测序的不同目标区域在检测到的稀有和丰富类群的数量上没有表现出显着差异。当针对V1-V3区域时，普雷沃氏菌属、梭杆菌属、链球菌属、颗粒菌属、拟杆菌属、卟啉单胞菌属和密螺旋体属丰富，而V4-V6引物产生的群落中链球菌属、密螺旋体属、普雷沃氏菌属、真杆菌属、卟啉单胞菌属、弯曲杆菌属和肠球菌属占主导地位，而V7-V9区域中的属数量最多群落包括韦荣球菌属、链球菌属、真杆菌属、肠球菌属、密螺旋体属、卡东氏菌属和硒单胞菌属。靶向V4-V6区域未能检测到梭杆菌属，而V7-V9引物对未能检测到硒单胞菌属、TM7和支原体类群。由简并和非简并引物生成的群落没有表现出显着差异。对 V1-V3 和 V7-V9 引物生成的群落指纹进行平均可提供与桑格测序相似的结果，同时允许比桑格测序更大的覆盖深度。因此，在所有深度测序工作中使用针对 16S rRNA 基因这两个区域的引物非常重要，以获得复杂微生物群落的代表性表征。
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, Campylobacer 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 providesd 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.
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影响因子：
12.3
作者：
Huse SM;Huber JA;Morrison HG;Sogin ML;Welch DM
通讯作者：
Welch DM
DOI：
10.1073/pnas.1002601107
发表时间：
2010-06-29
影响因子：
11.1
作者：
Dominguez-Bello, Maria G.;Costello, Elizabeth K.;Knight, Rob
通讯作者：
Knight, Rob
影响因子：
9.4
作者：
Kumar, Purnima S.;Leys, Eugene J.;Griffen, Ann L.
通讯作者：
Griffen, Ann L.
影响因子：
9.4
作者：
Kumar, PS;Griffen, AL;Leys, EJ
通讯作者：
Leys, EJ
影响因子：
--
作者：
de Lillo, A;Ashley, FP;Wade, WG
通讯作者：
Wade, WG