Development of a prokaryotic universal primer for simultaneous analysis of Bacteria and Archaea using next-generation sequencing.
Development of a prokaryotic universal primer for simultaneous analysis of Bacteria and Archaea using next-generation sequencing.
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DOI：
10.1371/journal.pone.0105592
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Nishijima M
中科院分区：
文献类型：
--
作者：
Takahashi S;Tomita J;Nishioka K;Hisada T;Nishijima M
对于基于16S rDNA序列多样性的微生物群落结构分析，灵敏且稳健的16S rDNA PCR扩增是关键步骤。为了获得准确的微生物成分数据，PCR扩增必须无偏差；然而，从含有多种微生物的样品中以同等效率扩增所有 16S rDNA 物种仍然具有挑战性。在这里，我们设计了基于原核16S rDNA V3-V4高变区的通用引物，用于使用Illumina MiSeq新一代测序仪同时检测杂交猪（长白×大白×杜洛克）粪便样本中的细菌和古细菌。计算机分析表明，新设计的通用原核引物与核糖体数据库项目数据库中大约 98.0% 的细菌和 94.6% 的古细菌 rRNA 基因序列匹配。对于使用原核通用引物进行的每个测序反应，平均获得 69,330 (±20,482) 个读数，其中古菌 rRNA 基因约占所有原核读数的 1.2% 至 3.2%。此外，在使用原核通用引物进行的NGS分析中，属于疣微菌门的细菌（包括疣微菌纲和Opitutae的成员）的检测频率高于使用细菌通用引物进行的NGS分析。重要的是，这种新的原核通用引物组的偏差明显低于大多数先前设计的通用引物。我们的研究结果表明，本研究中设计的原核通用引物组将允许同时检测细菌和古细菌，因此将允许更全面地了解环境样品中的微生物群落结构。
For the analysis of microbial community structure based on 16S rDNA sequence diversity, sensitive and robust PCR amplification of 16S rDNA is a critical step. To obtain accurate microbial composition data, PCR amplification must be free of bias; however, amplifying all 16S rDNA species with equal efficiency from a sample containing a large variety of microorganisms remains challenging. Here, we designed a universal primer based on the V3-V4 hypervariable region of prokaryotic 16S rDNA for the simultaneous detection of Bacteria and Archaea in fecal samples from crossbred pigs (Landrace×Large white×Duroc) using an Illumina MiSeq next-generation sequencer. In-silico analysis showed that the newly designed universal prokaryotic primers matched approximately 98.0% of Bacteria and 94.6% of Archaea rRNA gene sequences in the Ribosomal Database Project database. For each sequencing reaction performed with the prokaryotic universal primer, an average of 69,330 (±20,482) reads were obtained, of which archaeal rRNA genes comprised approximately 1.2% to 3.2% of all prokaryotic reads. In addition, the detection frequency of Bacteria belonging to the phylum Verrucomicrobia, including members of the classes Verrucomicrobiae and Opitutae, was higher in the NGS analysis using the prokaryotic universal primer than that performed with the bacterial universal primer. Importantly, this new prokaryotic universal primer set had markedly lower bias than that of most previously designed universal primers. Our findings demonstrate that the prokaryotic universal primer set designed in the present study will permit the simultaneous detection of Bacteria and Archaea, and will therefore allow for a more comprehensive understanding of microbial community structures in environmental samples.
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影响因子：
14.9
作者：
Klindworth A;Pruesse E;Schweer T;Peplies J;Quast C;Horn M;Glöckner FO
通讯作者：
Glöckner FO
影响因子：
9.7
作者：
Eilers, Kathryn G.;Debenport, Spencer;Fierer, Noah
通讯作者：
Fierer, Noah
影响因子：
11
作者：
Bowman, Jeff S.;Rasmussen, Simon;Sicheritz-Ponten, Thomas
通讯作者：
Sicheritz-Ponten, Thomas
DOI：
10.1073/pnas.1000080107
发表时间：
2011-03-15
影响因子：
11.1
作者：
Caporaso, J. Gregory;Lauber, Christian L.;Knight, Rob
通讯作者：
Knight, Rob
通过变性梯度凝胶电泳和 16S rDNA 测序分析揭示沼气池中细菌和古菌群落的结构
影响因子：
4
作者：
Liu, F. H.;Wang, S. B.;Zhou, Z. H.
通讯作者：
Zhou, Z. H.