Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies.
Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies.
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DOI：
10.1093/nar/gks808
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发表时间：
2013-01-07
影响因子：
14.9
通讯作者：
Glöckner FO
中科院分区：
文献类型：
--
作者：
Klindworth A;Pruesse E;Schweer T;Peplies J;Quast C;Horn M;Glöckner FO
16S核糖体RNA基因(RDNA)扩增分析仍然是独立于培养的微生物多样性研究的标准方法。这些分析的准确性在很大程度上取决于引物的选择。根据Silva 16S/18S rDNA非冗余参考数据集(SSURef 108 NR)，在电子计算机上评估了175个引物和512个引物对的总覆盖率和类群光谱。在此评价的基础上，为细菌和古生菌提供了三个扩增片段大小类别(100-400,400-1000，≥1000bp)的最佳可用引物对。通过比较16S rDNA扩增片段的分类分布和直接测序后的16S rDNA片段，对最有希望的细菌引物对(S-D-BA-0341-b-S-17/S-D-BA-0785-a-A-21)进行了实验评价。这项研究的结果可以作为选择具有最佳总覆盖率和类群光谱的特定应用的引物对的指导方针，从而减少基于PCR的微生物多样性研究中的偏差。
16S ribosomal RNA gene (rDNA) amplicon analysis remains the standard approach for the cultivation-independent investigation of microbial diversity. The accuracy of these analyses depends strongly on the choice of primers. The overall coverage and phylum spectrum of 175 primers and 512 primer pairs were evaluated in silico with respect to the SILVA 16S/18S rDNA non-redundant reference dataset (SSURef 108 NR). Based on this evaluation a selection of ‘best available’ primer pairs for Bacteria and Archaea for three amplicon size classes (100–400, 400–1000, ≥1000 bp) is provided. The most promising bacterial primer pair (S-D-Bact-0341-b-S-17/S-D-Bact-0785-a-A-21), with an amplicon size of 464 bp, was experimentally evaluated by comparing the taxonomic distribution of the 16S rDNA amplicons with 16S rDNA fragments from directly sequenced metagenomes. The results of this study may be used as a guideline for selecting primer pairs with the best overall coverage and phylum spectrum for specific applications, therefore reducing the bias in PCR-based microbial diversity studies.