Max Planck Institute for Marine Microbiology, Microbial Genomics and Bioinformatics Research Group, Celsiusstr 1, 28359 Bremen, Germany.
Nucleic Acids Res. 2013 Jan 7;41(1):e1. doi: 10.1093/nar/gks808. Epub 2012 Aug 28.
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.
16S 核糖体 RNA 基因（rDNA）扩增子分析仍然是微生物多样性非培养研究的标准方法。这些分析的准确性强烈依赖于引物的选择。使用 SILVA 16S/18S rDNA 非冗余参考数据集（SSURef 108 NR），对 175 个引物和 512 对引物的总体覆盖度和门谱进行了计算机评估。在此评估的基础上，为三个扩增子大小类别（100-400、400-1000 和≥1000 bp）提供了用于细菌和古菌的“最佳可用”引物对的选择。最有前途的细菌引物对（S-D-Bact-0341-b-S-17/S-D-Bact-0785-a-A-21），扩增子大小为 464 bp，通过将 16S rDNA 扩增子的分类分布与直接测序的宏基因组中 16S rDNA 片段进行比较，对其进行了实验评估。本研究的结果可作为选择具有最佳总体覆盖度和门谱的引物对特定应用的指南，从而减少基于 PCR 的微生物多样性研究中的偏倚。
