Evaluation of general 16S ribosomal RNA gene PCR primers for classical an next generation sequencing based diversity analysisqu

16S ribosomal RNA gene (rDNA) analysis remains the standard approach for the cultivation independent inverstigationof microbial diversity. A amplicon-based high throughput sequencing offers a fast and cost effective way to generate massive amounts of 16S rDNA fragments. However, accurateness of such biodiversity analyses strongly depends on the choice of primers. The overall coverage and phylum spectrum of 54 primers and 95 selected pairs of primers generating fragment length <1000 bases were evaluated in silico based on the SILVA 16S rDNA reference dataset. Arch20_F/Parch519_R and Arch20_F/A519_R satisfy as archaeal primer pairs. For Bacteria, the primer pair Bakt_341_F/Bakt_805_R provide the beste balance between coverage and a large phylum spectrum. The in silico evaluation was experimentally verified by comparing the taxonomic distribution of pyrosequenced 16S rDNA amplicons of marine samples with 16S rDNA fragments from directly sequenced metagenomes as well as quantitative data from single cell fluorescent in situ hybridization (FISH) studies. Additionally, 54 pairs of primers resulting in fragments >1000 bases were evaluated in silico. This study provides validated sets of 16S rDNA targeting PCR primers to successfully reveal the complexity of archaeal and bacterial diversity using classical, clone library based methods as well as next-generation sequencing methods.