Design and Experimental Application of aNovel Non-Degenerate Universal Primer Set that Amplifies Prokaryotic 16S rRNAGenes with a Low Possibility to Amplify Eukaryotic rRNA Genes

Thedeepsequencingof16SrRNAgenes amplifiedbyuniversalprimershas revolutionizedourunderstand-ing of microbial communities by allowing the characterization of the diversity of the uncultured majority. However, some universal primers also amplify eukaryotic rRNA genes, leading to a decrease in the efficiency of sequencing of prokaryotic 16S rRNAgeneswithpossiblemischaracterizationof thediversity in themicro-bial community. In this study, we compared 16S rRNA gene sequences from genome-sequenced strains and identified candidates for non-degenerate universal primers that could be used for the amplification of pro-karyotic 16S rRNA genes. The 50 identified candidates were investigated to calculate their coverage for pro-karyotic and eukaryotic rRNA genes, including those fromuncultured taxa and eukaryotic organelles, and a novel universal primer set, 342F-806R, coveringmanyprokaryotic, butnot eukaryotic, rRNAgeneswas iden-tified. This primer set was validated by the amplification of 16S rRNA genes from a soilmetagenomic sampl