PCR Primer Design for 16S rRNA Genes

To identify the candidate primer sequences for the PCR amplification of prokaryotic 16S rRNA genes, such genes from genome-sequenced strains were used as references because they have been accurately sequenced, are full-length genes, have well-defined taxonomic information. Bacterial and archaeal genomic sequences were obtained from the NCBI Genome Database (ftp://ftp.ncbi.nih.gov/genbank/genomes/Bacteria/, accessed on 11 November 2013) in November 2008. The 16S rRNA gene sequences in each strain were identified by RNAmmer.^{25 (link)} Then, one 16S rRNA gene sequence per species was randomly chosen because slight sequence differences exist among the 16S rRNA genes from strains of the same species,^{26 (link)} and among the gene copies within a genome.^{27 (link)} A total of 531 16S rRNA gene sequences were chosen. Their taxonomic information was obtained from the NCBI Taxonomy Database (http://www.ncbi.nlm.nih.gov/Taxonomy/taxonomyhome.html/, accessed on 11 November 2013). A multiple sequence alignment of the 531 16S rRNA gene sequences was constructed using MAFFT version 6.713 with default parameters.^{28 (link)}To find out the candidate sequences described above, highly conserved regions identified in the reference alignment were chosen as follows. Generally, the primer lengths for the PCR-amplification of 16S rRNA genes are more than 15 nt;^{9 (link)} therefore, we used a sliding window of 15 nt with a step size of 1 nt across the reference alignment. For each window, we calculated the frequency of each 15-nt sequence with one mismatch allowed. The 15-nt sequences that included gaps were also considered when calculating the frequencies. The consensus sequence for each window was defined as the 15-nt sequence that was found most frequently within one mismatch among strains. The coverage rate for a consensus sequence in each phylum was defined as the percentage of matched sequences among genome-sequenced strains within one mismatch.