Nonribosomal Peptide Synthetase Specific Genome Amplification Using Rolling Circle Amplification for Targeted Gene Sequencing.

Next-generation sequencing has transformed the acquisition of vast amounts of genomic information, including the rapid identification of target gene sequences in metagenomic databases. However, dominant species can sometimes hinder the detection of rare bacterial species. Therefore, a highly sensitive amplification technique that can selectively amplify bacterial genomes containing target genes of interest was developed in this study. The rolling circle amplification (RCA) method can initiate amplification from a single locus using a specific single primer to amplify a specific whole genome. A mixed cell suspension was prepared using ATCC17400 (targeting nonribosomal peptide synthetase [NRPS]) and (non-target), and a specific primer designed for the was used for the RCA reaction. The resulting RCA product (RCP) amplified only the genome. The was successfully amplified using RCP as a template from even five cells, indicating that the single-priming RCA technique can specifically enrich the target genome using gene-specific primers. Ultimately, this specific genome RCA technique was applied to metagenomes extracted from sponge-associated bacteria, and sequences were successfully obtained from an unknown sponge-associated bacterium. Therefore, this method could be effective for accessing species-specific sequences of in unknown bacteria, including viable but non-culturable bacteria.