Amplification of small subunit (SSU) rRNA genes with universal primers is a common method used to assess microbial populations in various environmental samples. However, owing to limitations in coverage of these universal primers, some microorganisms remain unidentified. The present study aimed to establish a method for amplifying nearly full-length SSU rRNA gene sequences of previously unidentified prokaryotes, using newly designed targeted primers via primer evaluation in meta-transcriptomic datasets.
Primer binding regions of universal primer 8F/Arch21F for bacteria or archaea were used for primer evaluation of SSU rRNA sequences in meta-transcriptomic datasets. Furthermore, targeted forward primers were designed based on SSU rRNA reads from unclassified groups unmatched with the universal primer 8F/Arch21F, and these primers were used to amplify nearly full-length special SSU rRNA gene sequences along with universal reverse primer 1492R. Similarity and phylogenetic analysis were used to confirm their novel status.