Abstract
BackgroundAmplification 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.MethodsPrimer 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.ResultsUsing this method, we identified unclassified SSU rRNA sequences that were not matched with universal primer 8F and Arch21F. A new group within the Asgard superphylum was amplified by the newly designed specific primer based on these unclassified SSU rRNA sequences by using mudflat samples.ConclusionWe showed that using specific primers designed based on universal primer evaluation from meta-transcriptomic datasets, identification of novel taxonomic groups from a specific environment is possible.
Highlights
Amplification of small subunit (SSU) ribosomal RNA (rRNA) genes with universal primers is a common method used to assess microbial populations in various environmental samples
To identify “rare biosphere” microbial dark matter from meta-transcriptomic datasets, we developed a method for designing new primers based on SSU rRNA datasets by using a modified meta-transcriptomic method [26] and amplified nearly full-length 16S rRNA genes of novel taxa by using these newly designed forward primers and universal reverse primer (Fig. 1)
Screening of SSU rRNA and primer evaluation From the meta-transcriptomic data obtained from mudflat sediments, 833,297 SSU rRNA sequences were detected using SSUsearch, accounting for 51.0% of the total metatranscriptomic sequences
Summary
Amplification of small subunit (SSU) rRNA genes with universal primers is a common method used to assess microbial populations in various environmental samples. Owing to limitations in coverage of these universal primers, some microorganisms remain unidentified. Through deep metagenome sequencing of ultra-small microbes, Anantharaman et al [14] reported 47 novel candidate phyla, of which 46 were not identified by 16S rRNA gene sequencing with universal primers. With the application of single cell genome and metagenome sequencing and assembly, the number of phyla in Archaea domain has increased to more than twenty, and these phyla clustered to Euryarchaota and three superphyla—TACK, DPANN, and Asgard [15]. According to the assembled genomes from metagenome sequencing, the Asgard superphylum embraced six phyla: Lokiarchaeota, Thorarchaeota, Heimdallarchaeota, Odinarchaeota, Helarchaeota, and Gerdarchaeota [16,17,18,19]. A Lokiarchaeota-related strain was firstly isolated [25]
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