Dataset Information

Design of targeted primers based on 16S rRNA sequences in meta-transcriptomic datasets and identification of a novel taxonomic group in the Asgard archaea.

ABSTRACT: BACKGROUND: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. METHODS: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. RESULTS:Using 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. CONCLUSION:We 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.

SUBMITTER: Zhang RY

PROVIDER: S-EPMC6998087 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Design of targeted primers based on 16S rRNA sequences in meta-transcriptomic datasets and identification of a novel taxonomic group in the Asgard archaea.

Zhang Ru-Yi RY Zou Bin B Yan Yong-Wei YW Jeon Che Ok CO Li Meng M Cai Mingwei M Quan Zhe-Xue ZX

BMC microbiology 20200203 1

<h4>Background</h4>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 me ...[more]

PMID: 32013868

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Project description:Based on comparative phylogenetic analysis of 16S rRNA gene sequences deposited in an RDP database, we constructed a local database of thaumarchaeotal 16S rRNA gene sequences and developed a novel PCR primer specific for the archaeal phylum Thaumarchaeota. Among 9,727 quality-filtered (chimeral-checked, size >1.2 kb) archaeal sequences downloaded from the RDP database, 1,549 thaumarchaeotal sequences were identified and included in our local database. In our study, Thaumarchaeota included archaeal groups MG-I, SAGMCG-I, SCG, FSCG, RC, and HWCG-III, forming a monophyletic group in the phylogenetic tree. Cluster analysis revealed 114 phylotypes for Thaumarchaeota. The majority of the phylotypes (66.7%) belonged to the MG-I and SCG, which together contained most (93.9%) of the thaumarchaeotal sequences in our local database. A phylum-directed primer was designed from a consensus sequence of the phylotype sequences, and the primer's specificity was evaluated for coverage and tolerance both in silico and empirically. The phylum-directed primer, designated THAUM-494, showed >90% coverage for Thaumarchaeota and <1% tolerance to non-target taxa, indicating high specificity. To validate this result experimentally, PCRs were performed with THAUM-494 in combination with a universal archaeal primer (ARC917R or 1017FAR) and DNAs from five environmental samples to construct clone libraries. THAUM-494 showed a satisfactory specificity in empirical studies, as expected from the in silico results. Phylogenetic analysis of 859 cloned sequences obtained from 10 clone libraries revealed that >95% of the amplified sequences belonged to Thaumarchaeota. The most frequently sampled thaumarchaeotal subgroups in our samples were SCG, MG-I, and SAGMCG-I. To our knowledge, THAUM-494 is the first phylum-level primer for Thaumarchaeota. Furthermore, the high coverage and low tolerance of THAUM-494 will make it a potentially valuable tool in understanding the phylogenetic diversity and ecological niche of Thaumarchaeota.

Project description:16S rRNA gene sequences are commonly analyzed for taxonomic and phylogenetic studies because they contain variable regions that can help distinguish different genera. However, intra-genus distinction using variable region homology is often impossible due to the high overall sequence identities among closely related species, even though some residues may be conserved within respective species. Using a computational method that included the allelic diversity within individual genomes, we discovered that certain Escherichia and Shigella species can be distinguished by a multi-allelic 16S rRNA variable region single nucleotide polymorphism (SNP). To evaluate the performance of 16S rRNAs with altered variable regions, we developed an in vivo system that measures the acceptance and distribution of variant 16S rRNAs into a large pool of natural versions supporting normal translation and growth. We found that 16S rRNAs containing evolutionarily disparate variable regions were underpopulated both in ribosomes and in active translation pools, even for an SNP. Overall, this study revealed that variable region sequences can substantially influence the performance of 16S rRNAs and that this biological constraint can be leveraged to justify refining taxonomic assignments of variable region sequence data. IMPORTANCE This study reevaluates the notion that 16S rRNA gene variable region sequences are uninformative for intra-genus classification and that single nucleotide variations within them have no consequence to strains that bear them. We demonstrated that the performance of 16S rRNAs in Escherichia coli can be negatively impacted by sequence changes in variable regions, even for single nucleotide changes that are native to closely related Escherichia and Shigella species; thus, biological performance is likely constraining the evolution of variable regions in bacteria. Further, the native nucleotide variations we tested occur in all strains of their respective species and across their multiple 16S rRNA gene copies, suggesting that these species evolved beyond what would be discerned from a consensus sequence comparison. Therefore, this work also reveals that the multiple 16S rRNA gene alleles found in most bacteria can provide more informative phylogenetic and taxonomic detail than a single reference allele.

Dataset Information

Design of targeted primers based on 16S rRNA sequences in meta-transcriptomic datasets and identification of a novel taxonomic group in the Asgard archaea.

Publications

Design of targeted primers based on 16S rRNA sequences in meta-transcriptomic datasets and identification of a novel taxonomic group in the Asgard archaea.

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