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Novel PCR Primers for the Archaeal Phylum Thaumarchaeota Designed Based on the Comparative Analysis of 16S rRNA Gene Sequences

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-chec...

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Autores principales: Hong, Jin-Kyung, Kim, Hye-Jin, Cho, Jae-Chang
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4013054/
https://www.ncbi.nlm.nih.gov/pubmed/24805255
http://dx.doi.org/10.1371/journal.pone.0096197
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author Hong, Jin-Kyung
Kim, Hye-Jin
Cho, Jae-Chang
author_facet Hong, Jin-Kyung
Kim, Hye-Jin
Cho, Jae-Chang
author_sort Hong, Jin-Kyung
collection PubMed
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.
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spelling pubmed-40130542014-05-09 Novel PCR Primers for the Archaeal Phylum Thaumarchaeota Designed Based on the Comparative Analysis of 16S rRNA Gene Sequences Hong, Jin-Kyung Kim, Hye-Jin Cho, Jae-Chang PLoS One Research Article 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. Public Library of Science 2014-05-07 /pmc/articles/PMC4013054/ /pubmed/24805255 http://dx.doi.org/10.1371/journal.pone.0096197 Text en © 2014 Hong et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Hong, Jin-Kyung
Kim, Hye-Jin
Cho, Jae-Chang
Novel PCR Primers for the Archaeal Phylum Thaumarchaeota Designed Based on the Comparative Analysis of 16S rRNA Gene Sequences
title Novel PCR Primers for the Archaeal Phylum Thaumarchaeota Designed Based on the Comparative Analysis of 16S rRNA Gene Sequences
title_full Novel PCR Primers for the Archaeal Phylum Thaumarchaeota Designed Based on the Comparative Analysis of 16S rRNA Gene Sequences
title_fullStr Novel PCR Primers for the Archaeal Phylum Thaumarchaeota Designed Based on the Comparative Analysis of 16S rRNA Gene Sequences
title_full_unstemmed Novel PCR Primers for the Archaeal Phylum Thaumarchaeota Designed Based on the Comparative Analysis of 16S rRNA Gene Sequences
title_short Novel PCR Primers for the Archaeal Phylum Thaumarchaeota Designed Based on the Comparative Analysis of 16S rRNA Gene Sequences
title_sort novel pcr primers for the archaeal phylum thaumarchaeota designed based on the comparative analysis of 16s rrna gene sequences
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4013054/
https://www.ncbi.nlm.nih.gov/pubmed/24805255
http://dx.doi.org/10.1371/journal.pone.0096197
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