In silico evaluation and selection of the best 16S rRNA gene primers for use in next-generation sequencing to detect oral bacteria and archaea

Background: Sequencing has been widely used to study the composition of the oral microbiome present in various health conditions. The extent of the coverage of the 16S rRNA gene primers employed for this purpose has not, however, been evaluated in silico using oral-specific databases. This paper ana...

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Autores: regueira Iglesias, Alba, Vázquez-González, L., Balsa Castro, Carlos, Vila Blanco, Nicolás, Blanco-Pintos, T., Tamames, J., Carreira Nouche, María José, Tomás Carmona, Inmaculada
Formato: artículo
Fecha de publicación:2023
País:España
Recursos:Servizo Galego de Saúde (SERGAS)
Repositorio:RUNA. Repositorio da Consellería de Sanidade e Sergas
OAI Identifier:oai:runa.sergas.gal:20.500.11940/21621
Acesso em linha:https://portalcientifico.sergas.gal//documentos/642b3759a1c8a315fd233181
http://hdl.handle.net/20.500.11940/21621
Access Level:acceso abierto
Palavra-chave:Humans
Archaea
RNA, Ribosomal, 16S
Genes, rRNA
DNA Primers
Bacteria
Microbiota
High-Throughput Nucleotide Sequencing
Phylogeny
AS Santiago
IDIS
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oai_identifier_str oai:runa.sergas.gal:20.500.11940/21621
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network_name_str España
repository_id_str
spelling In silico evaluation and selection of the best 16S rRNA gene primers for use in next-generation sequencing to detect oral bacteria and archaearegueira Iglesias, AlbaVázquez-González, L.Balsa Castro, CarlosVila Blanco, NicolásBlanco-Pintos, T.Tamames, J.Carreira Nouche, María JoséTomás Carmona, InmaculadaHumansArchaeaRNA, Ribosomal, 16SGenes, rRNADNA PrimersBacteriaMicrobiotaHigh-Throughput Nucleotide SequencingPhylogenyAS SantiagoIDISAS SantiagoIDISAS SantiagoIDISIDISBackground: Sequencing has been widely used to study the composition of the oral microbiome present in various health conditions. The extent of the coverage of the 16S rRNA gene primers employed for this purpose has not, however, been evaluated in silico using oral-specific databases. This paper analyses these primers using two databases containing 16S rRNA sequences from bacteria and archaea found in the human mouth and describes some of the best primers for each domain. Results: A total of 369 distinct individual primers were identified from sequencing studies of the oral microbiome and other ecosystems. These were evaluated against a database reported in the literature of 16S rRNA sequences obtained from oral bacteria, which was modified by our group, and a self-created oral archaea database. Both databases contained the genomic variants detected for each included species. Primers were evaluated at the variant and species levels, and those with a species coverage (SC) ?75.00% were selected for the pair analyses. All possible combinations of the forward and reverse primers were identified, with the resulting 4638 primer pairs also evaluated using the two databases. The best bacteria-specific pairs targeted the 3-4, 4-7, and 3-7 16S rRNA gene regions, with SC levels of 98.83-97.14%; meanwhile, the optimum archaea-specific primer pairs amplified regions 5-6, 3-6, and 3-6, with SC estimates of 95.88%. Finally, the best pairs for detecting both domains targeted regions 4-5, 3-5, and 5-9, and produced SC values of 95.71-94.54% and 99.48-96.91% for bacteria and archaea, respectively. Conclusions: Given the three amplicon length categories (100-300, 301-600, and >600 base pairs), the primer pairs with the best coverage values for detecting oral bacteria were as follows: KP_F048-OP_R043 (region 3-4; primer pair position for Escherichia coli J01859.1: 342-529), KP_F051-OP_R030 (4-7; 514-1079), and KP_F048-OP_R030 (3-7; 342-1079). For detecting oral archaea, these were as follows: OP_F066-KP_R013 (5-6; 784-undefined), KP_F020-KP_R013 (3-6; 518-undefined), and OP_F114-KP_R013 (3-6; 340-undefined). Lastly, for detecting both domains jointly they were KP_F020-KP_R032 (4-5; 518-801), OP_F114-KP_R031 (3-5; 340-801), and OP_F066-OP_R121 (5-9; 784-1405). The primer pairs with the best coverage identified herein are not among those described most widely in the oral microbiome literature. [MediaObject not available: see fulltext.]This study has been funded by Instituto de Salud Carlos III (ISCIII) through the project PI21/00588 and co-funded by the European Union; Conselleria de Cultura, Educacion e Ordenacion Universitaria (accreditation 2019-2022 ED431G-2019/04, group with growth potential ED431B 2020-2022 GPC2020/27; A. Regueira-Iglesias support ED481A-2017/233) and the ERDF, which acknowledges the CiTIUS-Research Center in Intelligent Technologies of the Universidade de Santiago de Compostela as a Research Center of the Galician University System. The funders had no role in study design, data collection, and analysis, decision to publish, or preparation of the manuscript.2023info:eu-repo/semantics/articlehttps://portalcientifico.sergas.gal//documentos/642b3759a1c8a315fd233181http://hdl.handle.net/20.500.11940/21621reponame:RUNA. Repositorio da Consellería de Sanidade e Sergasinstname:Servizo Galego de Saúde (SERGAS)Ingléshttp://creativecommons.org/licenses/by/4.0/info:eu-repo/semantics/openAccessoai:runa.sergas.gal:20.500.11940/216212026-06-12T08:40:47Z
dc.title.none.fl_str_mv In silico evaluation and selection of the best 16S rRNA gene primers for use in next-generation sequencing to detect oral bacteria and archaea
title In silico evaluation and selection of the best 16S rRNA gene primers for use in next-generation sequencing to detect oral bacteria and archaea
spellingShingle In silico evaluation and selection of the best 16S rRNA gene primers for use in next-generation sequencing to detect oral bacteria and archaea
regueira Iglesias, Alba
Humans
Archaea
RNA, Ribosomal, 16S
Genes, rRNA
DNA Primers
Bacteria
Microbiota
High-Throughput Nucleotide Sequencing
Phylogeny
AS Santiago
IDIS
AS Santiago
IDIS
AS Santiago
IDIS
IDIS
title_short In silico evaluation and selection of the best 16S rRNA gene primers for use in next-generation sequencing to detect oral bacteria and archaea
title_full In silico evaluation and selection of the best 16S rRNA gene primers for use in next-generation sequencing to detect oral bacteria and archaea
title_fullStr In silico evaluation and selection of the best 16S rRNA gene primers for use in next-generation sequencing to detect oral bacteria and archaea
title_full_unstemmed In silico evaluation and selection of the best 16S rRNA gene primers for use in next-generation sequencing to detect oral bacteria and archaea
title_sort In silico evaluation and selection of the best 16S rRNA gene primers for use in next-generation sequencing to detect oral bacteria and archaea
dc.creator.none.fl_str_mv regueira Iglesias, Alba
Vázquez-González, L.
Balsa Castro, Carlos
Vila Blanco, Nicolás
Blanco-Pintos, T.
Tamames, J.
Carreira Nouche, María José
Tomás Carmona, Inmaculada
author regueira Iglesias, Alba
author_facet regueira Iglesias, Alba
Vázquez-González, L.
Balsa Castro, Carlos
Vila Blanco, Nicolás
Blanco-Pintos, T.
Tamames, J.
Carreira Nouche, María José
Tomás Carmona, Inmaculada
author_role author
author2 Vázquez-González, L.
Balsa Castro, Carlos
Vila Blanco, Nicolás
Blanco-Pintos, T.
Tamames, J.
Carreira Nouche, María José
Tomás Carmona, Inmaculada
author2_role author
author
author
author
author
author
author
dc.subject.none.fl_str_mv Humans
Archaea
RNA, Ribosomal, 16S
Genes, rRNA
DNA Primers
Bacteria
Microbiota
High-Throughput Nucleotide Sequencing
Phylogeny
AS Santiago
IDIS
AS Santiago
IDIS
AS Santiago
IDIS
IDIS
topic Humans
Archaea
RNA, Ribosomal, 16S
Genes, rRNA
DNA Primers
Bacteria
Microbiota
High-Throughput Nucleotide Sequencing
Phylogeny
AS Santiago
IDIS
AS Santiago
IDIS
AS Santiago
IDIS
IDIS
description Background: Sequencing has been widely used to study the composition of the oral microbiome present in various health conditions. The extent of the coverage of the 16S rRNA gene primers employed for this purpose has not, however, been evaluated in silico using oral-specific databases. This paper analyses these primers using two databases containing 16S rRNA sequences from bacteria and archaea found in the human mouth and describes some of the best primers for each domain. Results: A total of 369 distinct individual primers were identified from sequencing studies of the oral microbiome and other ecosystems. These were evaluated against a database reported in the literature of 16S rRNA sequences obtained from oral bacteria, which was modified by our group, and a self-created oral archaea database. Both databases contained the genomic variants detected for each included species. Primers were evaluated at the variant and species levels, and those with a species coverage (SC) ?75.00% were selected for the pair analyses. All possible combinations of the forward and reverse primers were identified, with the resulting 4638 primer pairs also evaluated using the two databases. The best bacteria-specific pairs targeted the 3-4, 4-7, and 3-7 16S rRNA gene regions, with SC levels of 98.83-97.14%; meanwhile, the optimum archaea-specific primer pairs amplified regions 5-6, 3-6, and 3-6, with SC estimates of 95.88%. Finally, the best pairs for detecting both domains targeted regions 4-5, 3-5, and 5-9, and produced SC values of 95.71-94.54% and 99.48-96.91% for bacteria and archaea, respectively. Conclusions: Given the three amplicon length categories (100-300, 301-600, and >600 base pairs), the primer pairs with the best coverage values for detecting oral bacteria were as follows: KP_F048-OP_R043 (region 3-4; primer pair position for Escherichia coli J01859.1: 342-529), KP_F051-OP_R030 (4-7; 514-1079), and KP_F048-OP_R030 (3-7; 342-1079). For detecting oral archaea, these were as follows: OP_F066-KP_R013 (5-6; 784-undefined), KP_F020-KP_R013 (3-6; 518-undefined), and OP_F114-KP_R013 (3-6; 340-undefined). Lastly, for detecting both domains jointly they were KP_F020-KP_R032 (4-5; 518-801), OP_F114-KP_R031 (3-5; 340-801), and OP_F066-OP_R121 (5-9; 784-1405). The primer pairs with the best coverage identified herein are not among those described most widely in the oral microbiome literature. [MediaObject not available: see fulltext.]
publishDate 2023
dc.date.none.fl_str_mv 2023
dc.type.none.fl_str_mv info:eu-repo/semantics/article
format article
dc.identifier.none.fl_str_mv https://portalcientifico.sergas.gal//documentos/642b3759a1c8a315fd233181
http://hdl.handle.net/20.500.11940/21621
url https://portalcientifico.sergas.gal//documentos/642b3759a1c8a315fd233181
http://hdl.handle.net/20.500.11940/21621
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.rights.none.fl_str_mv http://creativecommons.org/licenses/by/4.0/
info:eu-repo/semantics/openAccess
rights_invalid_str_mv http://creativecommons.org/licenses/by/4.0/
eu_rights_str_mv openAccess
dc.source.none.fl_str_mv reponame:RUNA. Repositorio da Consellería de Sanidade e Sergas
instname:Servizo Galego de Saúde (SERGAS)
instname_str Servizo Galego de Saúde (SERGAS)
reponame_str RUNA. Repositorio da Consellería de Sanidade e Sergas
collection RUNA. Repositorio da Consellería de Sanidade e Sergas
repository.name.fl_str_mv
repository.mail.fl_str_mv
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score 15,811543

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