Impact of 16S rRNA Gene Redundancy and Primer Pair Selection on the Quantification and Classification of Oral Microbiota in Next-Generation Sequencing

This study aimed to evaluate the number of 16S rRNA genes in the complete genomes of the bacterial and archaeal species inhabiting the human mouth and to assess how the use of different primer pairs would affect the detection and classification of redundant amplicons and matching amplicons (MAs) fro...

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Autores: regueira Iglesias, Alba, Vázquez-González, L., Balsa Castro, Carlos, Blanco-Pintos, T., Vila Blanco, Nicolás, Carreira Nouche, María José, Tomás Carmona, Inmaculada
Tipo de documento: artigo
Data de publicação:2023
País:España
Recursos:Servizo Galego de Saúde (SERGAS)
Repositório:RUNA. Repositorio da Consellería de Sanidade e Sergas
OAI Identifier:oai:runa.sergas.gal:20.500.11940/21601
Acesso em linha:https://portalcientifico.sergas.gal//documentos/64609f16c6d6be6c90fc607a
http://hdl.handle.net/20.500.11940/21601
Access Level:Acceso aberto
Palavra-chave:AS Santiago
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spelling Impact of 16S rRNA Gene Redundancy and Primer Pair Selection on the Quantification and Classification of Oral Microbiota in Next-Generation Sequencingregueira Iglesias, AlbaVázquez-González, L.Balsa Castro, CarlosBlanco-Pintos, T.Vila Blanco, NicolásCarreira Nouche, María JoséTomás Carmona, InmaculadaAS SantiagoIDISAS SantiagoIDISAS SantiagoIDISIDISThis study aimed to evaluate the number of 16S rRNA genes in the complete genomes of the bacterial and archaeal species inhabiting the human mouth and to assess how the use of different primer pairs would affect the detection and classification of redundant amplicons and matching amplicons (MAs) from different taxa. A total of 518 oral-bacterial and 191 oral-Archaeal complete genomes were downloaded from the NCBI database, and their complete 16S rRNA genes were extracted. The numbers of genes and variants per genome were calculated. Next, 39 primer pairs were used to search for matches in the genomes and obtain amplicons. For each primer, we calculated the number of gene amplicons, variants, genomes, and species detected and the percentage of coverage at the species level with no MAs (SC-NMA). The results showed that 94.09% of oral bacteria and 52.59% of oral archaea had more than one intragenomic 16S rRNA gene. From 1.29% to 46.70% of bacterial species and from 4.65% to 38.89% of archaea detected by the primers had MAs. The best primers were the following (SC-NMA; region; position for Escherichia coli [GenBank version no. J01859 .1]): KPF048-OPR030 for bacteria (93.55%; V3 to V7; 342 to 1079), KPF018-KPR063 for archaea (89.63%; V3 to V9; undefined to 1506), and OPF114-OPR121 for both domains (92.52%; V3 to V9; 340 to 1405). In addition to 16S rRNA gene redundancy, the presence of MAs must be controlled to ensure an accurate interpretation of microbial diversity data. The SC-NMA is a more useful parameter than the conventional coverage percentage for selecting the best primer pairs. The pairs used the most in the oral microbiome literature were not among the best performers. IMPORTANCE Hundreds of publications have studied the oral microbiome through 16S rRNA gene sequencing. However, none have assessed the number of 16S rRNA genes in the genomes of oral microbes, or how the use of primer pairs targeting different regions affects the detection of MAs from different taxa. Here, we found that almost all oral bacteria and more than half of oral archaea have more than one intragenomic 16S rRNA gene. The performance of the primer pairs in not detecting MAs increases as the length of the amplicon augments. As none of those most employed in the oral literature were among the best performers, we selected a series of primers to detect bacteria and/or archaea based on their percentage of species detected without MAs. The intragenomic 16S rRNA gene redundancy and the presence of MAs between distinct taxa need to be considered to ensure an accurate interpretation of microbial diversity data.This study was funded by Instituto de Salud Carlos III (ISCIII) through project PI21/00588 and cofunded by the European Union, the Conselleria de Cultura, Educacion e Ordenacion Universitaria de la Xunta de Galicia (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 Santiago de Compostela University as a Research Center of the Galician University System.2023info:eu-repo/semantics/articlehttps://portalcientifico.sergas.gal//documentos/64609f16c6d6be6c90fc607ahttp://hdl.handle.net/20.500.11940/21601reponame: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:20.500.11940/216012026-06-12T08:40:47Z
dc.title.none.fl_str_mv Impact of 16S rRNA Gene Redundancy and Primer Pair Selection on the Quantification and Classification of Oral Microbiota in Next-Generation Sequencing
title Impact of 16S rRNA Gene Redundancy and Primer Pair Selection on the Quantification and Classification of Oral Microbiota in Next-Generation Sequencing
spellingShingle Impact of 16S rRNA Gene Redundancy and Primer Pair Selection on the Quantification and Classification of Oral Microbiota in Next-Generation Sequencing
regueira Iglesias, Alba
AS Santiago
IDIS
AS Santiago
IDIS
AS Santiago
IDIS
IDIS
title_short Impact of 16S rRNA Gene Redundancy and Primer Pair Selection on the Quantification and Classification of Oral Microbiota in Next-Generation Sequencing
title_full Impact of 16S rRNA Gene Redundancy and Primer Pair Selection on the Quantification and Classification of Oral Microbiota in Next-Generation Sequencing
title_fullStr Impact of 16S rRNA Gene Redundancy and Primer Pair Selection on the Quantification and Classification of Oral Microbiota in Next-Generation Sequencing
title_full_unstemmed Impact of 16S rRNA Gene Redundancy and Primer Pair Selection on the Quantification and Classification of Oral Microbiota in Next-Generation Sequencing
title_sort Impact of 16S rRNA Gene Redundancy and Primer Pair Selection on the Quantification and Classification of Oral Microbiota in Next-Generation Sequencing
dc.creator.none.fl_str_mv regueira Iglesias, Alba
Vázquez-González, L.
Balsa Castro, Carlos
Blanco-Pintos, T.
Vila Blanco, Nicolás
Carreira Nouche, María José
Tomás Carmona, Inmaculada
author regueira Iglesias, Alba
author_facet regueira Iglesias, Alba
Vázquez-González, L.
Balsa Castro, Carlos
Blanco-Pintos, T.
Vila Blanco, Nicolás
Carreira Nouche, María José
Tomás Carmona, Inmaculada
author_role author
author2 Vázquez-González, L.
Balsa Castro, Carlos
Blanco-Pintos, T.
Vila Blanco, Nicolás
Carreira Nouche, María José
Tomás Carmona, Inmaculada
author2_role author
author
author
author
author
author
dc.subject.none.fl_str_mv AS Santiago
IDIS
AS Santiago
IDIS
AS Santiago
IDIS
IDIS
topic AS Santiago
IDIS
AS Santiago
IDIS
AS Santiago
IDIS
IDIS
description This study aimed to evaluate the number of 16S rRNA genes in the complete genomes of the bacterial and archaeal species inhabiting the human mouth and to assess how the use of different primer pairs would affect the detection and classification of redundant amplicons and matching amplicons (MAs) from different taxa. A total of 518 oral-bacterial and 191 oral-Archaeal complete genomes were downloaded from the NCBI database, and their complete 16S rRNA genes were extracted. The numbers of genes and variants per genome were calculated. Next, 39 primer pairs were used to search for matches in the genomes and obtain amplicons. For each primer, we calculated the number of gene amplicons, variants, genomes, and species detected and the percentage of coverage at the species level with no MAs (SC-NMA). The results showed that 94.09% of oral bacteria and 52.59% of oral archaea had more than one intragenomic 16S rRNA gene. From 1.29% to 46.70% of bacterial species and from 4.65% to 38.89% of archaea detected by the primers had MAs. The best primers were the following (SC-NMA; region; position for Escherichia coli [GenBank version no. J01859 .1]): KPF048-OPR030 for bacteria (93.55%; V3 to V7; 342 to 1079), KPF018-KPR063 for archaea (89.63%; V3 to V9; undefined to 1506), and OPF114-OPR121 for both domains (92.52%; V3 to V9; 340 to 1405). In addition to 16S rRNA gene redundancy, the presence of MAs must be controlled to ensure an accurate interpretation of microbial diversity data. The SC-NMA is a more useful parameter than the conventional coverage percentage for selecting the best primer pairs. The pairs used the most in the oral microbiome literature were not among the best performers. IMPORTANCE Hundreds of publications have studied the oral microbiome through 16S rRNA gene sequencing. However, none have assessed the number of 16S rRNA genes in the genomes of oral microbes, or how the use of primer pairs targeting different regions affects the detection of MAs from different taxa. Here, we found that almost all oral bacteria and more than half of oral archaea have more than one intragenomic 16S rRNA gene. The performance of the primer pairs in not detecting MAs increases as the length of the amplicon augments. As none of those most employed in the oral literature were among the best performers, we selected a series of primers to detect bacteria and/or archaea based on their percentage of species detected without MAs. The intragenomic 16S rRNA gene redundancy and the presence of MAs between distinct taxa need to be considered to ensure an accurate interpretation of microbial diversity data.
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/64609f16c6d6be6c90fc607a
http://hdl.handle.net/20.500.11940/21601
url https://portalcientifico.sergas.gal//documentos/64609f16c6d6be6c90fc607a
http://hdl.handle.net/20.500.11940/21601
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
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