Establishment and assessment of an amplicon sequencing method targeting the 16S-ITS-23S rRNA operon for analysis of the equine gut microbiome

Abstract Microbial communities are commonly studied by using amplicon sequencing of part of the 16S rRNA gene. Sequencing of the full-length 16S rRNA gene can provide higher taxonomic resolution and accuracy. To obtain even higher taxonomic resolution, with as few false-positives as possible, we ass...

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Autores principales:	Yuta Kinoshita, Hidekazu Niwa, Eri Uchida-Fujii, Toshio Nukada
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/8ec6fb58f9d04f3c9e092a705acc5a44
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spelling	oai:doaj.org-article:8ec6fb58f9d04f3c9e092a705acc5a442021-12-02T17:51:29ZEstablishment and assessment of an amplicon sequencing method targeting the 16S-ITS-23S rRNA operon for analysis of the equine gut microbiome10.1038/s41598-021-91425-72045-2322https://doaj.org/article/8ec6fb58f9d04f3c9e092a705acc5a442021-06-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-91425-7https://doaj.org/toc/2045-2322Abstract Microbial communities are commonly studied by using amplicon sequencing of part of the 16S rRNA gene. Sequencing of the full-length 16S rRNA gene can provide higher taxonomic resolution and accuracy. To obtain even higher taxonomic resolution, with as few false-positives as possible, we assessed a method using long amplicon sequencing targeting the rRNA operon combined with a CCMetagen pipeline. Taxonomic assignment had > 90% accuracy at the species level in a mock sample and at the family level in equine fecal samples, generating similar taxonomic composition as shotgun sequencing. The rRNA operon amplicon sequencing of equine fecal samples underestimated compositional percentages of bacterial strains containing unlinked rRNA genes by a fourth to a third, but unlinked rRNA genes had a limited effect on the overall results. The rRNA operon amplicon sequencing with the A519F + U2428R primer set was able to detect some kind of archaeal genomes such as Methanobacteriales and Methanomicrobiales, whereas full-length 16S rRNA with 27F + 1492R could not. Therefore, we conclude that amplicon sequencing targeting the rRNA operon captures more detailed variations of equine microbiota.Yuta KinoshitaHidekazu NiwaEri Uchida-FujiiToshio NukadaNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-12 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Yuta Kinoshita Hidekazu Niwa Eri Uchida-Fujii Toshio Nukada Establishment and assessment of an amplicon sequencing method targeting the 16S-ITS-23S rRNA operon for analysis of the equine gut microbiome
description	Abstract Microbial communities are commonly studied by using amplicon sequencing of part of the 16S rRNA gene. Sequencing of the full-length 16S rRNA gene can provide higher taxonomic resolution and accuracy. To obtain even higher taxonomic resolution, with as few false-positives as possible, we assessed a method using long amplicon sequencing targeting the rRNA operon combined with a CCMetagen pipeline. Taxonomic assignment had > 90% accuracy at the species level in a mock sample and at the family level in equine fecal samples, generating similar taxonomic composition as shotgun sequencing. The rRNA operon amplicon sequencing of equine fecal samples underestimated compositional percentages of bacterial strains containing unlinked rRNA genes by a fourth to a third, but unlinked rRNA genes had a limited effect on the overall results. The rRNA operon amplicon sequencing with the A519F + U2428R primer set was able to detect some kind of archaeal genomes such as Methanobacteriales and Methanomicrobiales, whereas full-length 16S rRNA with 27F + 1492R could not. Therefore, we conclude that amplicon sequencing targeting the rRNA operon captures more detailed variations of equine microbiota.
format	article
author	Yuta Kinoshita Hidekazu Niwa Eri Uchida-Fujii Toshio Nukada
author_facet	Yuta Kinoshita Hidekazu Niwa Eri Uchida-Fujii Toshio Nukada
author_sort	Yuta Kinoshita
title	Establishment and assessment of an amplicon sequencing method targeting the 16S-ITS-23S rRNA operon for analysis of the equine gut microbiome
title_short	Establishment and assessment of an amplicon sequencing method targeting the 16S-ITS-23S rRNA operon for analysis of the equine gut microbiome
title_full	Establishment and assessment of an amplicon sequencing method targeting the 16S-ITS-23S rRNA operon for analysis of the equine gut microbiome
title_fullStr	Establishment and assessment of an amplicon sequencing method targeting the 16S-ITS-23S rRNA operon for analysis of the equine gut microbiome
title_full_unstemmed	Establishment and assessment of an amplicon sequencing method targeting the 16S-ITS-23S rRNA operon for analysis of the equine gut microbiome
title_sort	establishment and assessment of an amplicon sequencing method targeting the 16s-its-23s rrna operon for analysis of the equine gut microbiome
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/8ec6fb58f9d04f3c9e092a705acc5a44
work_keys_str_mv	AT yutakinoshita establishmentandassessmentofanampliconsequencingmethodtargetingthe16sits23srrnaoperonforanalysisoftheequinegutmicrobiome AT hidekazuniwa establishmentandassessmentofanampliconsequencingmethodtargetingthe16sits23srrnaoperonforanalysisoftheequinegutmicrobiome AT eriuchidafujii establishmentandassessmentofanampliconsequencingmethodtargetingthe16sits23srrnaoperonforanalysisoftheequinegutmicrobiome AT toshionukada establishmentandassessmentofanampliconsequencingmethodtargetingthe16sits23srrnaoperonforanalysisoftheequinegutmicrobiome
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Establishment and assessment of an amplicon sequencing method targeting the 16S-ITS-23S rRNA operon for analysis of the equine gut microbiome

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