Improved Bacterial 16S rRNA Gene (V4 and V4-5) and Fungal Internal Transcribed Spacer Marker Gene Primers for Microbial Community Surveys

ABSTRACT Designing primers for PCR-based taxonomic surveys that amplify a broad range of phylotypes in varied community samples is a difficult challenge, and the comparability of data sets amplified with varied primers requires attention. Here, we examined the performance of modified 16S rRNA gene a...

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Autores principales:	William Walters, Embriette R. Hyde, Donna Berg-Lyons, Gail Ackermann, Greg Humphrey, Alma Parada, Jack A. Gilbert, Janet K. Jansson, J. Gregory Caporaso, Jed A. Fuhrman, Amy Apprill, Rob Knight
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2016
Materias:	microbial ecology marker genes primers 16S ITS Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/45bcc9711b4a4111b1ae30133e5925a4
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spelling	oai:doaj.org-article:45bcc9711b4a4111b1ae30133e5925a42021-12-02T19:45:29ZImproved Bacterial 16S rRNA Gene (V4 and V4-5) and Fungal Internal Transcribed Spacer Marker Gene Primers for Microbial Community Surveys10.1128/mSystems.00009-152379-5077https://doaj.org/article/45bcc9711b4a4111b1ae30133e5925a42016-02-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSystems.00009-15https://doaj.org/toc/2379-5077ABSTRACT Designing primers for PCR-based taxonomic surveys that amplify a broad range of phylotypes in varied community samples is a difficult challenge, and the comparability of data sets amplified with varied primers requires attention. Here, we examined the performance of modified 16S rRNA gene and internal transcribed spacer (ITS) primers for archaea/bacteria and fungi, respectively, with nonaquatic samples. We moved primer bar codes to the 5′ end, allowing for a range of different 3′ primer pairings, such as the 515f/926r primer pair, which amplifies variable regions 4 and 5 of the 16S rRNA gene. We additionally demonstrated that modifications to the 515f/806r (variable region 4) 16S primer pair, which improves detection of Thaumarchaeota and clade SAR11 in marine samples, do not degrade performance on taxa already amplified effectively by the original primer set. Alterations to the fungal ITS primers did result in differential but overall improved performance compared to the original primers. In both cases, the improved primers should be widely adopted for amplicon studies. IMPORTANCE We continue to uncover a wealth of information connecting microbes in important ways to human and environmental ecology. As our scientific knowledge and technical abilities improve, the tools used for microbiome surveys can be modified to improve the accuracy of our techniques, ensuring that we can continue to identify groundbreaking connections between microbes and the ecosystems they populate, from ice caps to the human body. It is important to confirm that modifications to these tools do not cause new, detrimental biases that would inhibit the field rather than continue to move it forward. We therefore demonstrated that two recently modified primer pairs that target taxonomically discriminatory regions of bacterial and fungal genomic DNA do not introduce new biases when used on a variety of sample types, from soil to human skin. This confirms the utility of these primers for maintaining currently recommended microbiome research techniques as the state of the art.William WaltersEmbriette R. HydeDonna Berg-LyonsGail AckermannGreg HumphreyAlma ParadaJack A. GilbertJanet K. JanssonJ. Gregory CaporasoJed A. FuhrmanAmy ApprillRob KnightAmerican Society for Microbiologyarticlemicrobial ecologymarker genesprimers16SITSMicrobiologyQR1-502ENmSystems, Vol 1, Iss 1 (2016)
institution	DOAJ
collection	DOAJ
language	EN
topic	microbial ecology marker genes primers 16S ITS Microbiology QR1-502
spellingShingle	microbial ecology marker genes primers 16S ITS Microbiology QR1-502 William Walters Embriette R. Hyde Donna Berg-Lyons Gail Ackermann Greg Humphrey Alma Parada Jack A. Gilbert Janet K. Jansson J. Gregory Caporaso Jed A. Fuhrman Amy Apprill Rob Knight Improved Bacterial 16S rRNA Gene (V4 and V4-5) and Fungal Internal Transcribed Spacer Marker Gene Primers for Microbial Community Surveys
description	ABSTRACT Designing primers for PCR-based taxonomic surveys that amplify a broad range of phylotypes in varied community samples is a difficult challenge, and the comparability of data sets amplified with varied primers requires attention. Here, we examined the performance of modified 16S rRNA gene and internal transcribed spacer (ITS) primers for archaea/bacteria and fungi, respectively, with nonaquatic samples. We moved primer bar codes to the 5′ end, allowing for a range of different 3′ primer pairings, such as the 515f/926r primer pair, which amplifies variable regions 4 and 5 of the 16S rRNA gene. We additionally demonstrated that modifications to the 515f/806r (variable region 4) 16S primer pair, which improves detection of Thaumarchaeota and clade SAR11 in marine samples, do not degrade performance on taxa already amplified effectively by the original primer set. Alterations to the fungal ITS primers did result in differential but overall improved performance compared to the original primers. In both cases, the improved primers should be widely adopted for amplicon studies. IMPORTANCE We continue to uncover a wealth of information connecting microbes in important ways to human and environmental ecology. As our scientific knowledge and technical abilities improve, the tools used for microbiome surveys can be modified to improve the accuracy of our techniques, ensuring that we can continue to identify groundbreaking connections between microbes and the ecosystems they populate, from ice caps to the human body. It is important to confirm that modifications to these tools do not cause new, detrimental biases that would inhibit the field rather than continue to move it forward. We therefore demonstrated that two recently modified primer pairs that target taxonomically discriminatory regions of bacterial and fungal genomic DNA do not introduce new biases when used on a variety of sample types, from soil to human skin. This confirms the utility of these primers for maintaining currently recommended microbiome research techniques as the state of the art.
format	article
author	William Walters Embriette R. Hyde Donna Berg-Lyons Gail Ackermann Greg Humphrey Alma Parada Jack A. Gilbert Janet K. Jansson J. Gregory Caporaso Jed A. Fuhrman Amy Apprill Rob Knight
author_facet	William Walters Embriette R. Hyde Donna Berg-Lyons Gail Ackermann Greg Humphrey Alma Parada Jack A. Gilbert Janet K. Jansson J. Gregory Caporaso Jed A. Fuhrman Amy Apprill Rob Knight
author_sort	William Walters
title	Improved Bacterial 16S rRNA Gene (V4 and V4-5) and Fungal Internal Transcribed Spacer Marker Gene Primers for Microbial Community Surveys
title_short	Improved Bacterial 16S rRNA Gene (V4 and V4-5) and Fungal Internal Transcribed Spacer Marker Gene Primers for Microbial Community Surveys
title_full	Improved Bacterial 16S rRNA Gene (V4 and V4-5) and Fungal Internal Transcribed Spacer Marker Gene Primers for Microbial Community Surveys
title_fullStr	Improved Bacterial 16S rRNA Gene (V4 and V4-5) and Fungal Internal Transcribed Spacer Marker Gene Primers for Microbial Community Surveys
title_full_unstemmed	Improved Bacterial 16S rRNA Gene (V4 and V4-5) and Fungal Internal Transcribed Spacer Marker Gene Primers for Microbial Community Surveys
title_sort	improved bacterial 16s rrna gene (v4 and v4-5) and fungal internal transcribed spacer marker gene primers for microbial community surveys
publisher	American Society for Microbiology
publishDate	2016
url	https://doaj.org/article/45bcc9711b4a4111b1ae30133e5925a4
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Improved Bacterial 16S rRNA Gene (V4 and V4-5) and Fungal Internal Transcribed Spacer Marker Gene Primers for Microbial Community Surveys

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