Highly Reproducible 16S Sequencing Facilitates Measurement of Host Genetic Influences on the Stickleback Gut Microbiome

ABSTRACT Multicellular organisms interact with resident microbes in important ways, and a better understanding of host-microbe interactions is aided by tools such as high-throughput 16S sequencing. However, rigorous evaluation of the veracity of these tools in a different context from which they wer...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	Clayton M. Small, Mark Currey, Emily A. Beck, Susan Bassham, William A. Cresko
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2019
Materias:	DNA isolation fish model host-microbe systems microbial ecology repeatability reproducibility Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/eadd0f761733448dbbfddb2ae162890c
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:eadd0f761733448dbbfddb2ae162890c
record_format	dspace
spelling	oai:doaj.org-article:eadd0f761733448dbbfddb2ae162890c2021-12-02T19:47:34ZHighly Reproducible 16S Sequencing Facilitates Measurement of Host Genetic Influences on the Stickleback Gut Microbiome10.1128/mSystems.00331-192379-5077https://doaj.org/article/eadd0f761733448dbbfddb2ae162890c2019-08-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSystems.00331-19https://doaj.org/toc/2379-5077ABSTRACT Multicellular organisms interact with resident microbes in important ways, and a better understanding of host-microbe interactions is aided by tools such as high-throughput 16S sequencing. However, rigorous evaluation of the veracity of these tools in a different context from which they were developed has often lagged behind. Our goal was to perform one such critical test by examining how variation in tissue preparation and DNA isolation could affect inferences about gut microbiome variation between two genetically divergent lines of threespine stickleback fish maintained in the same laboratory environment. Using careful experimental design and intensive sampling of individuals, we addressed technical and biological sources of variation in 16S-based estimates of microbial diversity. After employing a two-tiered bead beating approach that comprised tissue homogenization followed by microbial lysis in subsamples, we found an extremely minor effect of DNA isolation protocol relative to among-host microbial diversity differences. Abundance estimates for rare operational taxonomic units (OTUs), however, showed much lower reproducibility. Gut microbiome composition was highly variable across fish—even among cohoused siblings—relative to technical replicates, but a subtle effect of host genotype (stickleback line) was nevertheless detected for some microbial taxa. IMPORTANCE Our findings demonstrate the importance of appropriately quantifying biological and technical variance components when attempting to understand major influences on high-throughput microbiome data. Our focus was on understanding among-host (biological) variance in community metrics and its magnitude in relation to within-host (technical) variance, because meaningful comparisons among individuals are necessary in addressing major questions in host-microbe ecology and evolution, such as heritability of the microbiome. Our study design and insights should provide a useful example for others desiring to quantify microbiome variation at biological levels in the face of various technical factors in a variety of systems.Clayton M. SmallMark CurreyEmily A. BeckSusan BasshamWilliam A. CreskoAmerican Society for MicrobiologyarticleDNA isolationfish modelhost-microbe systemsmicrobial ecologyrepeatabilityreproducibilityMicrobiologyQR1-502ENmSystems, Vol 4, Iss 4 (2019)
institution	DOAJ
collection	DOAJ
language	EN
topic	DNA isolation fish model host-microbe systems microbial ecology repeatability reproducibility Microbiology QR1-502
spellingShingle	DNA isolation fish model host-microbe systems microbial ecology repeatability reproducibility Microbiology QR1-502 Clayton M. Small Mark Currey Emily A. Beck Susan Bassham William A. Cresko Highly Reproducible 16S Sequencing Facilitates Measurement of Host Genetic Influences on the Stickleback Gut Microbiome
description	ABSTRACT Multicellular organisms interact with resident microbes in important ways, and a better understanding of host-microbe interactions is aided by tools such as high-throughput 16S sequencing. However, rigorous evaluation of the veracity of these tools in a different context from which they were developed has often lagged behind. Our goal was to perform one such critical test by examining how variation in tissue preparation and DNA isolation could affect inferences about gut microbiome variation between two genetically divergent lines of threespine stickleback fish maintained in the same laboratory environment. Using careful experimental design and intensive sampling of individuals, we addressed technical and biological sources of variation in 16S-based estimates of microbial diversity. After employing a two-tiered bead beating approach that comprised tissue homogenization followed by microbial lysis in subsamples, we found an extremely minor effect of DNA isolation protocol relative to among-host microbial diversity differences. Abundance estimates for rare operational taxonomic units (OTUs), however, showed much lower reproducibility. Gut microbiome composition was highly variable across fish—even among cohoused siblings—relative to technical replicates, but a subtle effect of host genotype (stickleback line) was nevertheless detected for some microbial taxa. IMPORTANCE Our findings demonstrate the importance of appropriately quantifying biological and technical variance components when attempting to understand major influences on high-throughput microbiome data. Our focus was on understanding among-host (biological) variance in community metrics and its magnitude in relation to within-host (technical) variance, because meaningful comparisons among individuals are necessary in addressing major questions in host-microbe ecology and evolution, such as heritability of the microbiome. Our study design and insights should provide a useful example for others desiring to quantify microbiome variation at biological levels in the face of various technical factors in a variety of systems.
format	article
author	Clayton M. Small Mark Currey Emily A. Beck Susan Bassham William A. Cresko
author_facet	Clayton M. Small Mark Currey Emily A. Beck Susan Bassham William A. Cresko
author_sort	Clayton M. Small
title	Highly Reproducible 16S Sequencing Facilitates Measurement of Host Genetic Influences on the Stickleback Gut Microbiome
title_short	Highly Reproducible 16S Sequencing Facilitates Measurement of Host Genetic Influences on the Stickleback Gut Microbiome
title_full	Highly Reproducible 16S Sequencing Facilitates Measurement of Host Genetic Influences on the Stickleback Gut Microbiome
title_fullStr	Highly Reproducible 16S Sequencing Facilitates Measurement of Host Genetic Influences on the Stickleback Gut Microbiome
title_full_unstemmed	Highly Reproducible 16S Sequencing Facilitates Measurement of Host Genetic Influences on the Stickleback Gut Microbiome
title_sort	highly reproducible 16s sequencing facilitates measurement of host genetic influences on the stickleback gut microbiome
publisher	American Society for Microbiology
publishDate	2019
url	https://doaj.org/article/eadd0f761733448dbbfddb2ae162890c
work_keys_str_mv	AT claytonmsmall highlyreproducible16ssequencingfacilitatesmeasurementofhostgeneticinfluencesonthesticklebackgutmicrobiome AT markcurrey highlyreproducible16ssequencingfacilitatesmeasurementofhostgeneticinfluencesonthesticklebackgutmicrobiome AT emilyabeck highlyreproducible16ssequencingfacilitatesmeasurementofhostgeneticinfluencesonthesticklebackgutmicrobiome AT susanbassham highlyreproducible16ssequencingfacilitatesmeasurementofhostgeneticinfluencesonthesticklebackgutmicrobiome AT williamacresko highlyreproducible16ssequencingfacilitatesmeasurementofhostgeneticinfluencesonthesticklebackgutmicrobiome
_version_	1718375997792845824

Highly Reproducible 16S Sequencing Facilitates Measurement of Host Genetic Influences on the Stickleback Gut Microbiome

Ejemplares similares