Impact of Sample Type and DNA Isolation Procedure on Genomic Inference of Microbiome Composition

ABSTRACT Explorations of complex microbiomes using genomics greatly enhance our understanding about their diversity, biogeography, and function. The isolation of DNA from microbiome specimens is a key prerequisite for such examinations, but challenges remain in obtaining sufficient DNA quantities re...

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Autores principales:	Berith E. Knudsen, Lasse Bergmark, Patrick Munk, Oksana Lukjancenko, Anders Priemé, Frank M. Aarestrup, Sünje J. Pamp
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2016
Materias:	16S rRNA gene profiling DNA isolation metagenomics microbial ecology microbiome next-generation sequencing Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/273653aaf8f349009176bfb5a979b751
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spelling	oai:doaj.org-article:273653aaf8f349009176bfb5a979b7512021-12-02T19:45:30ZImpact of Sample Type and DNA Isolation Procedure on Genomic Inference of Microbiome Composition10.1128/mSystems.00095-162379-5077https://doaj.org/article/273653aaf8f349009176bfb5a979b7512016-10-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSystems.00095-16https://doaj.org/toc/2379-5077ABSTRACT Explorations of complex microbiomes using genomics greatly enhance our understanding about their diversity, biogeography, and function. The isolation of DNA from microbiome specimens is a key prerequisite for such examinations, but challenges remain in obtaining sufficient DNA quantities required for certain sequencing approaches, achieving accurate genomic inference of microbiome composition, and facilitating comparability of findings across specimen types and sequencing projects. These aspects are particularly relevant for the genomics-based global surveillance of infectious agents and antimicrobial resistance from different reservoirs. Here, we compare in a stepwise approach a total of eight commercially available DNA extraction kits and 16 procedures based on these for three specimen types (human feces, pig feces, and hospital sewage). We assess DNA extraction using spike-in controls and different types of beads for bead beating, facilitating cell lysis. We evaluate DNA concentration, purity, and stability and microbial community composition using 16S rRNA gene sequencing and for selected samples using shotgun metagenomic sequencing. Our results suggest that inferred community composition was dependent on inherent specimen properties as well as DNA extraction method. We further show that bead beating or enzymatic treatment can increase the extraction of DNA from Gram-positive bacteria. Final DNA quantities could be increased by isolating DNA from a larger volume of cell lysate than that in standard protocols. Based on this insight, we designed an improved DNA isolation procedure optimized for microbiome genomics that can be used for the three examined specimen types and potentially also for other biological specimens. A standard operating procedure is available from https://dx.doi.org/10.6084/m9.figshare.3475406 . IMPORTANCE Sequencing-based analyses of microbiomes may lead to a breakthrough in our understanding of the microbial worlds associated with humans, animals, and the environment. Such insight could further the development of innovative ecosystem management approaches for the protection of our natural resources and the design of more effective and sustainable solutions to prevent and control infectious diseases. Genome sequence information is an organism (pathogen)-independent language that can be used across sectors, space, and time. Harmonized standards, protocols, and workflows for sample processing and analysis can facilitate the generation of such actionable information. In this study, we assessed several procedures for the isolation of DNA for next-generation sequencing. Our study highlights several important aspects to consider in the design and conduct of sequence-based analysis of microbiomes. We provide a standard operating procedure for the isolation of DNA from a range of biological specimens particularly relevant in clinical diagnostics and epidemiology.Berith E. KnudsenLasse BergmarkPatrick MunkOksana LukjancenkoAnders PrieméFrank M. AarestrupSünje J. PampAmerican Society for Microbiologyarticle16S rRNA gene profilingDNA isolationmetagenomicsmicrobial ecologymicrobiomenext-generation sequencingMicrobiologyQR1-502ENmSystems, Vol 1, Iss 5 (2016)
institution	DOAJ
collection	DOAJ
language	EN
topic	16S rRNA gene profiling DNA isolation metagenomics microbial ecology microbiome next-generation sequencing Microbiology QR1-502
spellingShingle	16S rRNA gene profiling DNA isolation metagenomics microbial ecology microbiome next-generation sequencing Microbiology QR1-502 Berith E. Knudsen Lasse Bergmark Patrick Munk Oksana Lukjancenko Anders Priemé Frank M. Aarestrup Sünje J. Pamp Impact of Sample Type and DNA Isolation Procedure on Genomic Inference of Microbiome Composition
description	ABSTRACT Explorations of complex microbiomes using genomics greatly enhance our understanding about their diversity, biogeography, and function. The isolation of DNA from microbiome specimens is a key prerequisite for such examinations, but challenges remain in obtaining sufficient DNA quantities required for certain sequencing approaches, achieving accurate genomic inference of microbiome composition, and facilitating comparability of findings across specimen types and sequencing projects. These aspects are particularly relevant for the genomics-based global surveillance of infectious agents and antimicrobial resistance from different reservoirs. Here, we compare in a stepwise approach a total of eight commercially available DNA extraction kits and 16 procedures based on these for three specimen types (human feces, pig feces, and hospital sewage). We assess DNA extraction using spike-in controls and different types of beads for bead beating, facilitating cell lysis. We evaluate DNA concentration, purity, and stability and microbial community composition using 16S rRNA gene sequencing and for selected samples using shotgun metagenomic sequencing. Our results suggest that inferred community composition was dependent on inherent specimen properties as well as DNA extraction method. We further show that bead beating or enzymatic treatment can increase the extraction of DNA from Gram-positive bacteria. Final DNA quantities could be increased by isolating DNA from a larger volume of cell lysate than that in standard protocols. Based on this insight, we designed an improved DNA isolation procedure optimized for microbiome genomics that can be used for the three examined specimen types and potentially also for other biological specimens. A standard operating procedure is available from https://dx.doi.org/10.6084/m9.figshare.3475406 . IMPORTANCE Sequencing-based analyses of microbiomes may lead to a breakthrough in our understanding of the microbial worlds associated with humans, animals, and the environment. Such insight could further the development of innovative ecosystem management approaches for the protection of our natural resources and the design of more effective and sustainable solutions to prevent and control infectious diseases. Genome sequence information is an organism (pathogen)-independent language that can be used across sectors, space, and time. Harmonized standards, protocols, and workflows for sample processing and analysis can facilitate the generation of such actionable information. In this study, we assessed several procedures for the isolation of DNA for next-generation sequencing. Our study highlights several important aspects to consider in the design and conduct of sequence-based analysis of microbiomes. We provide a standard operating procedure for the isolation of DNA from a range of biological specimens particularly relevant in clinical diagnostics and epidemiology.
format	article
author	Berith E. Knudsen Lasse Bergmark Patrick Munk Oksana Lukjancenko Anders Priemé Frank M. Aarestrup Sünje J. Pamp
author_facet	Berith E. Knudsen Lasse Bergmark Patrick Munk Oksana Lukjancenko Anders Priemé Frank M. Aarestrup Sünje J. Pamp
author_sort	Berith E. Knudsen
title	Impact of Sample Type and DNA Isolation Procedure on Genomic Inference of Microbiome Composition
title_short	Impact of Sample Type and DNA Isolation Procedure on Genomic Inference of Microbiome Composition
title_full	Impact of Sample Type and DNA Isolation Procedure on Genomic Inference of Microbiome Composition
title_fullStr	Impact of Sample Type and DNA Isolation Procedure on Genomic Inference of Microbiome Composition
title_full_unstemmed	Impact of Sample Type and DNA Isolation Procedure on Genomic Inference of Microbiome Composition
title_sort	impact of sample type and dna isolation procedure on genomic inference of microbiome composition
publisher	American Society for Microbiology
publishDate	2016
url	https://doaj.org/article/273653aaf8f349009176bfb5a979b751
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Impact of Sample Type and DNA Isolation Procedure on Genomic Inference of Microbiome Composition

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