Effect of DNA extraction methods and sampling techniques on the apparent structure of cow and sheep rumen microbial communities.

Molecular microbial ecology techniques are widely used to study the composition of the rumen microbiota and to increase understanding of the roles they play. Therefore, sampling and DNA extraction methods that result in adequate yields of microbial DNA that also accurately represents the microbial c...

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Autores principales:	Gemma Henderson, Faith Cox, Sandra Kittelmann, Vahideh Heidarian Miri, Michael Zethof, Samantha J Noel, Garry C Waghorn, Peter H Janssen
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2013
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/3be68243b80d4c8b837070780571d56a
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spelling	oai:doaj.org-article:3be68243b80d4c8b837070780571d56a2021-11-18T08:55:40ZEffect of DNA extraction methods and sampling techniques on the apparent structure of cow and sheep rumen microbial communities.1932-620310.1371/journal.pone.0074787https://doaj.org/article/3be68243b80d4c8b837070780571d56a2013-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/24040342/pdf/?tool=EBIhttps://doaj.org/toc/1932-6203Molecular microbial ecology techniques are widely used to study the composition of the rumen microbiota and to increase understanding of the roles they play. Therefore, sampling and DNA extraction methods that result in adequate yields of microbial DNA that also accurately represents the microbial community are crucial. Fifteen different methods were used to extract DNA from cow and sheep rumen samples. The DNA yield and quality, and its suitability for downstream PCR amplifications varied considerably, depending on the DNA extraction method used. DNA extracts from nine extraction methods that passed these first quality criteria were evaluated further by quantitative PCR enumeration of microbial marker loci. Absolute microbial numbers, determined on the same rumen samples, differed by more than 100-fold, depending on the DNA extraction method used. The apparent compositions of the archaeal, bacterial, ciliate protozoal, and fungal communities in identical rumen samples were assessed using 454 Titanium pyrosequencing. Significant differences in microbial community composition were observed between extraction methods, for example in the relative abundances of members of the phyla Bacteroidetes and Firmicutes. Microbial communities in parallel samples collected from cows by oral stomach-tubing or through a rumen fistula, and in liquid and solid rumen digesta fractions, were compared using one of the DNA extraction methods. Community representations were generally similar, regardless of the rumen sampling technique used, but significant differences in the abundances of some microbial taxa such as the Clostridiales and the Methanobrevibacter ruminantium clade were observed. The apparent microbial community composition differed between rumen sample fractions, and Prevotellaceae were most abundant in the liquid fraction. DNA extraction methods that involved phenol-chloroform extraction and mechanical lysis steps tended to be more comparable. However, comparison of data from studies in which different sampling techniques, different rumen sample fractions or different DNA extraction methods were used should be avoided.Gemma HendersonFaith CoxSandra KittelmannVahideh Heidarian MiriMichael ZethofSamantha J NoelGarry C WaghornPeter H JanssenPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 8, Iss 9, p e74787 (2013)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Gemma Henderson Faith Cox Sandra Kittelmann Vahideh Heidarian Miri Michael Zethof Samantha J Noel Garry C Waghorn Peter H Janssen Effect of DNA extraction methods and sampling techniques on the apparent structure of cow and sheep rumen microbial communities.
description	Molecular microbial ecology techniques are widely used to study the composition of the rumen microbiota and to increase understanding of the roles they play. Therefore, sampling and DNA extraction methods that result in adequate yields of microbial DNA that also accurately represents the microbial community are crucial. Fifteen different methods were used to extract DNA from cow and sheep rumen samples. The DNA yield and quality, and its suitability for downstream PCR amplifications varied considerably, depending on the DNA extraction method used. DNA extracts from nine extraction methods that passed these first quality criteria were evaluated further by quantitative PCR enumeration of microbial marker loci. Absolute microbial numbers, determined on the same rumen samples, differed by more than 100-fold, depending on the DNA extraction method used. The apparent compositions of the archaeal, bacterial, ciliate protozoal, and fungal communities in identical rumen samples were assessed using 454 Titanium pyrosequencing. Significant differences in microbial community composition were observed between extraction methods, for example in the relative abundances of members of the phyla Bacteroidetes and Firmicutes. Microbial communities in parallel samples collected from cows by oral stomach-tubing or through a rumen fistula, and in liquid and solid rumen digesta fractions, were compared using one of the DNA extraction methods. Community representations were generally similar, regardless of the rumen sampling technique used, but significant differences in the abundances of some microbial taxa such as the Clostridiales and the Methanobrevibacter ruminantium clade were observed. The apparent microbial community composition differed between rumen sample fractions, and Prevotellaceae were most abundant in the liquid fraction. DNA extraction methods that involved phenol-chloroform extraction and mechanical lysis steps tended to be more comparable. However, comparison of data from studies in which different sampling techniques, different rumen sample fractions or different DNA extraction methods were used should be avoided.
format	article
author	Gemma Henderson Faith Cox Sandra Kittelmann Vahideh Heidarian Miri Michael Zethof Samantha J Noel Garry C Waghorn Peter H Janssen
author_facet	Gemma Henderson Faith Cox Sandra Kittelmann Vahideh Heidarian Miri Michael Zethof Samantha J Noel Garry C Waghorn Peter H Janssen
author_sort	Gemma Henderson
title	Effect of DNA extraction methods and sampling techniques on the apparent structure of cow and sheep rumen microbial communities.
title_short	Effect of DNA extraction methods and sampling techniques on the apparent structure of cow and sheep rumen microbial communities.
title_full	Effect of DNA extraction methods and sampling techniques on the apparent structure of cow and sheep rumen microbial communities.
title_fullStr	Effect of DNA extraction methods and sampling techniques on the apparent structure of cow and sheep rumen microbial communities.
title_full_unstemmed	Effect of DNA extraction methods and sampling techniques on the apparent structure of cow and sheep rumen microbial communities.
title_sort	effect of dna extraction methods and sampling techniques on the apparent structure of cow and sheep rumen microbial communities.
publisher	Public Library of Science (PLoS)
publishDate	2013
url	https://doaj.org/article/3be68243b80d4c8b837070780571d56a
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Effect of DNA extraction methods and sampling techniques on the apparent structure of cow and sheep rumen microbial communities.

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