Diversity and community composition of methanogenic archaea in the rumen of Scottish upland sheep assessed by different methods

Timothy J. Snelling; Buğra Genc; Nest McKain; Mick Watson; Sinead M. Waters; Christopher J. Creevy; R. John Wallace

doi:10.1371/journal.pone.0106491

Diversity and community composition of methanogenic archaea in the rumen of Scottish upland sheep assessed by different methods

Timothy J. Snelling, Buğra Genc, Nest McKain, Mick Watson, Sinead M. Waters, Christopher J. Creevy, R. John Wallace^* (Corresponding Author)

^*Corresponding author for this work

The Rowett Institute of Nutrition and Health

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Abstract

Ruminal archaeomes of two mature sheep grazing in the Scottish uplands were analysed by different sequencing and analysis methods in order to compare the apparent archaeal communities. All methods revealed that the majority of methanogens belonged to the Methanobacteriales order containing the Methanobrevibacter, Methanosphaera and Methanobacteria genera. Sanger sequenced 1.3 kb 16S rRNA gene amplicons identified the main species of Methanobrevibacter present to be a SGMT Clade member Mbb. millerae (≥91% of OTUs); Methanosphaera comprised the remainder of the OTUs. The primers did not amplify ruminal Thermoplasmatales-related 16S rRNA genes. Illumina sequenced V6–V8 16S rRNA gene amplicons identified similar Methanobrevibacter spp. and Methanosphaera clades and also identified the Thermoplasmatales-related order as 13% of total archaea. Unusually, both methods concluded that Mbb. ruminantium and relatives from the same clade (RO) were almost absent. Sequences mapping to rumen 16S rRNA and mcrA gene references were extracted from Illumina metagenome data. Mapping of the metagenome data to16S rRNA gene references produced taxonomic identification to Order level including 2–3% Thermoplasmatales, but was unable to discriminate to species level. Mapping of the metagenome data to mcrA gene references resolved 69% to unclassified Methanobacteriales. Only 30% of sequences were assigned to species level clades: of the sequences assigned to Methanobrevibacter, most mapped to SGMT (16%) and RO (10%) clades. The Sanger 16S amplicon and Illumina metagenome mcrA analyses showed similar species richness (Chao1 Index 19–35), while Illumina metagenome and amplicon 16S rRNA analysis gave lower richness estimates (10–18). The values of the Shannon Index were low in all methods, indicating low richness and uneven species distribution. Thus, although much information may be extracted from the other methods, Illumina amplicon sequencing of the V6–V8 16S rRNA gene would be the method of choice for studying rumen archaeal communities.

Original language	English
Article number	e106491
Number of pages	9
Journal	PloS ONE
Volume	9
Issue number	9
DOIs	https://doi.org/10.1371/journal.pone.0106491
Publication status	Published - 24 Sept 2014

Bibliographical note

Acknowledgments
We thank Bob Mayes and Dave Hamilton of the James Hutton Institute for their permission and help in sampling the sheep digesta. Gillian Campbell and Pauline Young provided an excellent DNA sequencing service. We also thank Dr Matthew McCabe for preparing V6–V8 amplicon libraries.

Author Contributions
Conceived and designed the experiments: RJW. Performed the experiments: RJW BG NM SMW CJC. Analyzed the data: TJS MW SMW CJC RJW. Contributed reagents/materials/analysis tools: NM RJW MW SMW CJC. Contributed to the writing of the manuscript: TJS MW SMW CJC RJW.

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Cite this

@article{ddb880e276814af18ddadb6fb097a059,

title = "Diversity and community composition of methanogenic archaea in the rumen of Scottish upland sheep assessed by different methods",

abstract = "Ruminal archaeomes of two mature sheep grazing in the Scottish uplands were analysed by different sequencing and analysis methods in order to compare the apparent archaeal communities. All methods revealed that the majority of methanogens belonged to the Methanobacteriales order containing the Methanobrevibacter, Methanosphaera and Methanobacteria genera. Sanger sequenced 1.3 kb 16S rRNA gene amplicons identified the main species of Methanobrevibacter present to be a SGMT Clade member Mbb. millerae (≥91% of OTUs); Methanosphaera comprised the remainder of the OTUs. The primers did not amplify ruminal Thermoplasmatales-related 16S rRNA genes. Illumina sequenced V6–V8 16S rRNA gene amplicons identified similar Methanobrevibacter spp. and Methanosphaera clades and also identified the Thermoplasmatales-related order as 13% of total archaea. Unusually, both methods concluded that Mbb. ruminantium and relatives from the same clade (RO) were almost absent. Sequences mapping to rumen 16S rRNA and mcrA gene references were extracted from Illumina metagenome data. Mapping of the metagenome data to16S rRNA gene references produced taxonomic identification to Order level including 2–3% Thermoplasmatales, but was unable to discriminate to species level. Mapping of the metagenome data to mcrA gene references resolved 69% to unclassified Methanobacteriales. Only 30% of sequences were assigned to species level clades: of the sequences assigned to Methanobrevibacter, most mapped to SGMT (16%) and RO (10%) clades. The Sanger 16S amplicon and Illumina metagenome mcrA analyses showed similar species richness (Chao1 Index 19–35), while Illumina metagenome and amplicon 16S rRNA analysis gave lower richness estimates (10–18). The values of the Shannon Index were low in all methods, indicating low richness and uneven species distribution. Thus, although much information may be extracted from the other methods, Illumina amplicon sequencing of the V6–V8 16S rRNA gene would be the method of choice for studying rumen archaeal communities.",

author = "Snelling, {Timothy J.} and Buğra Genc and Nest McKain and Mick Watson and Waters, {Sinead M.} and Creevy, {Christopher J.} and Wallace, {R. John}",

note = "Acknowledgments We thank Bob Mayes and Dave Hamilton of the James Hutton Institute for their permission and help in sampling the sheep digesta. Gillian Campbell and Pauline Young provided an excellent DNA sequencing service. We also thank Dr Matthew McCabe for preparing V6–V8 amplicon libraries. Author Contributions Conceived and designed the experiments: RJW. Performed the experiments: RJW BG NM SMW CJC. Analyzed the data: TJS MW SMW CJC RJW. Contributed reagents/materials/analysis tools: NM RJW MW SMW CJC. Contributed to the writing of the manuscript: TJS MW SMW CJC RJW.",

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T1 - Diversity and community composition of methanogenic archaea in the rumen of Scottish upland sheep assessed by different methods

AU - Snelling, Timothy J.

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AU - McKain, Nest

AU - Watson, Mick

AU - Waters, Sinead M.

AU - Creevy, Christopher J.

AU - Wallace, R. John

N1 - Acknowledgments We thank Bob Mayes and Dave Hamilton of the James Hutton Institute for their permission and help in sampling the sheep digesta. Gillian Campbell and Pauline Young provided an excellent DNA sequencing service. We also thank Dr Matthew McCabe for preparing V6–V8 amplicon libraries. Author Contributions Conceived and designed the experiments: RJW. Performed the experiments: RJW BG NM SMW CJC. Analyzed the data: TJS MW SMW CJC RJW. Contributed reagents/materials/analysis tools: NM RJW MW SMW CJC. Contributed to the writing of the manuscript: TJS MW SMW CJC RJW.

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N2 - Ruminal archaeomes of two mature sheep grazing in the Scottish uplands were analysed by different sequencing and analysis methods in order to compare the apparent archaeal communities. All methods revealed that the majority of methanogens belonged to the Methanobacteriales order containing the Methanobrevibacter, Methanosphaera and Methanobacteria genera. Sanger sequenced 1.3 kb 16S rRNA gene amplicons identified the main species of Methanobrevibacter present to be a SGMT Clade member Mbb. millerae (≥91% of OTUs); Methanosphaera comprised the remainder of the OTUs. The primers did not amplify ruminal Thermoplasmatales-related 16S rRNA genes. Illumina sequenced V6–V8 16S rRNA gene amplicons identified similar Methanobrevibacter spp. and Methanosphaera clades and also identified the Thermoplasmatales-related order as 13% of total archaea. Unusually, both methods concluded that Mbb. ruminantium and relatives from the same clade (RO) were almost absent. Sequences mapping to rumen 16S rRNA and mcrA gene references were extracted from Illumina metagenome data. Mapping of the metagenome data to16S rRNA gene references produced taxonomic identification to Order level including 2–3% Thermoplasmatales, but was unable to discriminate to species level. Mapping of the metagenome data to mcrA gene references resolved 69% to unclassified Methanobacteriales. Only 30% of sequences were assigned to species level clades: of the sequences assigned to Methanobrevibacter, most mapped to SGMT (16%) and RO (10%) clades. The Sanger 16S amplicon and Illumina metagenome mcrA analyses showed similar species richness (Chao1 Index 19–35), while Illumina metagenome and amplicon 16S rRNA analysis gave lower richness estimates (10–18). The values of the Shannon Index were low in all methods, indicating low richness and uneven species distribution. Thus, although much information may be extracted from the other methods, Illumina amplicon sequencing of the V6–V8 16S rRNA gene would be the method of choice for studying rumen archaeal communities.

AB - Ruminal archaeomes of two mature sheep grazing in the Scottish uplands were analysed by different sequencing and analysis methods in order to compare the apparent archaeal communities. All methods revealed that the majority of methanogens belonged to the Methanobacteriales order containing the Methanobrevibacter, Methanosphaera and Methanobacteria genera. Sanger sequenced 1.3 kb 16S rRNA gene amplicons identified the main species of Methanobrevibacter present to be a SGMT Clade member Mbb. millerae (≥91% of OTUs); Methanosphaera comprised the remainder of the OTUs. The primers did not amplify ruminal Thermoplasmatales-related 16S rRNA genes. Illumina sequenced V6–V8 16S rRNA gene amplicons identified similar Methanobrevibacter spp. and Methanosphaera clades and also identified the Thermoplasmatales-related order as 13% of total archaea. Unusually, both methods concluded that Mbb. ruminantium and relatives from the same clade (RO) were almost absent. Sequences mapping to rumen 16S rRNA and mcrA gene references were extracted from Illumina metagenome data. Mapping of the metagenome data to16S rRNA gene references produced taxonomic identification to Order level including 2–3% Thermoplasmatales, but was unable to discriminate to species level. Mapping of the metagenome data to mcrA gene references resolved 69% to unclassified Methanobacteriales. Only 30% of sequences were assigned to species level clades: of the sequences assigned to Methanobrevibacter, most mapped to SGMT (16%) and RO (10%) clades. The Sanger 16S amplicon and Illumina metagenome mcrA analyses showed similar species richness (Chao1 Index 19–35), while Illumina metagenome and amplicon 16S rRNA analysis gave lower richness estimates (10–18). The values of the Shannon Index were low in all methods, indicating low richness and uneven species distribution. Thus, although much information may be extracted from the other methods, Illumina amplicon sequencing of the V6–V8 16S rRNA gene would be the method of choice for studying rumen archaeal communities.

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DO - 10.1371/journal.pone.0106491

M3 - Article

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JF - PloS ONE

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M1 - e106491

ER -

Diversity and community composition of methanogenic archaea in the rumen of Scottish upland sheep assessed by different methods

Abstract

Bibliographical note

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