TY - JOUR
T1 - Diversity and Community Composition of Methanogenic Archaea in the Rumen of Scottish Upland Sheep Assessed by Different Methods
AU - Snelling, Timothy J.
AU - Genç, Buğra
AU - McKain, Nest
AU - Watson, Mick
AU - Waters, Sinéad M.
AU - Creevey, Christopher J.
AU - Wallace, R. John
N1 - The Rowett Institute of Nutrition and Health is funded by the Rural and Environment Science and Analytical Services Division (RESAS) of the Scottish Government. Research at The Roslin Institute was supported by the Biotechnology and Biological Sciences Research Council (BBSRC; BB/J004243/1, BB/J004235/1), and by the Technology Strategy Board (TS/J000108/1, TS/J000116/1). BG thanks the Turkish Higher Education Council for the award of a travelling fellowship. CJC was supported by the BBSRC Institute Strategic Programme Grant, Rumen Systems Biology (BB/J004413/1). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
PY - 2014/9/24
Y1 - 2014/9/24
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.
U2 - 10.1371/journal.pone.0106491
DO - 10.1371/journal.pone.0106491
M3 - Article
SN - 1932-6203
VL - 9
JO - PloS ONE
JF - PloS ONE
IS - 9
M1 - e106491
ER -