Rumen samples were obtained from the animals when they were alive (n = 50) and after slaughter (n = 68). Rumen samples were taken from live animals within 2 hours of leaving the respiration chambers. Approximately 50 mL rumen contents were taken by inserting a stomach tube (16 × 2700 mm Equivet Stomach Tube, JørgenKruuse A/S, Langeskov, Denmark) nasally and aspirating manually. Between 3 to 17 days after leaving the respiration chamber the animals were slaughtered in a commercial abattoir where two rumen fluid samples (approximately 50 mL) were taken immediately after the rumen was opened to be drained. The slaughter process results in well mixed samples of rumen contents. DNA was extracted from the rumen samples and subjected to qPCR for the 16S rRNA genes as described in [14 (link)] to determine the abundance of archaea and bacteria and their ratio.
Eight extreme animals (4 high and 4 low) for methane emissions, balanced for breed type and diet, were used in a metagenomic study, in which deep sequencing was applied. Illumina TruSeq libraries were prepared from genomic DNA and sequenced on an Illumina HiSeq 2500 instrument by Edinburgh Genomics. Paired-end reads (2 × 100 bp) were generated, resulting in between 8.6 and 14.5 GB per sample (between 43.4 and 72.7 million paired reads). The genomic reads were aligned to the KEGG genes database. Parameters were adjusted such that all hits were reported that were equal in quality to the best hit for each genomic read. The read and best hits have to be more than 90% identical and have to be belonging to a single KEGG orthologue group to be kept in the data. If the best hits are spread over more than one KEGG orthologue group, the read were disregarded. Read counts for KEGG orthologues were summed and normalised to the total number of hits.
Eight extreme animals (4 high and 4 low) for methane emissions, balanced for breed type and diet, were used in a metagenomic study, in which deep sequencing was applied. Illumina TruSeq libraries were prepared from genomic DNA and sequenced on an Illumina HiSeq 2500 instrument by Edinburgh Genomics. Paired-end reads (2 × 100 bp) were generated, resulting in between 8.6 and 14.5 GB per sample (between 43.4 and 72.7 million paired reads). The genomic reads were aligned to the KEGG genes database. Parameters were adjusted such that all hits were reported that were equal in quality to the best hit for each genomic read. The read and best hits have to be more than 90% identical and have to be belonging to a single KEGG orthologue group to be kept in the data. If the best hits are spread over more than one KEGG orthologue group, the read were disregarded. Read counts for KEGG orthologues were summed and normalised to the total number of hits.
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