Computational protocol: Molecular Genetic Diversity and Quantitation of Methanogen in Ruminal Fluid of Buffalo (Bubalus bubalis) Fed Ration (Wheat Straw and Concentrate Mixture Diet)

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Protocol publication

[…] Sequencing performed for all the clones with an ABI Prism 310 Genetic Analyser (Applied Biosystems Inc., CA) using BigDye Terminator (version 3.1) cycle sequencing kit (ABI, USA) at the Animal Biotechnology Laboratory, AAU, Anand, Gujarat, India.All reference sequences were obtained from the Genbank/EMBL []. Sequences from the current study were mainly trimmed to remove low-quality base calls from the start and end of DNA sequences and further analysed by the CHECK_CHIMERA program [] to remove any chimera rDNA clone. The similarity searches for sequences were carried out by BLAST (, and alignment was perform using CLUSTAL W ( The phylogenetic relatedness was estimated using the neighbor-joining method []. All positions containing gaps and missing data were eliminated from the dataset (complete deletion option). One thousand bootstrap replications were performed to place the confidence estimates on the major groups resolved in the tree. The bootstrap consensus tree inferred from 1,000 replicates represents the evolutionary history of the sequences analyzed []. The phylogenetic analysis was carried out using MEGA software version 4.0 []. […]

Pipeline specifications

Software tools Clustal W, MEGA
Application Phylogenetics
Organisms Bubalus bubalis, Triticum aestivum
Chemicals Methane