Computational protocol: Phylogenetic relationships between Bacillus species and related genera inferred from 16s rDNA sequences

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Protocol publication

[…] Bacillus species, type species of the genera of Bacillaceae and type species of some families in Bacillales were selected for the phylogenetic study (LPSN updated date September 04, 2007). The 16S rDNA sequences of the type strains of the bacteria mentioned above were downloaded from the GenBank. If several 16S rDNA sequences from the type strain(s) of the same species were available, the longest one with the least non-AGTC characters would be selected. All the rectifiable ambiguous nucleotides in the selected sequences were corrected according to the homologous sequences searched by BLAST ( and/or other 16S rDNA sequences of the type strain(s).Nucleotide sequence alignments were made using ClustalX 1.83 () and optimized using Tune ClustalX (Hall 2004, by modifying multiple alignment parameters. Then Bioedit 7 () was used for refining the entire alignment by eye. Calculations of pairwise 16S rDNA sequences similarity were achieved using the EzTaxon server ( (). Escherichia coli was used as the outgroup. The optimal models of nucleotide substitutions were estimated by the program Modeltest 3.7 (), using hierarchical likelihood ratio tests (hLRT) and the Akaike Information Criterion (AIC).Neighbor-joining (NJ), maximum-parsimony (MP) and minimum-evolution (ME) analyses were performed with MEGA 4 (). NJ and ME analyses were performed using the maximum composite likelihood method and 1000 bootstrap replications. Maximum likelihood (ML)-based phylogenetic analyses were performed with RAxML-VI-HPC 4 ( using default parameters (bootstrap=100). Bayesian trees were inferred using MrBayes 3.1.2 () according to the MrBayes 3.1 Manual (2005). All the Markov chain Monte Carlo searches were run with four chains for 4,300,000 generations, with trees being sampled every 100 generations. The first 30000 trees were discarded as “burnin”, keeping only trees generated well after those parameters stabilized. […]

Pipeline specifications

Software tools Clustal W, BioEdit, ModelTest-NG, MEGA, RAxML, MrBayes
Databases EzBioCloud LPSN
Application Phylogenetics
Organisms Bacillus subtilis, Hemibarbus labeo, Kyrpidia tusciae, Paenibacillus mucilaginosus