Computational protocol: Bacillus Strains Most Closely Related to Bacillus nealsonii Are Not Effectively Circumscribed within the Taxonomic Species Definition

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Protocol publication

[…] Amplification of 16S rRNA gene sequences from Bacillus strains, sequencing of the approximately 1500 bp long products, fragment assemblies and alignment of 16S rRNA gene sequences from type strains of selected Bacillus species were performed as previously reported []; an additional primer, 534R, was employed in some amplifications. Identification of phylogenetic neighbors was carried out by BLAST 2.2.20+ [] and megaBLAST (discontinuous option) [] searches of GenBank []. Calculation of pairwise sequence similarity to nearest neighbors used the EzTaxon global alignment algorithm []. Alignments of 16S rDNA sequences were also made using the Infernal secondary structure based aligner and SeqMatch scores (S_ab) calculated with RDP10 at the Ribosomal Database Project website []. Nucleotide (nt) positions in the hypervariable regions V1–V3 were identified in the 16S sequence of strain CBD 118 by alignment with conserved regions at nt positions 48–70, 346–366, and 490–511 in rrnE of B. subtilis subsp. subtilis strain 168T (NC_000964.2, Locus tag: BSUr022, GeneID: 2914197, updated 3/2010 to NC_000964.3, Locus tag: BSU_rRNA_30, GeneID: 8303085) []. In the E. coli numbering system, regions V1–V3 correspond to nt positions 69–99, 137–242 and 433–497, respectively []. Using GenBank bl2seg, pairwise alignments of 461 bp from strain CBD 118 (nt 26–461 corresponding to rrnE nt positions 48–511) were made to 16S rDNA sequences from closely related strains. Presumptive signature sequences (PSS) within the V1–V3 region were identified and compared to all GenBank sequences using BLASTN 2.2.20+ with parameters adjusted for short input sequences []. Dendrograms were constructed from approximately 1390 bp or 448 bp using neighbor-joining, maximum parsimony and maximum likelihood algorithms (PHYLIP v. 3.6.80) [] with 1000 bootstrap replications performed to estimate support for each branch. […]

Pipeline specifications

Software tools BLASTN, GeneID, PHYLIP
Application Phylogenetics
Organisms Cell fusing agent virus
Diseases Sprains and Strains
Chemicals Carbohydrates