Computational protocol: Addition of plant growth promoting Bacillus subtilis PTS 394 on tomato rhizosphere has no durable impact on composition of root microbiome

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Protocol publication

[…] Phylogenomic analysis of strain PTS-394 was performed taking advantage of availability of the whole genome sequence []. JSpeciesWS ( was used to determine ANIb (average nucleotide identity based on BLAST+) and ANIm (average nucleotide identity based on MUMmer) values by pairwise genome comparisons. Correlation indexes of their Tetra-nucleotide signatures (TETRA) were determined by using the JSpeciesWS software (Richter et al., 2015). Electronic DNA-DNA hybridization (dDDH) is useful to mimic the wet-lab DDH and can be used for genome-based species delineation and genome-based subspecies delineation [, ] and was applied to finally defined PTS-394 whether belong to B. subtilis subsp. subtilis. Three formulas are applicable for the calculation: Formula: 1 (HSP length / total length), formula: 2 (identities / HSP length) and formula 3 (identities / total length). Formula 2, which is especially appropriate to analyze draft genomes, was used. [...] Sequences were assigned to different samples according to sample-specific barcodes and processed using QIIME (version 1.17). Data were selected based on the following criteria: (i) an almost perfect match with barcode and primers; (ii) a length of at least 200 nucleotides (barcodes and primers excluded); (iii) an average quality score of >25, with no ambiguous bases or homopolymers longer than six bp, and without any primer mismatches. After this procedure, 67,520 and 140,099 high-quality sequences were obtained for ITS and 16S rDNA reads, respectively. The number of clean reads per samples are listed in Additional file : Table S1 and Additional file : Table S2.Operational Taxonomic Units (OTUs) were clustered using a 97% similarity cutoff by UPARSE (version 7.1 and chimeric sequences were identified and removed using UCHIME. The phylogenetic affiliation of each 16S rRNA gene and ITS sequence were analyzed by RDP Classifier ( against the SILVA (SSU111) 16S/18S rRNA database. Taxonomic assignment from phylum level to strain level based on hits, and these were used to plot abundance graphs. […]

Pipeline specifications

Software tools JSpeciesWS, MUMmer, QIIME, UPARSE, UCHIME, RDP Classifier
Applications Phylogenetics, 16S rRNA-seq analysis, Nucleotide sequence alignment
Organisms Bacillus subtilis, Solanum lycopersicum, Bacillus amyloliquefaciens