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

Similar protocols

Protocol publication

[…] Phylogenomic analysis of strain PTS-394 was performed taking advantage of availability of the whole genome sequence []. JSpeciesWS (http://jspecies.ribohost.com/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 http://drive5.com/uparse/) 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 (http://rdp.cme.msu.edu/) 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