Computational protocol: Origin and Evolution of Nitrogen Fixation Genes on Symbiosis Islands and Plasmid in Bradyrhizobium

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Protocol publication

[…] Strains DOA1 and MAFF107635 were cultured to the stationary phase at 30°C in HM salt medium () containing 0.1% arabinose and 0.025% yeast extract. The cells were harvested by centrifugation, and total DNA was prepared as previously described (). The 8-kb paired-end 454 libraries of strains DOA1 and MAFF107635 were then constructed from total DNA by Takara Bio (Shiga, Japan). Genome sequencing was performed using a 454 GS FLX+ sequencer (Roche Diagnostics; Tokyo, Japan). The generated sequences were assembled using Roche GS De Novo Assembler version 2.5p1 (Roche Diagnostics). Two potentially contaminated regions from primer or adaptor sequences used in 454 sequencing were detected in the strain DOA1 genome at positions 1625647–1625666 and 6325190–6325209, both of which were located at the edge of contigs. The nucleotides of these regions were changed into Ns. Predictions of gene regions and annotations were performed using NCBI Prokaryotic Genome Automatic Annotation Pipeline (PGAP) (). A circular genome map showing the GC skew and GC content was created using arcWithColor version 1.3.8 (http://www.ige.tohoku.ac.jp/joho/gmProject/gmdownload.html). GC content and skew were calculated with sliding windows along the genome. The window size was set to 20,000 bases and the step size to 500 bases. The chromosome sequences of strains DOA1 and MAFF107635 containing runs of Ns that represent gaps were used as inputs. All predicted gap lengths were less than 5 kb. Organizations of nif–clusters were compared using GenomeMatcher version 1.861 (). [...] 16S rRNA gene sequences were aligned using the CLUSTAL W program in MEGA 6.06 (). The phylogenetic tree was constructed using nucleotide sequences via the maximum likelihood method with 1,000 bootstrap replicates in MEGA 6.06. The model with the lowest Bayesian Information Criterion (BIC) score by the “Find Best DNA/Protein Models” procedure of MEGA 6.06 was applied. The same analyses were conducted using the concatenated nucleotide sequences of four housekeeping genes (rpoB, recA, gyrB, and atpD). […]

Pipeline specifications

Software tools Newbler, PGAP, GenomeMatcher, Clustal W, MEGA
Applications Phylogenetics, Genome data visualization
Chemicals Cytosine, Guanine, Nitrogen