Computational protocol: Genotypic comparison of Pantoea agglomerans plant and clinical strains

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Protocol publication

[…] PCR amplicons were purified from the PCR mix by washing twice with 50 μl of double-distilled water (ddH2O) on a MultiScreen PCR Plate (Millipore, Molsheim, France), resuspended in 30 μl of ddH2O, and quantified spectrophotometrically as described above. The cycle-sequencing reaction was performed with 20-40 ng of purified PCR product using the ABI PRISM BigDye Terminators v1.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, U.S.A.) according to the manufacturer instructions employing the same primers used for PCR amplification. For 16S rRNA gene sequencing, additional primers 16S-609R and 16S-533R (Table ) were used to obtain complete coverage of the amplicon. Cycle sequencing products were cleaned through water-swelled Sephadex G-50 columns (Amersham Biosciences, Uppsala, Sweden) on MultiScreen HV plates (Millipore) and sequenced on an ABI PRISM 3100 Genetic Analyzer. Obtained sequences were assembled using the Sequencher software (version 4.0.5; Gene Codes Corporation, Ann Arbor, MI, U.S.A.). [...] Phylogenetic trees were generated on the basis of partial 16S rDNA, gyrB and pagRI sequences without choosing any outgroup. DNA sequences were aligned with ClustalW []. Sites presenting alignment gaps were excluded from analysis. The Molecular Evolutionary Genetics Analysis (MEGA) program version 4.0 [] was used to calculate evolutionary distances and to infer trees based on the Minimum Evolution (ME) method using the Maximum Composite Likelihood (MCL) formula. Nodal robustness of the inferred trees was assessed by 1000-bootstrap replicates. […]

Pipeline specifications

Software tools Sequencher, Clustal W, MEGA
Applications Phylogenetics, 16S rRNA-seq analysis
Organisms Pantoea agglomerans, Malus domestica, Homo sapiens
Diseases Mycoses