Computational protocol: Distribution of Diverse Escherichia coli between Cattle and Pasture

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Protocol publication

[…] Genomic DNA was extracted from overnight LB agar cultures suspended in 10 mM phosphate buffer (pH 7.0) using the genomic DNA Quick Prep Kit (Zymo Research), and stored at –20°C. The uidA and mutS genes were amplified by PCR using primers described previously () (), with E. coli MG1655 as a positive control. PCR reactions (25 μL) were set up as follows: 2.5 μL reaction buffer (10X) (New England Biolab), 0.5 μL MgCl2 (25 mM), 0.5 μL dNTPs (40 mM), 0.1 μL forward primer and 0.1 μL reverse primer (100 μmol), 0.125 μL of Taq polymerase (NE Biolab), 0.5 μL of a DNA template, and 20.7 μL of sterile nano pure water. The amplification cycle was initiated with 95°C for 2 min, followed by 30 cycles of denaturing at 95°C for 30 s, annealing at 56°C for 30 s, and extension at 72°C for 1 min, with a final extension at 72°C for 5 min. DNA sequences were elucidated by the dideoxy chain termination method (Beckman Coulter Genomic Center at Denver, MA). uidA sequences were submitted to GenBank (http://www.ncbi.nlm.nih.gov/genbank/) under the BankIt number 1841773 (accession numbers KT311394–KT311756), and mutS sequences as the BankIt number 1841687 (accession numbers KT311004–KT311366). DNA sequences were aligned using ClustalW (), and overhangs were trimmed using SeAl (Rambaut, A. 2002. http://tree.bio.ed.ac.uk/software/seal/). The uidA and mutS sequences for all isolates and reference strains () were concatenated using SeAl. A maximum likelihood analysis using model GTR+G+I with 1,000 bootstrap replicates was performed in the program MEGA6.06 (). The tree was then annotated and visualized using the ITOL online tool (). […]

Pipeline specifications

Software tools Clustal W, MEGA, iTOL
Application Phylogenetics
Organisms Escherichia coli, Bos taurus