Computational protocol: Multi-Locus Sequence Typing of Enteroaggregative Escherichia coli Isolates from Nigerian Children Uncovers Multiple Lineages

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Protocol publication

[…] Gene fragments from the adk, fumC, gyrB, icd, mdh, purA and recA were amplified as described by Wirth et al . Amplified DNA was prepared for sequencing using the “ExoSAP” method (Amersham Biosciences UK Ltd). Cleaned fragments were sequenced from both ends using the di-deoxy chain terminator method , with V3.1 Bigdye terminator chemistry . Both strands of each fragment were sequenced at least once. The resulting sequencing reactions were analyzed on 3700 or 3730 ABI sequencing machines (Applied Biosystems, USA). Allele and sequence type (ST) assignments were made at the publicly accessible E. coli MLST database at http://mlst.ucc.ie/mlst/dbs/Ecoli/.Phylogenetic inferences about ancestral allelic profiles and strain interrelatedness were made using eBURST version 3 http://eburst.mlst.net/ and ClonalFrame version 1.1 http://www.xavierdidelot.xtreemhost.com/clonalframe.htm , . Sequence type complexes were defined using eBURST as groups sharing at least six identical alleles and bootstrapping with 1000 samplings. EAEC strains in this study were mapped onto the phylogeny of the E. coli species as a whole using MLST data available from http://mlst.ucc.ie/mlst/dbs/Ecoli/. EAEC MLST data was further analyzed by ClonalFrame to investigate relationships among different sequence type complexes. ClonalFrame is a Bayesian method of constructing evolutionary histories that takes both mutation and recombination into account. For each analysis, four independent runs of the Markov chain were employed and runs were compared by the method of Gelman and Rubin . Calculated Gelman-Rubin statistics for all parameters were below 1.20, indicating satisfactory convergence between tree replicates. A 75% consensus tree was created for the EAEC isolates. Mutation and recombination rates were computed using ClonalFrame as described by Didelot and Falush . […]

Pipeline specifications

Software tools BURST, ClonalFrame
Databases MLST.net
Applications Phylogenetics, WGS analysis
Organisms Escherichia coli, Homo sapiens