Computational protocol: Genomic Comparison of Translocating and Non-Translocating Escherichia coli

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Protocol publication

[…] Genomic DNA of all six strains was sequenced at the Australian Genome Research Facility (AGRF) using an Illumina MiSeq to produce paired-end 101-bp reads. Read quality was checked with FASTQC and Spades 3.0.0 was used to assemble the E. coli genomes with k-mer values of 15, 21, 33, 51 and 71 [] The evolutionary relationship of the E. coli strains sequenced in this study compared to 29 complete E. coli and one E. fergusonnii genome was predicted by phylogenetic analysis using concatenated nucleotide sequences of seven housekeeping genes (adk, fumC, gyrB, icd, mdh, purA and recA), as previously described []. Sequences were aligned in Muscle v3.8.31 with default settings. The Neighbour-Joining method of MEGA5 was used to infer the evolutionary history, with distances computed by the Jukes-Cantor method. The phylogenetic tree was rooted using E. fergusonnii as an out-group. [...] The Rapid Annotation using Subsystems Technology (RAST) server provided annotation for the six E. coli genomes. Manual curation was also performed to ensure the accuracy of the annotation with particular attention to regions of difference, prophage regions and genomic islands using Artemis []. BLAST ring image generator (BRIG) [], Easyfig [] and Artemis Comparison Tool (ACT) [] were used to visualise the comparison of the six E. coli genomes sequenced in this study and 41 previously completed E. coli genomes (). The presence/absence of chaperone-usher (CU) fimbriae gene clusters were determined with BLAST using 38 CU fimbrial operons defined in Wurpel et al [] and visualized with BRIG. […]

Pipeline specifications

Software tools FastQC, SPAdes, RAST, BRIG, Easyfig, ACT
Applications Phylogenetics, Genome data visualization
Organisms Sus scrofa, Escherichia coli, Rattus norvegicus, Homo sapiens