Computational protocol: Separate F-Type Plasmids Have Shaped the Evolution of the H30 Subclone of Escherichia coli Sequence Type 131

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Protocol publication

[…] Genome assemblies from PacBio data were created using HGAP 3 as part of SMRTAnalysis version 2.2. Assemblies were subjected to three rounds of polishing with Quiver. Completed and circular chromosomes or plasmids were reoriented to their replicon (for plasmids) or the origin of replication (for chromosomes) and subjected to a final round of polishing with Quiver. Genome annotation was performed using the National Center for Biotechnology Information (NCBI) Prokaryotic Genomes Automatic Annotation Pipeline (PGAAP; http://www.ncbi.nlm.nih.gov/genome/annotation_prok/) followed by manual curation for F-type plasmids. XPlasMap was used to construct plasmid maps (http://www.iayork.com/Widgets.shtml#XPlasMap). Resistance genes were identified using the CARD antibiotic resistance database (). Plasmid types were identified using PlasmidFinder (). F plasmid sequence types were identified using pMLST (). [...] Comparisons of the completed sequences of ST131 plasmids with the existing collection of 104 ST131 draft genome assemblies were performed using the CGView comparison tool (). Similarities between plasmid genomes were calculated using nucleotide BLAST with a 1,000-bp sliding window and an E value of 1 × 10−6. Circular comparison maps were then generated. Completed plasmids and reference sequences were aligned using MAUVE (). Core single nucleotide variants from this alignment were extracted from nonrepetitive regions and analyzed using maximum parsimony methods with 1,000 bootstrap replicates in MEGA5 (). […]

Pipeline specifications

Software tools SMRT-Analysis, PGAP, PlasmidFinder, pMLST, CCT, Mauve, MEGA
Applications WGS analysis, Nucleotide sequence alignment, Genome data visualization
Organisms Escherichia coli, Homo sapiens
Diseases Infection
Chemicals Fluoroquinolones