Computational protocol: Extragenic suppressor mutations in ΔripA disrupt stability and function of LpxA

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Protocol publication

[…] Genomic DNA was purified from 10 ml cultures of F. tularensis grown in CDM using the MasterPure DNA purification kit (Epicentre). DNA was dissolved in 200 μl of EB buffer at 50 ng μl−1 and submitted for whole genome sequencing at the High-Throughput Sequencing Facility at the University of North Carolina at Chapel Hill. Using the Genome Analyzer IIx (Illumina) we produced 36 bp single-end reads that were mapped to the annotated genome on NCBI (NC_007880). By aligning the reads from the suppressor mutant strain to this reference we could identify the suppressor mutation. Alignments were made using SOAP with default parameters. Average sequence coverage was over 50 for the 1.89 Mb genome []. Single nucleotide polymorphisms, deletions and insertions were located using SOAP and BLAT []. A total of 4 mutations and 55 zero coverage regions were identified. Mutations also present in our wild type laboratory strain and the ΔripA strain were discarded as background mutations, leaving one unique mutation present in S102. To verify all polymorphisms detected by Illumina sequencing we PCR amplified the regions of interest and sequenced all strains including wild type F. tularensis, ΔripA, and all suppressors (Genewiz, Inc.). Following further confirmatory sequencing all but one mutation were eliminated as background mutations. […]

Pipeline specifications

Software tools SOAP, BLAT
Application WGS analysis
Organisms Francisella tularensis, Homo sapiens
Chemicals Uridine Diphosphate