Computational protocol: Candidate Essential Genes in Burkholderia cenocepacia J2315 Identified by Genome-Wide TraDIS

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Protocol publication

[…] Sequence reads from the Illumina FASTQ files were filtered for 10 bases matching the 3′ end of the transposon (TAAGAGACAG) with one mismatch allowed. The transposon tag was removed from the matching reads and the reads were then mapped to the Bcc J2315 reference sequence (EMBL accession numbers AM747720, AM747721, AM747722, and AM747723) using SMALT-0.7.2. The precise transposon insertion site (TIS) was determined using Bio::Tradis. Gene essentiality was assessed as previously described (). Briefly, an insertion index (number of insertion sites divided by gene length) was calculated for each gene. Insertion indexes were observed to fit a bimodal distribution corresponding to two essential and non-essential sets, each of the modes was modeled using a gamma distribution or an exponential distribution to fit genes with no observed insertion sites. Log2 likelihood ratios (LLR) were calculated between the essential and non-essential models and a gene was classified as essential if it had an LLR of < -2. Similarly, a gene was assigned as non-essential if it had an LLR of >2.The search against available essential genes available in the database of essential genes (DEG; version 13.3, updated on January 7, 2016; ) was performed using BLASTP with the default parameters provided in DEG. BLAST similarities at protein-protein level that resulted in E-values of 10-10 or less, were considered matches. B. pseudomallei and B. thailandensis orthologs of B. cenocepacia genes were obtained from the Burkholderia Genome Database (). For growth curves, doubling time (g) was calculated from the exponential phase using the formula: g = t log 2/(log Nt – log N0) where N0 = number of CFU at a point during log phase, Nt = number of CFU at a different time point during log phase and t = time interval between N0 and Nt. The data were expressed as mean ± standard error of the mean (SE) from two independent assays. Statistical analyses were performed using the unpaired, two-tailed Student’s t-test. […]

Pipeline specifications

Software tools SMALT, Bio-Tradis, BLASTP
Databases DEG
Application Tn-seq analysis
Organisms Burkholderia cenocepacia J2315, Burkholderia cenocepacia
Diseases Cystic Fibrosis, Lymphatic Diseases, Burkholderia Infections