A large-scale whole-genome sequencing analysis reveals false positives of bacterial essential genes.

Essential genes are crucial for bacterial viability and represent attractive targets for novel anti-pathogen drug discovery. However, essential genes determined by the transposon insertion sequencing (Tn-seq) approach often contain many false positives. We hypothesized that some of those false positives are genes that are actually deleted from the genome, so they do not present any transposon insertion in the course of Tn-seq analysis. Based on this assumption, we performed a large-scale whole-genome sequencing analysis for the bacterium of interest. Our analysis revealed that some "essential genes" are indeed removed from the analyzed bacterial genomes. Since these genes were kicked out by bacteria, they should not be defined as essential. Our work showed that gene deletion is one of the false positive sources of essentiality determination, which is apparently underestimated in previous studies. We suggest subtracting the genome backgrounds before the evaluation of Tn-seq, and created a list of false positive gene essentiality as a reference for the downstream application. KEY POINTS: • Discovery of false positives of essential genes defined previously through the analyses of a large scale of whole-genome sequencing data • These false positives are the results of gene deletions in the studied genomes • Sequencing the target genome before Tn-seq analysis is of importance while some studies neglected it.