Gene classifications for S.

The identification of essential genes in Transposon Directed Insertion Site Sequencing (TraDIS) data relies on the assumption that transposon insertions occur randomly in non-essential regions, leaving essential genes largely insertion-free. While intragenic insertion-free sequences have been considered as a reliable indicator for gene essentiality, so far, no exact probability distribution for these sequences has been proposed. Further, many methods require setting thresholds or parameter values a priori without providing any statistical basis, limiting the comparability of results. Here, we introduce Consecutive Non-Insertion Sites (ConNIS), a novel method for gene essentiality determination. ConNIS provides an analytic solution for the probability of observing insertion-free sequences within genes of given length and considers variation in insertion density across the genome. Based on an extensive simulation study and different real-world scenarios, ConNIS was found to be superior to prevalent state-of-the-art methods, particularly when libraries had only a low or medium insertion density. In addition, our results showed that the precision of existing methods can be improved by incorporating a simple weighting factor for the genome-wide insertion density. To set methodically embedded parameter and threshold values of TraDIS methods a subsample-based instability criterion was developed. Application of this criterion in real and synthetic data settings demonstrated its effectiveness in selecting well-suited parameter/threshold values across methods. An R package and an interactive web application are provided to facilitate application and reproducibility.