AbstractBackgroundStandard protocols for Pulsed-field gel electrophoresis (PFGE) were adopted in a global scale for molecular sub-typing and outbreak investigations for many pathogenic bacterial species. Whole genome sequencing (WGS) is playing significant rules for the same mentioned purposes and much more. But the results of the two typing methods are being compared separately. In PFGE; matched bands are considered regardless of co-migration of different DNA fragments. A new algorithm for PFGE image analysis that enabled calculating the number of DNA fragments in each PFGE band is described. The algorithm is based on pixel densities of single DNA fragments. Reliability of the method was assessed in-silico using computer simulation approach. 931 whole chromosome sequences (representing 5 bacterial species) were digested by 11 restriction enzymes for each species as recommended by PulseNet international. The simulation was carried out at two levels of accuracy named critical co-migration thresholds (CCTs) assuming that at the first CCT; DNA fragments differ by >= 10% are separated while at the second CCT, fragments differ by >= 5% are separated.ResultsTotal number of bands at 10% CCT was 21,395 PFGE band but at 5% CCT the figure increased to 29,381 bands (by 37.33%). Single bands were found to comprise 64.2% and 45.8% at 5% and 10% CCT respectively. PFGE coverage of >= 95% of whole chromosome sizes comprised 75.26%. While, 83.17% of all digestion models have more than ten and less than 30 bands. A prototype database for comparing PFGE results to whole chromosome enzyme digestion models is deployed (web link: geltowgs.uofk.edu).ConclusionsThe described PFGE image analysis algorithm reveals number of DNA fragments rather than just determine whether a band exists or none exists. Our simulation results have shown reasonable similarities when compared to previously published wet-lab PFGE results. Linking PFGE data to WGS digestion models may further enrich our knowledge about bacterial evolution, population dynamics in addition to reducing the high costs of WGS. Simulation approaches presented in this work may provide a better framework for in-silico evaluation of currently used or future PFGE protocols.

The author describes a new image analysis algorithm that enables identification of how many DNA fragments comigrate during PFGE. The method is named factor of co-migration based on exponential correlation between single-fragment bands and their pixel densities "FCM-ECSB".

The author describes a new image analysis algorithm that enables identification of how many DNA fragments co-migrate during PFGE. The method is named factor of co-migration based on exponential correlation between single-fragment bands and their pixel densities "FCM-ECSB".