High-Precision, Whole-Genome Sequencing of Laboratory Strains Facilitates Genetic Studies

Abstract

Whole-genome sequencing is a powerful technique for obtaining the reference sequence information of multiple organisms. Its use can be dramatically expanded to rapidly identify genomic variations, which can be linked with phenotypes to obtain biological insights. We explored these potential applications using the emerging next-generation sequencing platform Solexa Genome Analyzer, and the well-characterized model bacterium Bacillus subtilis. Combining sequencing with experimental verification, we first improved the accuracy of the published sequence of the B. subtilis reference strain 168, then obtained sequences of multiple related laboratory strains and different isolates of each strain. This provides a framework for comparing the divergence between different laboratory strains and between their individual isolates. We also demonstrated the power of Solexa sequencing by using its results to predict a defect in the citrate signal transduction pathway of a common laboratory strain, which we verified experimentally. Finally, we examined the molecular nature of spontaneously generated mutations that suppress the growth defect caused by deletion of the stringent response mediator relA. Using whole-genome sequencing, we rapidly mapped these suppressor mutations to two small homologs of relA. Interestingly, stable suppressor strains had mutations in both genes, with each mutation alone partially relieving the relA growth defect. This supports an intriguing three-locus interaction module that is not easily identifiable through traditional suppressor mapping. We conclude that whole-genome sequencing can drastically accelerate the identification of suppressor mutations and complex genetic interactions, and it can be applied as a standard tool to investigate the genetic traits of model organisms.

Author Summary

In this manuscript, we describe novel applications of the newly developed Solexa sequencing technology. We aim to provide insights into the following questions: (1) Can whole-genome sequencing, while rapidly surveying mega-bases of genome information, also reliably identify variations at the base-pair resolution? (2) Can it be used to identify the differences between isolates of the same laboratory strain and between different laboratory strains? (3) Can it be used as a genetic tool to predict phenotypes and identify suppressors? To this end, we performed whole-genome shotgun sequencing of several related strains of the widely studied model bacterium Bacillus subtilis, we identified genomic variations that potentially underlie strain-specific phenotypes, which occur frequently in biological studies, and we found multiple suppressor mutations within a single strain that are difficult to discern through traditional methods. We conclude that whole-genome sequencing can be directly used to guide genetic studies.