DNA sequencing: Methods, strategies and protocols

DNA sequencing is a common and requisite practice for molecular biologists today working in all areas of biology, including microbiology. A number of nucleotide and amino acid-based sequence typing methods are now widely recognized and have transformed the way in which complex evolutionary and epidemiological relationships are elucidated among bacterial strains. Recent advances in DNA sequencing underpin a vast array of bacterial subtyping methods and have become an integral component to traceback investigations of foodborne outbreaks caused by specific bacterial pathogenic strains. Direct DNA sequence-based approaches include multi-locus sequence analysis (MLSA), which simultaneously analyzes the nucleotide substitutions found within concatenated sequence alignments from multiple housekeeping genes. This collective and simultaneous analysis of nascent single nucleotide polymorphisms (SNPs) is effective in differentiating highly homogeneous strain clusters including the closely related strains and serovars of Salmonella enterica. In addition to the extraordinary power this approach offers in molecular evolution/epidemiology, the study of concatenated gene sequences has been instrumental in defining a new paradigm for horizontal gene transfer in the genetic structure of many bacterial species. Facile variations of MLSA are now being developed that rely on the culling of SNP-based targets from relatively uncharted and under-exploited regions of the bacterial genome. As an example, SNPs that reside in the selectively neutral intergenic non-transcribed spacer regions of the genome hold great promise in resolving bacterial strains down to a level of epidemiological relevance. In parallel with these advances in nucleotide sequence analysis, the development of rapid and highly accurate SNP discovery and detection tools is and includes methods such as Pyrosequencing, Luminex probe recognition technology, and single-basepair primer extension assays to name but a few. Together, these successes clearly position DNA sequence-based strategies at the helm for future discoveries in the genetics, evolution, and epidemiology of bacterial strains and species.