Bacterial genetics and molecular pathogenesis in the age of high throughput DNA sequencing
Graphical abstract
Section snippets
Whole genome sequencing
The options for microbial whole genome sequencing (WGS) include platforms for shorts reads, primarily Illumina, as well as ‘third generation’ DNA sequencing platforms, such as PacBio’s single molecule real-time sequencing (SMRT) [1] and Oxford Nanopore Technology (ONT) [2••]. Illumina sequencing reads can be used for de novo genome assemblies; however, its short reads can fail to assemble repetitive or transposed regions of a genome. In contrast, SMRT and ONT produce long reads that span
Transposon insertion sequencing
Genomic sequences provide insight into gene content, but gene function often relies on homologies to genes previously characterized in model organisms. A powerful tool for functional analysis of genes is transposon mutagenesis. Typically, transposon (Tn) mutants are selected or screened for a particular altered function and the location of the Tn insertion in the genome is then determined using various PCR-based methods. Tn-insertion sequencing (abbreviated TIS herein) can be used to identify
Chemical mutagenesis and whole genome sequencing
For many microbes the necessary genetic tools or DNA delivery mechanisms for Tn mutagenesis do not exist. One method to circumvent the need for molecular genetic tools is to induce mutagenesis through DNA damaging agents and mapping the resulting mutations by WGS [65]. Mutagens such as UV radiation and the DNA alkylating compounds ethyl methanesulfonate (EMS) and N-ethyl-N-nitrosourea (ENU) introduce point mutations randomly throughout the genome, which can inactivate or truncate a gene. As in
Large scale transcriptional profiling (RNA-seq)
High throughput sequencing has had a particularly transformative impact on the analysis of gene expression. RNA-seq has long replaced microarray as the standard method to assess global patterns of gene expression. While alternative methods such as direct sequencing of RNA molecules with ONT [38], and cDNA sequencing with SMRT [72] have recently been described, Illumina sequencing of cDNAs remains the most approach for RNA-seq. With high levels of sequencing depth, RNA-seq can to deliver
Future directions
Next generation DNA sequencing has transformed bacterial genetics and opened previously intractable organisms to genetic analysis. As technologies develop, costs decrease and more robust bioinformatic analysis tools emerge, we expect that increased applications for WGS in single bacterial cell analysis, to reveal heterogeneity in populations, and to facilitate analysis of unculturable organisms. For example, single bacteria WGS is possible [84] and technical advances in cell isolation and
Conflict of interest statement
RHV is a member of the Scientific Advisory Board of Meridian Bioscience. Meridian had no input in the writing of thus manuscript.
References and recommended reading
Papers of particular interest, published within the period of review, have been highlighted as:
• of special interest
•• of outstanding interest
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