Our work in this area has two broad areas of focus: (1) application of nanopore sequencing technologies to MDR plasmids from clinical isolates, and (2) application of RNA-seq to understand gene expression from these plasmids. In this research, we seek both to evaluate sequencing methods that can be used practically in a clinical microbiology lab and to study the biology of plasmid gene expression. A longstanding problem in the sequencing of bacterial isolates is the difficulty (or impossibility) of assembling plasmids to closure using short read sequencing technologies. Given that the virulence factors and resistance genes constituting the most clinically important parts of pathogen genomes are carried on plasmids, a practical, bench-top solution to plasmid sequencing would be valuable for infectious disease diagnostics and epidemiology in the clinical microbiology lab. Benchtop nanopore sequencing technologies offer a potential practical solution to this problem given their very long-read sequencing capabilities. Work done during the current fiscal year involved developing and optimizing SOPs for the nanopore sequencing device and benchmarking performance against known reference sequences. Global gene expression analysis using NGS technology (RNA-seq) offers a powerful approach to study the large-scale structure of transcription and transcriptional regulation. In these experiments, we are employing strand-specific methods to distinguish antisense from overlapping sense expression, and regulatory antisense RNA expression will be studied with this technique. Work done during the past fiscal year involved developing and optimizing RNA-seq SOPs. Work during the current fiscal year has produced rich RNA-seq data sets from plasmids from a number of clinical isolates, currently under analysis.
