UC San Diego

Antimicrobial resistance (AMR) is becoming an increasingly important issue in healthcare, and a slowdown in discovery of new antibiotics necessitates an understanding of the development of this resistance. While there are various methods with which to study drug resistance, and various subtopics in this broad field, we have focused on understanding genetic mutations within bacterial pathogens. Here, we analyzed longitudinal isolates of bacterial pathogens within patients, as the patients are being treated with antibiotics. The first step in finding mutations is creating a high-quality reference genome. Reference genomes are often made with solely short-read DNA sequencing, but utilizing long-read DNA sequencing in tandem with short-read sequencing can create a higher-quality genome. We employed long-read fragments to create a scaffold with which we aligned the short reads more accurately. Using the relatively new Oxford Nanopore MinION device, we began doing long-read sequencing in-house and found it to be an effective substitute for the more expensive PacBio long-read sequencing. We have slowly been developing a protocol for both wet and dry lab methods in acquiring and utilizing long-reads. For our longitudinal isolates, we used Illumina sequencing on all the samples and MinION sequencing on the base, or first, sample of each set of isolates. After creating a reference genome of the base strain, we compared short-read sequences from the subsequent isolates to the base strain. Collectively, these tools and methods allowed us to develop a pipeline for determining genetic mutations in longitudinal isolates of bacterial pathogens. This pipeline provides a starting point for understanding the pathogen’s phenotype and the relationship between antibiotics and bacterial evolution.