High-throughput genotyping and fingerprinting of mycobacterium tuberculosis multi-drug resistant strains

Thesis (Ph.D.)--Boston UniversityPLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you.Multiple drug resistance in Mycobacterium tuberculosis poses both significant treatment and epidemiological challenges. Measuring drug resistance in clinical settings is time consuming and prone to errors, problems that can lead to suboptimal treatments and the selection of further resistance to an increased number of antibacterial drugs. A fast and accurate genotyping assay, directed at mutations that are highly associated with drug-resistance, would improve response time and the choice of drugs used to treat multiple drug resistant tuberculosis. From an epidemiologic perspective, tracking the origin and dynamics of drug resistant strains in outbreaks is also a challenge and existing methods fall short because they lack resolution (spoligtyping) or are too expensive or labor-intensive to implement on a large scale (RFLP, MIRU-VNTR). In this work, I developed methods to adapt and expand a high throughput targeted resequencing method based on molecular inversion probes and subsequent Illumina sequencing, to cover 28 protein and rRNA-coding genes described previously as primary and secondary actors in drug resistance. I validated the method on a control set, compared it with traditional Sanger sequencing and whole-genome Illumina sequencing and applied it to a collection of 1200 drug resistant Mycobacterium tuberculosis strains from all over the world. This project was funded by the Bill and Melinda Gates Foundation and the result of our work will be freely available as a resource to the research community through a website hosted by the Broad Institute. For this project, I have written, tested and optimized algorithms for large-scale molecular inversion probe design (MIPDesigner), for next-generation sequence data processing (MIPCleaner), for SNP filtering, and for quality-control metric computation. Molecular inversion probes also provide a mechanism for rapid, high-throughput, molecular fingerprinting of Mycobacterium tuberculosis strains, that can be performed simultaneously with the detection drug resistance mutations. I used my optimized MIPs pipeline to design and test a "virtual spoligotyping" method based on the capture and sequencing of the spacers in the CRISPR locus with a molecular inversion probe. This new method expands the resolution and power of the classical spoligotyping assay and provides a mechanism for the continuous improvement of Mycobacterium tuberculosis fingerprinting.2031-01-0