New approaches to sequence genomes of Mycobacterium leprae from clinical and field specimens

Project Summary Culture-based diagnostics for the leprosy-causing agent Mycobacterium leprae are challenged by our inability to culture the pathogen in vitro. Moreover, obtaining a sufficient quantity of bacilli relies on in vivo models such as mice or armadillos in a process that requires significant resources and time because of the slow growth of the pathogen. This extended timeframe, coupled with our inability to engineer recombinant M. leprae strains in vitro, presents significant obstacles to investigating the mechanisms behind its pathogenicity and drug resistance. Furthermore, to thoroughly study M. leprae's epidemiology, transmission, and evolution, it is imperative to sequence M. leprae stains from all clinical and field origins. However, obtaining sufficient bacterial DNA from human and animal samples is challenging. Over the past decade, extensive efforts have been dedicated to enhancing our ability to sequence M. leprae genomes. Currently, two methods are employed for this purpose, but they are limited in either their sensitivity, applicability to different tissue samples, or high cost. As a result, these limitations are currently impeding the widespread use of genome sequencing in leprosy research. We propose to develop a new method based on the selective whole genome amplification (SWGA) of a minuscule quantity of M. leprae directly from complex samples. Our preliminary data on DNA collected from laboratory-infected armadillos shows that SWGA could combine sensitive, accurate, easy-to-implement, and cost-effective characteristics to successfully sequence M. leprae from complex samples. This exploratory project aims to develop the SWGA method and compare it with the gold standard bait capture to validate its use to sequence the genome of M. leprae from infected humans and animals. Aim 1 will evaluate the sensitivity and accuracy of SWGA combined with short-read sequencing and its applicability to long-read sequencing on M. leprae DNA extracted from laboratory and naturally infected armadillo tissues. Aim 2 will generate and validate the necessary reagents to perform SWGA on M. leprae DNA extracted from invasive (skin biopsy) and non-invasive (nasal swab) human samples.