Culture-free genome-wide locus sequence typing (GLST) provides new perspectives on Trypanosoma cruzi dispersal and infection complexity

Autoři: Philipp Schwabl aff001;  Jalil Maiguashca Sánchez aff002;  Jaime A. Costales aff002;  Sofía Ocaña-Mayorga aff002;  Maikell Segovia aff003;  Hernán J. Carrasco aff003;  Carolina Hernández aff004;  Juan David Ramírez aff004;  Michael D. Lewis aff005;  Mario J. Grijalva aff002;  Martin S. Llewellyn aff001
Působiště autorů: Institute of Biodiversity, Animal Health & Comparative Medicine, University of Glasgow, Glasgow, United Kingdom aff001;  Centro de Investigación para la Salud en América Latina, Pontificia Universidad Católica del Ecuador, Quito, Ecuador aff002;  Laboratorio de Biología Molecular de Protozoarios, Instituto de Medicina Tropical, Universidad Central de Venezuela, Caracas, Venezuela aff003;  Grupo de Investigaciones Microbiológicas-UR (GIMUR), Departamento de Biología, Facultad de Ciencias Naturales, Universidad del Rosario, Bogotá, Colombia aff004;  London School of Hygiene & Tropical Medicine, Keppel Street, London, United Kingdom aff005;  Infectious and Tropical Disease Institute, Biomedical Sciences Department, Heritage College of Osteopathic Medicine, Ohio University, Athens, OH, United States of America aff006
Vyšlo v časopise: Culture-free genome-wide locus sequence typing (GLST) provides new perspectives on Trypanosoma cruzi dispersal and infection complexity. PLoS Genet 16(12): e1009170. doi:10.1371/journal.pgen.1009170
Kategorie: Research Article
doi: 10.1371/journal.pgen.1009170


Analysis of genetic polymorphism is a powerful tool for epidemiological surveillance and research. Powerful inference from pathogen genetic variation, however, is often restrained by limited access to representative target DNA, especially in the study of obligate parasitic species for which ex vivo culture is resource-intensive or bias-prone. Modern sequence capture methods enable pathogen genetic variation to be analyzed directly from host/vector material but are often too complex and expensive for resource-poor settings where infectious diseases prevail. This study proposes a simple, cost-effective ‘genome-wide locus sequence typing’ (GLST) tool based on massive parallel amplification of information hotspots throughout the target pathogen genome. The multiplexed polymerase chain reaction amplifies hundreds of different, user-defined genetic targets in a single reaction tube, and subsequent agarose gel-based clean-up and barcoding completes library preparation at under 4 USD per sample. Our study generates a flexible GLST primer panel design workflow for Trypanosoma cruzi, the parasitic agent of Chagas disease. We successfully apply our 203-target GLST panel to direct, culture-free metagenomic extracts from triatomine vectors containing a minimum of 3.69 pg/μl T. cruzi DNA and further elaborate on method performance by sequencing GLST libraries from T. cruzi reference clones representing discrete typing units (DTUs) TcI, TcIII, TcIV, TcV and TcVI. The 780 SNP sites we identify in the sample set repeatably distinguish parasites infecting sympatric vectors and detect correlations between genetic and geographic distances at regional (< 150 km) as well as continental scales. The markers also clearly separate TcI, TcIII, TcIV and TcV + TcVI and appear to distinguish multiclonal infections within TcI. We discuss the advantages, limitations and prospects of our method across a spectrum of epidemiological research.

Klíčová slova:

Cloning – DNA cloning – Heterozygosity – Polymerase chain reaction – Satellite DNA – Single nucleotide polymorphisms – Trypanosoma cruzi – Variant genotypes


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