Schwabl, P. et al. (2020) Culture-free genome-wide locus sequence typing (GLST) provides new perspectives on Trypanosoma cruzi dispersal and infection complexity. PLoS Genetics, 16(12), e1009170. (doi: 10.1371/journal.pgen.1009170) (PMID:33326438) (PMCID:PMC7743988)
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Abstract
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.
| Item Type: | Articles |
|---|---|
| Status: | Published |
| Refereed: | Yes |
| Glasgow Author(s) Enlighten ID: | Schwabl, Philipp and Llewellyn, Professor Martin |
| Creator Roles: | Schwabl, P.Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration Llewellyn, M. S.Conceptualization, Funding acquisition, Methodology, Project administration, Supervision |
| Authors: | Schwabl, P., Maiguashca Sánchez, J., Costales, J. A., Ocaña-Mayorga, S., Segovia, M., Carrasco, H. J., Hernández, C., Ramírez, J. D., Lewis, M. D., Grijalva, M. J., and Llewellyn, M. S. |
| College/School: | College of Medical Veterinary and Life Sciences > School of Biodiversity, One Health & Veterinary Medicine |
| Journal Name: | PLoS Genetics |
| Publisher: | Public Library of Science |
| ISSN: | 1553-7390 |
| ISSN (Online): | 1553-7404 |
| Published Online: | 16 December 2020 |
| Copyright Holders: | Copyright © 2020 Schwabl et al. |
| First Published: | First published in PLoS Genetics 16(12): e1009170 |
| Publisher Policy: | Reproduced under a Creative Commons License |
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Funder and Project Information
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