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. Author summary This study details a rapid and cost-effective amplicon sequencing-based approach to measuring genome-wide DNA polymorphism in pathogenic microorganisms. Library preparation is completed in two simple polymerase chain reactions and thus avoids significant costs and biases of cell purification and culturing procedures typically involved prior to the sequencing of obligate parasite genomes. An emphasis on reaction multiplexability during primer panel design enables efficient genome-wide target amplification directly from infection source DNA. We provide proof-of-principle by genotyping hundreds of single-nucleotide polymorphisms in the Chagas disease agent Trypanosoma cruzi using metagenomic DNA extracts from infected triatomine (kissing bug) intestinal material collected in Colombia, Venezuela and Ecuador. We also evaluate method performance using reference clone DNA. Results distinguish T . cruzi population structure and diversity patterns from within-city to cross-country scales and recapitulate ancestral relationships among the sub-lineages TcI, TcIII, TcIV, TcV and TcVI. Unbalanced alternate allele frequency distributions repeatedly measured in a subset of samples also suggest potential to distinguish co-infection by multiple TcI strains. We discuss further applications as well as possibilities for method refinement.
【초록키워드】 Infectious diseases, Sequencing, Genome, Genetic, Infection, polymorphism, Infectious disease, parasites, polymerase chain reaction, amplification, DNA, Culture, pathogen, Surveillance, vector, Genetic variation, Research, Co-infection, Single-nucleotide polymorphisms, SNP, target, epidemiological, genomes, epidemiological surveillance, Ecuador, correlation, distribution, information, Spectrum, genotyping, lineages, Strains, marker, Genetic polymorphism, Pathogenic microorganisms, Chagas disease, Venezuela, capture, Chain Reaction, Trypanosoma cruzi, polymerase chain reactions, infection source, frequency distributions, Ex vivo, locus, principle, city, complex, sequence, correlations, powerful tool, limitation, primer, biases, library, minimum, purification, clone, parasite, hotspot, agarose gel, Trypanosoma, infecting, Complete, approach, Cell, intestinal, polymerase chain, flexible, Result, analyzed, identify, detect, collected, evaluate, involved, subsequent, generate, amplify, representing, subset, alternate allele frequency, capture method, metagenomic, single-nucleotide, target pathogen, 【제목키워드】 Infection, dispersal, locus, Perspective, sequence, provide,