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.
Highlights
Genome-wide single nucleotide polymorphism (SNP) analysis is a powerful and increasingly common approach in the study and surveillance of infectious disease
Library preparation is completed in two simple polymerase chain reactions and avoids significant costs and biases of cell purification and culturing procedures typically involved prior to the sequencing of obligate parasite genomes
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 intestinal material collected in Colombia, Venezuela and Ecuador
Summary
Genome-wide single nucleotide polymorphism (SNP) analysis is a powerful and increasingly common approach in the study and surveillance of infectious disease. Sequencing is rarely viable directly from the infection source and studies have often found it necessary to isolate and culture the target organism to higher densities before extracting DNA. These additional steps, are resource-intensive and bias-prone. Genomic sequencing data on the protozoan parasite Leishmania infantum, for example, has for such reasons come to exhibit considerable selection bias towards aggressive strains isolated by invasive sampling from canine hosts. Karyotypic changes arise during T. cruzi micromanipulation and axenic growth [13,14] These effects in culture have confounded efforts to associate genetic variability and sub-lineage taxonomy to important clinical and eco-epidemiological traits (see further below) [15]
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