Abstract
BackgroundAnalysis of single nucleotide polymorphisms (SNPs) derived from whole-genome studies allows for rapid evaluation of genome-wide diversity, and genomic epidemiology studies of Plasmodium falciparum provide insights into parasite population structure, gene flow, drug resistance and vaccine development. In areas with adequate cold chain facilities, large volumes of leukocyte-depleted patient blood can be frozen for use in parasite genomic analyses. In more remote endemic areas smaller volumes of infected blood are taken by finger prick, and dried and stored on filter paper. These dried blood spots do not generally yield enough concentrated parasite DNA for whole-genome sequencing.ResultsA DNA microarray was designed for use on field samples to type a genome-wide set of SNPs which prior sequencing had shown to be variable in Africa, Southeast Asia, and Papua New Guinea. An algorithm was designed to call SNPs in samples with low parasite DNA. With this new algorithm SNP-calling accuracy of 98% was measured by hybridizing purified DNA from malaria lab strains and comparing calls with SNPs called from full genome sequences. An average accuracy of >98% was likewise obtained for DNA extracted from malaria field samples collected in studies in Southeast Asia, with an average call rate of > 82%.ConclusionThis new high-density microarray provided high quality SNP calls from a wide range of parasite DNA quantities, and represents a robust tool for genome-wide analysis of malaria parasites in diverse settings.
Highlights
Analysis of single nucleotide polymorphisms (SNPs) derived from whole-genome studies allows for rapid evaluation of genome-wide diversity, and genomic epidemiology studies of Plasmodium falciparum provide insights into parasite population structure, gene flow, drug resistance and vaccine development
Microarrays have been developed for genotyping Plasmodium falciparum, using a variety of platforms that detect single nucleotide polymorphisms (SNPs) at loci that had been described at the time of platform design [9,10,11,12]
Samples with low parasitemia (
Summary
Analysis of single nucleotide polymorphisms (SNPs) derived from whole-genome studies allows for rapid evaluation of genome-wide diversity, and genomic epidemiology studies of Plasmodium falciparum provide insights into parasite population structure, gene flow, drug resistance and vaccine development. In more remote endemic areas smaller volumes of infected blood are taken by finger prick, and dried and stored on filter paper These dried blood spots do not generally yield enough concentrated parasite DNA for whole-genome sequencing. Recent advances in genome sequencing technology offer high-throughput methods for obtaining full genomic data [7,8] These technologies still require relatively large quantities of high quality DNA. Field samples unsuitable for Microarrays have been developed for genotyping Plasmodium falciparum, using a variety of platforms that detect single nucleotide polymorphisms (SNPs) at loci that had been described at the time of platform design [9,10,11,12]. We chose to use a custom NimbleGen 4.2 million probe design in multi-plex format This platform is comprised of 12 identical arrays on one slide, each capable of genotyping 33,716 loci within the P. falciparum genome. When dual-color labeling is used, two samples can be hybridized to a single array yielding 33,716 SNPs for 24 samples in a single 2-day experiment, where several such slides can be run simultaneously, making this approach relatively highthroughput and inexpensive
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