Person: Volkman, Sarah
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Volkman
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Sarah
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Volkman, Sarah
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Publication De Novo Mutations Resolve Disease Transmission Pathways in Clonal Malaria(Oxford University Press, 2018) Redmond, Seth N; MacInnis, Bronwyn M; Bopp, Selina; Bei, Amy; Ndiaye, Daouda; Hartl, Daniel; Wirth, Dyann; Volkman, Sarah; Neafsey, DanielAbstract Detecting de novo mutations in viral and bacterial pathogens enables researchers to reconstruct detailed networks of disease transmission and is a key technique in genomic epidemiology. However, these techniques have not yet been applied to the malaria parasite, Plasmodium falciparum, in which a larger genome, slower generation times, and a complex life cycle make them difficult to implement. Here, we demonstrate the viability of de novo mutation studies in P. falciparum for the first time. Using a combination of sequencing, library preparation, and genotyping methods that have been optimized for accuracy in low-complexity genomic regions, we have detected de novo mutations that distinguish nominally identical parasites from clonal lineages. Despite its slower evolutionary rate compared with bacterial or viral species, de novo mutation can be detected in P. falciparum across timescales of just 1–2 years and evolutionary rates in low-complexity regions of the genome can be up to twice that detected in the rest of the genome. The increased mutation rate allows the identification of separate clade expansions that cannot be found using previous genomic epidemiology approaches and could be a crucial tool for mapping residual transmission patterns in disease elimination campaigns and reintroduction scenarios.Publication Long read assemblies of geographically dispersed Plasmodium falciparum isolates reveal highly structured subtelomeres(F1000 Research Limited, 2018) Otto, Thomas D.; Böhme, Ulrike; Sanders, Mandy; Reid, Adam; Bruske, Ellen I.; Duffy, Craig W.; Bull, Pete C.; Pearson, Richard D.; Abdi, Abdirahman; Dimonte, Sandra; Stewart, Lindsay B.; Campino, Susana; Kekre, Mihir; Hamilton, William L.; Claessens, Antoine; Volkman, Sarah; Ndiaye, Daouda; Amambua-Ngwa, Alfred; Diakite, Mahamadou; Fairhurst, Rick M.; Conway, David J.; Franck, Matthias; Newbold, Chris I.; Berriman, MattBackground:: Although thousands of clinical isolates of Plasmodium falciparum are being sequenced and analysed by short read technology, the data do not resolve the highly variable subtelomeric regions of the genomes that contain polymorphic gene families involved in immune evasion and pathogenesis. There is also no current standard definition of the boundaries of these variable subtelomeric regions. Methods:: Using long-read sequence data (Pacific Biosciences SMRT technology), we assembled and annotated the genomes of 15 P. falciparum isolates, ten of which are newly cultured clinical isolates. We performed comparative analysis of the entire genome with particular emphasis on the subtelomeric regions and the internal var genes clusters. Results:: The nearly complete sequence of these 15 isolates has enabled us to define a highly conserved core genome, to delineate the boundaries of the subtelomeric regions, and to compare these across isolates. We found highly structured variable regions in the genome. Some exported gene families purportedly involved in release of merozoites show copy number variation. As an example of ongoing genome evolution, we found a novel CLAG gene in six isolates. We also found a novel gene that was relatively enriched in the South East Asian isolates compared to those from Africa. Conclusions:: These 15 manually curated new reference genome sequences with their nearly complete subtelomeric regions and fully assembled genes are an important new resource for the malaria research community. We report the overall conserved structure and pattern of important gene families and the more clearly defined subtelomeric regions.Publication Malaria Life Cycle Intensifies Both Natural Selection and Random Genetic Drift(Proceedings of the National Academy of Sciences, 2013) Chang, Hsiao-Han; Moss, Eli L.; Park, Daniel John; Ndiaye, Daouda; Mboup, Souleymane; Volkman, Sarah; Sabeti, Pardis; Wirth, Dyann; Neafsey, Daniel; Hartl, DanielAnalysis of genome sequences of 159 isolates of Plasmodium falciparum from Senegal yields an extraordinarily high proportion (26.85%) of protein-coding genes with the ratio of nonsynonymous to synonymous polymorphism greater than one. This proportion is much greater than observed in other organisms. Also unusual is that the site-frequency spectra of synonymous and nonsynonymous polymorphisms are virtually indistinguishable. We hypothesized that the complicated life cycle of malaria parasites might lead to qualitatively different population genetics from that predicted from the classical Wright-Fisher (WF) model, which assumes a single random-mating population with a finite and constant population size in an organism with nonoverlapping generations. This paper summarizes simulation studies of random genetic drift and selection in malaria parasites that take into account their unusual life history. Our results show that random genetic drift in the malaria life cycle is more pronounced than under the WF model. Paradoxically, the efficiency of purifying selection in the malaria life cycle is also greater than under WF, and the relative efficiency of positive selection varies according to conditions. Additionally, the site-frequency spectrum under neutrality is also more skewed toward low-frequency alleles than expected with WF. These results highlight the importance of considering the malaria life cycle when applying existing population genetic tools based on the WF model. The same caveat applies to other species with similarly complex life cycles.Publication Sequence-Based Association and Selection Scans Identify Drug Resistance Loci in the Plasmodium Falciparum Malaria Parasite(Proceedings of the National Academy of Sciences, 2012) Park, Daniel John; Lukens, Amanda; Neafsey, Daniel; Schaffner, Stephen; Chang, Hsiao-Han; Valim, Clarissa; Ribacke, Ulf; Van tyne, Daria; Galinsky, Kevin; Galligan, Meghan; Becker, Justin S.; Ndiaye, Daouda; Mboup, Souleymane; Wiegand, Roger; Hartl, Daniel; Sabeti, Pardis; Wirth, Dyann; Volkman, SarahThrough rapid genetic adaptation and natural selection, the Plasmodium falciparum parasite—the deadliest of those that cause malaria—is able to develop resistance to antimalarial drugs, thwarting present efforts to control it. Genome-wide association studies (GWAS) provide a critical hypothesis-generating tool for understanding how this occurs. However, in P. falciparum, the limited amount of linkage disequilibrium hinders the power of traditional array-based GWAS. Here, we demonstrate the feasibility and power improvements gained by using whole-genome sequencing for association studies. We analyzed data from 45 Senegalese parasites and identified genetic changes associated with the parasites’ in vitro response to 12 different antimalarials. To further increase statistical power, we adapted a common test for natural selection, XP-EHH (cross-population extended haplotype homozygosity), and used it to identify genomic regions associated with resistance to drugs. Using this sequence-based approach and the combination of association and selection-based tests, we detected several loci associated with drug resistance. These loci included the previously known signals at pfcrt, dhfr, and pfmdr1, as well as many genes not previously implicated in drug-resistance roles, including genes in the ubiquitination pathway. Based on the success of the analysis presented in this study, and on the demonstrated shortcomings of array-based approaches, we argue for a complete transition to sequence-based GWAS for small, low linkage-disequilibrium genomes like that of P. falciparum.Publication Modeling malaria genomics reveals transmission decline and rebound in Senegal(Proceedings of the National Academy of Sciences, 2015) Daniels, Rachel; Schaffner, Stephen; Wenger, Edward A.; Proctor, Joshua L.; Chang, Hsiao-Han; Wong, Wesley; Baro, Nicholas; Ndiaye, Daouda; Fall, Fatou Ba; Ndiop, Medoune; Ba, Mady; Milner, Danny; Taylor, Terrie E.; Neafsey, Daniel; Volkman, Sarah; Eckhoff, Philip A.; Hartl, Daniel; Wirth, DyannTo study the effects of malaria-control interventions on parasite population genomics, we examined a set of 1,007 samples of the malaria parasite Plasmodium falciparum collected in Thiès, Senegal between 2006 and 2013. The parasite samples were genotyped using a molecular barcode of 24 SNPs. About 35% of the samples grouped into subsets with identical barcodes, varying in size by year and sometimes persisting across years. The barcodes also formed networks of related groups. Analysis of 164 completely sequenced parasites revealed extensive sharing of genomic regions. In at least two cases we found first-generation recombinant offspring of parents whose genomes are similar or identical to genomes also present in the sample. An epidemiological model that tracks parasite genotypes can reproduce the observed pattern of barcode subsets. Quantification of likelihoods in the model strongly suggests a reduction of transmission from 2006-2010 with a significant rebound in 2012-2013. The reduced transmission and rebound were confirmed directly by incidence data from Thiès. These findings imply that intensive intervention to control malaria results in rapid and dramatic changes in parasite population genomics. The results also suggest that genomics combined with epidemiological modeling may afford prompt, continuous, and cost-effective tracking of progress toward malaria elimination.Publication Human Cerebral Malaria and Plasmodium falciparum Genotypes in Malawi(BioMed Central, 2012) Milner, Danny; Vareta, Jimmy; Valim, Clarissa; Montgomery, Jacqui; Daniels, Rachel; Volkman, Sarah; Neafsey, Daniel; Park, Daniel John; Schaffner, Stephen; Mahesh, Nira C; Barnes, Kayla G; Rosen, David M; Lukens, Amanda; Van-Tyne, Daria; Wiegand, Roger; Sabeti, Pardis; Seydel, Karl B; Glover, Simon J; Kamiza, Steve; Molyneux, Malcolm E; Taylor, Terrie E; Wirth, DyannBackground: Cerebral malaria, a severe form of Plasmodium falciparum infection, is an important cause of mortality in sub-Saharan African children. A Taqman 24 Single Nucleotide Polymorphisms (SNP) molecular barcode assay was developed for use in laboratory parasites which estimates genotype number and identifies the predominant genotype. Methods The 24 SNP assay was used to determine predominant genotypes in blood and tissues from autopsy and clinical patients with cerebral malaria. Results: Single genotypes were shared between the peripheral blood, the brain, and other tissues of cerebral malaria patients, while malaria-infected patients who died of non-malarial causes had mixed genetic signatures in tissues examined. Children with retinopathy-positive cerebral malaria had significantly less complex infections than those without retinopathy (OR = 3.7, 95% CI [1.51-9.10]).The complexity of infections significantly decreased over the malaria season in retinopathy-positive patients compared to retinopathy-negative patients. Conclusions: Cerebral malaria patients harbour a single or small set of predominant parasites; patients with incidental parasitaemia sustain infections involving diverse genotypes. Limited diversity in the peripheral blood of cerebral malaria patients and correlation with tissues supports peripheral blood samples as appropriate for genome-wide association studies of parasite determinants of pathogenicity.Publication Genetic Surveillance Detects Both Clonal and Epidemic Transmission of Malaria following Enhanced Intervention in Senegal(Public Library of Science, 2013) Daniels, Rachel; Chang, Hsiao-Han; Séne, Papa Diogoye; Park, Danny C.; Neafsey, Daniel; Schaffner, Stephen; Hamilton, Elizabeth; Lukens, Amanda; Van tyne, Daria; Mboup, Souleymane; Sabeti, Pardis; Ndiaye, Daouda; Wirth, Dyann; Hartl, Daniel; Volkman, SarahUsing parasite genotyping tools, we screened patients with mild uncomplicated malaria seeking treatment at a clinic in Thiès, Senegal, from 2006 to 2011. We identified a growing frequency of infections caused by genetically identical parasite strains, coincident with increased deployment of malaria control interventions and decreased malaria deaths. Parasite genotypes in some cases persisted clonally across dry seasons. The increase in frequency of genetically identical parasite strains corresponded with decrease in the probability of multiple infections. Further, these observations support evidence of both clonal and epidemic population structures. These data provide the first evidence of a temporal correlation between the appearance of identical parasite types and increased malaria control efforts in Africa, which here included distribution of insecticide treated nets (ITNs), use of rapid diagnostic tests (RDTs) for malaria detection, and deployment of artemisinin combination therapy (ACT). Our results imply that genetic surveillance can be used to evaluate the effectiveness of disease control strategies and assist a rational global malaria eradication campaign.Publication Rapid, Field-Deployable Method for Genotyping and Discovery of Single-Nucleotide Polymorphisms Associated with Drug Resistance in Plasmodium falciparum(American Society for Microbiology, 2012) Daniels, Rachel; Ndiaye, Daouda; Wall, Mikeal; McKinney, Jason; Séne, Papa Diogoye; Sabeti, Pardis; Volkman, Sarah; Mboup, Souleymane; Wirth, DyannDespite efforts to reduce malaria morbidity and mortality, drug-resistant parasites continue to evade control strategies. Recently, emphasis has shifted away from control and toward regional elimination and global eradication of malaria. Such a campaign requires tools to monitor genetic changes in the parasite that could compromise the effectiveness of antimalarial drugs and undermine eradication programs. These tools must be fast, sensitive, unambiguous, and cost-effective to offer real-time reports of parasite drug susceptibility status across the globe. We have developed and validated a set of genotyping assays using high-resolution melting (HRM) analysis to detect molecular biomarkers associated with drug resistance across six genes in Plasmodium falciparum. We improved on existing technical approaches by developing refinements and extensions of HRM, including the use of blocked probes (LunaProbes) and the mutant allele amplification bias (MAAB) technique. To validate the sensitivity and accuracy of our assays, we compared our findings to sequencing results in both culture-adapted lines and clinical isolates from Senegal. We demonstrate that our assays (i) identify both known and novel polymorphisms, (ii) detect multiple genotypes indicative of mixed infections, and (iii) distinguish between variants when multiple copies of a locus are present. These rapid and inexpensive assays can track drug resistance and detect emerging mutations in targeted genetic loci in P. falciparum. They pro- vide tools for monitoring molecular changes associated with changes in drug response across populations and for determining whether parasites present after drug treatment are the result of recrudescence or reinfection in clinical settings.Publication A Global Transcriptional Analysis of Plasmodium Falciparum Malaria Reveals A Novel Family of Telomere-Associated lncRNAs(BioMed Central, 2011) Broadbent, Kate Mariel; Park, Daniel John; Wolf, Ashley Robin; Van tyne, Daria; Sims, Jennifer Sung; Ribacke, Ulf; Volkman, Sarah; Duraisingh, Manoj; Wirth, Dyann; Sabeti, Pardis; Rinn, JohnBackground: Mounting evidence suggests a major role for epigenetic feedback in Plasmodium falciparum transcriptional regulation. Long non-coding RNAs (lncRNAs) have recently emerged as a new paradigm in epigenetic remodeling. We therefore set out to investigate putative roles for lncRNAs in P. falciparum transcriptional regulation. Results: We used a high-resolution DNA tiling microarray to survey transcriptional activity across 22.6% of the P. falciparum strain 3D7 genome. We identified 872 protein-coding genes and 60 putative P. falciparum lncRNAs under developmental regulation during the parasite's pathogenic human blood stage. Further characterization of lncRNA candidates led to the discovery of an intriguing family of lncRNA telomere-associated repetitive element transcripts, termed lncRNA-TARE. We have quantified lncRNA-TARE expression at 15 distinct chromosome ends and mapped putative transcriptional start and termination sites of lncRNA-TARE loci. Remarkably, we observed coordinated and stage-specific expression of lncRNA-TARE on all chromosome ends tested, and two dominant transcripts of approximately 1.5 kb and 3.1 kb transcribed towards the telomere. Conclusions: We have characterized a family of 22 telomere-associated lncRNAs in P. falciparum. Homologous lncRNA-TARE loci are coordinately expressed after parasite DNA replication, and are poised to play an important role in P. falciparum telomere maintenance, virulence gene regulation, and potentially other processes of parasite chromosome end biology. Further study of lncRNA-TARE and other promising lncRNA candidates may provide mechanistic insight into P. falciparum transcriptional regulation.Publication Identification and Functional Validation of the Novel Antimalarial Resistance Locus PF10_0355 in Plasmodium falciparum(Public Library of Science, 2011) Van tyne, Daria; Park, Daniel John; Schaffner, Stephen; Neafsey, Daniel; Angelino, Elaine Lee; Cortese, Joseph F.; Barnes, Kayla G.; Rosen, David M.; Lukens, Amanda; Daniels, Rachel; Milner, Danny; Johnson, Charles A.; Shlyakhter, Ilya; Grossman, Sharon; Becker, Justin S.; Yamins, Daniel Louis Kanef; Karlsson, Elinor K; Ndiaye, Daouda; Sarr, Ousmane; Mboup, Souleymane; Happi, Christian; Furlotte, Nicholas A.; Eskin, Eleazar; Kang, Hyun Min; Hartl, Daniel; Birren, Bruce W.; Wiegand, Roger; Lander, Eric; Wirth, Dyann; Volkman, Sarah; Sabeti, PardisThe Plasmodium falciparum parasite's ability to adapt to environmental pressures, such as the human immune system and antimalarial drugs, makes malaria an enduring burden to public health. Understanding the genetic basis of these adaptations is critical to intervening successfully against malaria. To that end, we created a high-density genotyping array that assays over 17,000 single nucleotide polymorphisms (~1 SNP/kb), and applied it to 57 culture-adapted parasites from three continents. We characterized genome-wide genetic diversity within and between populations and identified numerous loci with signals of natural selection, suggesting their role in recent adaptation. In addition, we performed a genome-wide association study (GWAS), searching for loci correlated with resistance to thirteen antimalarials; we detected both known and novel resistance loci, including a new halofantrine resistance locus, PF10_0355. Through functional testing we demonstrated that PF10_0355 overexpression decreases sensitivity to halofantrine, mefloquine, and lumefantrine, but not to structurally unrelated antimalarials, and that increased gene copy number mediates resistance. Our GWAS and follow-on functional validation demonstrate the potential of genome-wide studies to elucidate functionally important loci in the malaria parasite genome.