Magnus Manske

Wellcome Sanger Institute · Malaria Programme

Malaria elimination strategies require surveillance of the parasite population for genetic changes that demand a public health response, such as new forms of drug resistance. Here we describe methods for the large-scale analysis of genetic variation in Plasmodium falciparum by deep sequencing of parasite DNA obtained from the blood of patients with...

We describe an analysis of genome variation in 825 P. falciparum samples from Asia and Africa that identifies an unusual pattern of parasite population structure at the epicenter of artemisinin resistance in western Cambodia. Within this relatively small geographic area, we have discovered several distinct but apparently sympatric parasite subpopul...

Sequencing a genome to great depth can be highly informative about heterogeneity within an individual or a population. Here we address the problem of how to visualize the multiple layers of information contained in deep sequencing data. We propose an interactive AJAX-based web viewer for browsing large data sets of aligned sequence reads. By enabli...

MalariaGEN is a data-sharing network that enables groups around the world to work together on the genomic epidemiology of malaria. Here we describe a new release of curated genome variation data on 7,000 Plasmodium falciparum samples from MalariaGEN partner studies in 28 malaria-endemic countries. High-quality genotype calls on 3 million single nuc...

MalariaGEN is a data-sharing network that enables groups around the world to work together on the genomic epidemiology of malaria. Here we describe a new release of curated genome variation data on 7,000 Plasmodium falciparum samples from MalariaGEN partner studies in 28 malaria-endemic countries. High-quality genotype calls on 3 million single nuc...

Invasion of human erythrocytes is essential for Plasmodium falciparum parasite survival and pathogenesis, and is also a complex phenotype. While some later steps in invasion appear to be invariant and essential, the earlier steps of recognition are controlled by a series of redundant, and only partially understood, receptor-ligand interactions. Rev...

Elucidation of the evolutionary history and interrelatedness of Plasmodium species that infect humans has been hampered by a lack of genetic information for three human-infective species: P. malariae and two P. ovale species (P. o. curtisi and P. o. wallikeri). These species are prevalent across most regions in which malaria is endemic and are ofte...

Background Translating genomic technologies into healthcare applications for the malaria parasite Plasmodium falciparum has been limited by the technical and logistical difficulties of obtaining high quality clinical samples from the field. Sampling by dried blood spot (DBS) finger-pricks can be performed safely and efficiently with minimal resourc...

Translating genomic technologies into healthcare applications for the malaria parasite Plasmodium falciparum has been limited by the technical and logistical difficulties of obtaining high quality clinical samples from the field. Sampling by dried blood spot (DBS) finger-pricks can be performed safely and efficiently with minimal resource and stora...

Background: Infection by the simian malaria parasite, Plasmodium knowlesi, can lead to severe and fatal disease in humans, and is the most common cause of malaria in parts of Malaysia. Despite being a serious public health concern, the geographical distribution of P. knowlesi malaria risk is poorly understood because the parasite is often misident...

S1 Fig. Covariates used in predicting the distribution of risk of Plasmodium knowlesi malaria. (TIF) A. Displays human population density. B-D. Show the relative environmental suitability for vector (the Leucosphyrus Group) and reservoir species (Macaca fascicularis and M. nemestrina) of P. knowlesi, respectively. E. Shows an index of temperature...

S4 Fig. Map of model uncertainty. (TIF) Standard deviation values for each pixel were calculated across the model ensemble. Areas from lower to higher standard deviation values are shown.

S1 File. Methods supplement. (DOCX)

S2 Fig. Land cover covariates used in predicting the distribution of risk of Plasmodium knowlesi malaria. (TIF) A-I. Displays proportional cover for 2012 of lands with croplands, croplands natural vegetation mosaics, open shrublands, permanent wetlands, grasslands, intact forest, disturbed forest, woody savannas and savannas, respectively. For det...

S6 Fig. Predicted disease risk values at locations with confirmed/unconfirmed presence and absence from outside Malaysia, Brunei and Singapore. (TIF) The black dots represent the predicted values of confirmed/unconfirmed P. knowlesi presence and absence points and violin plots showing the density of points at each predicted value are shown in grey...

S7 Fig. Marginal effect plots for the most influential covariates. (TIF) The black line represents the mean marginal effect and grey envelopes the associated 95% quantiles. The mean relative contribution is displayed in the top left corner of each plot.

S2 File. Database of Plasmodium knowlesi infections recorded in humans, macaque reservoirs and vectors. (XLSX)

S3 Fig. Fine-scale map of presumed limits of zoonotic Plasmodium knowlesi transmission. (TIF) Areas of spatial co-occurrence of known or putative reservoir (at least one of M. fascicularis, M. nemestrina or M. leonina) and vector species (members of the Leucosphyrus Group) are indicated, as well as areas where either reservoir or vector species ar...

S5 Fig. Unmasked mean model output. (TIF) Suitability for zoonotic Plasmodium knowlesi transmission from known reservoir and vector species from relative low to high suitability.

Unmasked mean model output. Suitability for zoonotic Plasmodium knowlesi transmission from known reservoir and vector species from relative low to high suitability. (TIF)

Methods supplement. (DOCX)

Land cover covariates used in predicting the distribution of risk of Plasmodium knowlesi malaria. A-I. Displays proportional cover for 2012 of lands with croplands, croplands natural vegetation mosaics, open shrublands, permanent wetlands, grasslands, intact forest, disturbed forest, woody savannas and savannas, respectively. For details of how eac...

Covariates used in predicting the distribution of risk of Plasmodium knowlesi malaria. A. Displays human population density. B-D. Show the relative environmental suitability for vector (the Leucosphyrus Group) and reservoir species (Macaca fascicularis and M. nemestrina) of P. knowlesi, respectively. E. Shows an index of temperature suitability for...

Fine-scale map of presumed limits of zoonotic Plasmodium knowlesi transmission. Areas of spatial co-occurrence of known or putative reservoir (at least one of M. fascicularis, M. nemestrina or M. leonina) and vector species (members of the Leucosphyrus Group) are indicated, as well as areas where either reservoir or vector species are absent. (TIF)

Predicted disease risk values at locations with confirmed/unconfirmed presence and absence from outside Malaysia, Brunei and Singapore. The black dots represent the predicted values of confirmed/unconfirmed P. knowlesi presence and absence points and violin plots showing the density of points at each predicted value are shown in grey. Reports of P....

Database of Plasmodium knowlesi infections recorded in humans, macaque reservoirs and vectors. (XLSX)

Map of model uncertainty. Standard deviation values for each pixel were calculated across the model ensemble. Areas from lower to higher standard deviation values are shown. (TIF)

Marginal effect plots for the most influential covariates. The black line represents the mean marginal effect and grey envelopes the associated 95% quantiles. The mean relative contribution is displayed in the top left corner of each plot. (TIF)

Genomic epidemiology of artemisinin resistant malaria MalariaGEN Plasmodium falciparum Community Project. Elife. 2016. Free PMC article The current epidemic of artemisinin resistant Plasmodium falciparum in Southeast Asia is the result of a soft selective sweep involving at least 20 independent kelch13 mutations. In a large global survey, we find...

The widespread distribution and relapsing nature of Plasmodium vivax infection present major challenges for the elimination of malaria. To characterize the genetic diversity of this parasite in individual infections and across the population, we performed deep genome sequencing of >200 clinical samples collected across the Asia-Pacific region and a...

Despite the huge international endeavor to understand the genomic basis of malaria biology, there remains a lack of information about two human-infective species: Plasmodium malariae and P. ovale . The former is prevalent across all malaria endemic regions and able to recrudesce decades after the initial infection. The latter is a dormant stage hyp...

The current epidemic of artemisinin resistant Plasmodium falciparum in Southeast Asia is the result of a soft selective sweep involving at least 20 independent kelch13 mutations. In a large global survey, we find that kelch13 mutations which cause resistance in Southeast Asia are present at low frequency in Africa. We show that African kelch13 muta...

The current epidemic of artemisinin resistant Plasmodium falciparum in Southeast Asia is the result of a soft selective sweep involving at least 20 independent kelch13 mutations. In a large global survey, we find that kelch13 mutations which cause resistance in Southeast Asia are present at low frequency in Africa. We show that African kelch13 muta...

The current epidemic of artemisinin resistant Plasmodium falciparum in Southeast Asia is the result of a soft selective sweep involving at least 20 independent kelch13 mutations. In a large global survey, we find that kelchl3 mutations which cause resistance in Southeast Asia are present at low frequency in Africa. We show that African kelch13 muta...

Artemisinin resistant Plasmodium falciparum is advancing across Southeast Asia in a soft selective sweep involving at least 20 independent kelch13 mutations. In a large global survey, we find that kelch13 mutations which cause resistance in Southeast Asia are present at low frequency in Africa. We show that African kelch13 mutations have originated...

We report a large multicenter genome-wide association study of Plasmodium falciparum resistance to artemisinin, the frontline antimalarial drug. Across 15 locations in Southeast Asia, we identified at least 20 mutations in kelch13 (PF3D7_1343700) affecting the encoded propeller and BTB/POZ domains, which were associated with a slow parasite clearan...

Pathogen genome sequencing directly from clinical samples is quickly gaining importance in genetic and medical research studies. However, low DNA yield from blood-borne pathogens is often a limiting factor. The problem worsens in extremely base-biased genomes such as the AT-rich Plasmodium falciparum. We present a strategy for whole-genome amplific...

Adaptive introgression can provide novel genetic variation to fuel rapid evolutionary responses, though it may be counterbalanced by potential for detrimental disruption of the recipient genomic background. We examine the extent and impact of recent introgression of a strongly selected insecticide-resistance mutation (Vgsc-1014F) located within one...

Drug resistance remains a chief concern for malaria control. In order to determine the genetic markers of drug resistant parasites, we tested the genome-wide associations (GWA) of sequence-based genotypes from 35 Kenyan P. falciparum parasites with the activities of 22 antimalarial drugs. Parasites isolated from children with acute febrile malaria...

Early identification of causal genetic variants underlying antimalarial drug resistance could provide robust epidemiological tools for timely public health interventions. Using a novel natural genetics strategy for mapping novel candidate genes we analyzed >75,000 high quality single nucleotide polymorphisms selected from high-resolution whole-geno...

Next-generation sequencing (NGS) is increasingly being adopted as the backbone of biomedical research. With the commercialization of various affordable desktop sequencers, NGS will be reached by increasing numbers of cellular and molecular biologists, necessitating community consensus on bioinformatics protocols to tackle the exponential increase i...

Wild-living chimpanzees and gorillas harbor a multitude of Plasmodium species, including six of the subgenus Laverania, one of which served as the progenitor of Plasmodium falciparum. Despite the magnitude of this reservoir, it is unknown whether apes represent a source of human infections. Here, we used Plasmodium species-specific PCR, single-geno...

The cost of whole-genome sequencing (WGS) is decreasing rapidly as next-generation sequencing technology continues to advance, and the prospect of making WGS available for public health applications is becoming a reality. So far, a number of studies have demonstrated the use of WGS as an epidemiological tool for typing and controlling outbreaks of...

Whole genome sequencing (WGS) of Plasmodium vivax is problematic due to the reliance on clinical isolates which are generally low in parasitaemia and sample volume. Furthermore, clinical isolates contain a significant contaminating background of host DNA which confounds efforts to map short read sequence of the target P. vivax DNA. Here, we discuss...

European Nucleotide Archive Sample Accession Numbers. For ethics purposes, all samples were subject to human read alignment filtration prior to submission to the ENA. (DOCX)

Coding and Non-coding Sequence Depth Distributions in 22 Independent, Pure P. vivax Isolates. Plots indicate the number of bases (y-axis) with a given sequence depth (x-axis) in coding (solid green line) and non-coding (dashed green line) regions of the P. vivax genome. Dashed grey line indicates the expected (nominal) sequence depth for each sampl...

Clinical and Laboratory Sample Properties. *Poor maturation during short-term culture. (DOCX)

Sequence Coverage Statistics. Pv = P. vivax; Pf = P. falciparum; a Multiplexes comprise 12 samples. b Quantitative real-time PCR estimate; c Reads overlapping P. vivax and P. falciparum. d Expected sequence depth at each position in the P. vivax genome – calculated as total number of bases sequenced divided by number of bases in the genome (based o...

Acquired immunity in vertebrates maintains polymorphisms in endemic pathogens, leading to identifiable signatures of balancing selection. To comprehensively survey for genes under such selection in the human malaria parasite Plasmodium falciparum, we generated paired-end short-read sequences of parasites in clinical isolates from an endemic Gambian...

Multiple-labelled immunoflourescence showing that the minority of parasites expressing MSPDBL2 (product of PF10_0355) also express other MSP3-like proteins. Three parasite lines are illustrated out of 12 tested, with parasites stained with DAPI (blue) for DNA, rhodamine (red) for antibodies to MSPDBL2 (N-terminal), FP642 (purple) for antibodies to...

Summary indices of polymorphic site frequency spectra in each of 2853 genes with ≥3 SNPs in a Gambian population sample of P. falciparum clinical isolates (n = 65) (XLSX)

Spectrum of values of Tajima's D and Fu & Li's F* indices for genes with 3 or more SNPs analysed separately for multiple clone isolates (n = 37) and single clone isolates (n = 28). The correlation for Tajima's D values across all genes between the two strata is highly significant (Spearman's ρ = 0.62, P<0.0001). The genes with the top 30 values of...

Recombinant proteins based on conserved sequences in the N-terminal and C-terminal regions of the PF10_0355 product MSPDBL2. A. The position of the sequences are shown as bars underneath the scheme of MSLDBL2 (black shading indicates the DBL-domain, grey shading the SPAM domain, and hatched shading the main repeat sequence). B. SDS-PAGE gel showing...

List of 65 Gambian P. falciparum isolates analysed with details of individual sample material, read coverage and accession numbers for archived short-read sequences. (XLSX)

Exact counts of mature schizonts positive for MSPDBL2 (antibody to N-terminal) by immunofluorescence in each parasite line. Similarly low proportions of parasites were seen reactive with antibodies to the C-terminal although fewer parasites were counted (data not shown). (PDF)

Sequences of primers and probes, and assay conditions used for quantitative real time PCR to assay transcript abundance of each of the six msp3-like genes. (PDF)

There is an immediate need for tools to both analyse and visualize in real-time single-nucleotide polymorphisms, insertions and deletions, and other structural variants from new sequence file formats. We have developed VarB software that can be used to visualize variant call format files in real time, as well as identify regions under balancing sel...

Studies on DNA from pathogenic organisms, within clinical samples, are often complicated by the presence of large amounts of host, e.g., human DNA. Isolation of pathogen DNA from these samples would improve the efficiency of next-generation sequencing (NGS) and pathogen identification. Here we describe a solution-based hybridisation method for isol...

Our understanding of the composition of multi-clonal malarial infections and the epidemiological factors which shape their diversity remain poorly understood. Traditionally within-host diversity has been defined in terms of the multiplicity of infection (MOI) derived by PCR-based genotyping. Massively parallel, single molecule sequencing technologi...

MSP-based MOI Estimates. (TIFF)

Read depth distribution at the typable SNP positions in the West African samples. Distribution of SNP coverage at read depth (number of sequenced nucleotides covering a given locus) thresholds of 1, 5, 10, 15, 20 and 25. (TIFF)

Distribution of Fws scores in the West African samples. Dashed lines indicate thresholds for highly diverse (Fws≤0.7) and moderately “clonal” (Fws≥0.95) samples. (TIFF)

Figure S1. Bioanalyzer quantification and analysis of libraries. Aliquots of the PE-adapter-ligated library (shown) was amplified using enzymes/conditions indicated. After purification with Agencourt Ampure XP beads, library products were analyzed using Bioanlyzer. Bioanalyzer traces show various yields obtained by different conditions and amplific...

Massively parallel sequencing technology is revolutionizing approaches to genomic and genetic research. Since its advent, the scale and efficiency of Next-Generation Sequencing (NGS) has rapidly improved. In spite of this success, sequencing genomes or genomic regions with extremely biased base composition is still a great challenge to the currentl...

Naturally acquired blood-stage infections of the malaria parasite Plasmodium falciparum typically harbour multiple haploid clones. The apparent number of clones observed in any single infection depends on the diversity of the polymorphic markers used for the analysis, and the relative abundance of rare clones, which frequently fail to be detected a...