Natural genetic variation and malaria

Under the leadership of Dominic Kwiatkowski, our team is addressing fundamental questions about malaria that require a deep understanding of the genetic diversity and co-evolution of host, parasite and mosquito populations.

We use state-of-the-art sequencing technologies to investigate Plasmodium, Anopheles and human genome variation in large clinical and epidemiological studies carried out with partners in over 20 malaria-endemic countries. Our goal is to understand basic questions about malaria biology that have practical implications for malaria control, such as how antimalarial drug resistance emerges and spreads, how the mosquito population evolves in response to malaria control efforts, and why some people resist malaria better than others.

More information on the Malaria programme.

[Susana Campino, Genome Research Limited]

Background

Malaria affects hundreds of millions of people each year and is one of the most important causes of child mortality, particularly in Africa. The disease is caused by Plasmodium parasites which invade and replicate in human erythrocytes. The parasites are transmitted from one person to another by blood-sucking Anopheles mosquitoes.

At the core of malaria biology is a longstanding evolutionary arms race between Plasmodium, Anopheles and human populations. This is still ongoing and has a major impact on disease control efforts. Plasmodium populations are continually evolving to resist antimalarial drugs and to evade the human immune system, while Anopheles populations are continually evolving to resist the insecticides used in vector control. The remarkable ability of both parasite and vector to thwart public health interventions by evolutionary adaptation represents a major obstacle to malaria elimination. The other side of the coin is that malaria has exerted strong selective pressure on the human genome and this has led to natural mechanisms of disease resistance that, if better understood, could provide critical insights into how to develop an effective vaccine.

Our research goal is to gain a deeper understanding of the genetic and molecular mechanisms that underlie these evolutionary processes, and to translate this scientific understanding into actionable knowledge that will lead to more effective and sustainable strategies for disease elimination and vaccine development.

Research

Malaria-infected Red Blood Cell.

Malaria-infected Red Blood Cell. [NIAID on Flickr]

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Understanding human genetic resistance to malaria

In areas where malaria is common, some people become gravely ill, while others show no sign of disease. This curious paradox arises from an ancient and on-going evolutionary arms race between human hosts and malaria-causing parasites. Well-documented examples of human adaptation to malaria include sickle-cell anaemia and G6PD enzyme deficiency.

We have two main approaches to understand the impact of genetic variation on host susceptibility to malaria. The first is large-scale genome-wide association (GWA) studies looking for association of known genetic markers across the human genome that correlate with resistance or susceptibility to malaria. We are also examining how signals of genetic association are affected by diversity in African population structure, and identifying regions of the genome under recent positive selection in malaria-endemic populations by accurate haplotype construction using family-based GWA data. The second approach involves deep re-sequencing of candidate resistance genes to identify new genetic variants that correlate with innate immunity to malaria within and between African populations.

This work involves close collaboration with the Sanger Institute's genotyping teams, and is being done through the MalariaGEN Consortium, a network of scientists in more than 20 countries, many in the most affected regions of the world.

MalariaGEN Consortial Projects include multi-centre case-control and family-based association studies of severe malaria, as well as large cohort studies examining the natural evolution of infection and immunity. The Consortium also has on-going genetic linkage studies in a number of populations, as well as investigations of ethnic groups that naturally have a high level of resistance to malaria.

Neighbour-joining tree of 825 P. falciparum samples illustrates the unique population structure of malaria parasites within a relatively small geographic range in Southeast Asia. Published in Miotto et al, 2013.

Neighbour-joining tree of 825 P. falciparum samples illustrates the unique population structure of malaria parasites within a relatively small geographic range in Southeast Asia. Published in Miotto et al, 2013.

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Biological consequences of natural variation in the Plasmodium genome

Studying the natural genetic diversity and population genetics of malaria-causing Plasmodium species can provide crucial insight into the parasites' extraordinary ability to evade the immune system and to develop resistance to antimalarial drugs.

Initially, Plasmodium genome sequencing at the Sanger Institute and elsewhere focused on laboratory-adapted parasites. In recent years, we've developed the experimental, epidemiological, and analytical tools to undertake characterisation of natural genome diversity in Plasmodium isolates from multiple malaria-endemic regions in Africa, Asia and Oceania.

Leveraging Solexa/Illumina high-throughput sequencing technology, we are developing shotgun genotyping as a cost-effective method for genome-wide analysis of natural variation in Plasmodium falciparum.

Understanding the complex population genetic structures that arise under different conditions of malaria transmission will revolutionise malaria biology, serving as the foundation for large-scale epidemiological studies of genotype-phenotype correlation for example for drug resistance or immune evasion and other parasite phenotypes, and informing malaria monitoring and control strategies in the field. Accordingly, we will use natural P. falciparum variation data to inform functional analysis of parasite biology in the laboratory.

Much of our work on Plasmodium parasites is undertaken through a series of MalariaGEN projects, the largest of which is the P. falciparum Community Project, which partners with researchers in malaria-endemic areas and has to date sequenced more than 5,000 field isolates (August 2014). This feat has driven methods development, including the establishment of a robust sequence analysis pipeline at the Sanger Institute, and laid the foundation for another multi-centre collaboration, Pf3k, which is being led jointly by the Sanger Institute, the Broad Institute and University of Oxford. Additionally, we have projects studying P. falciparum genetic crosses and genome variation in P. vivax.

We work closely with Chris Newbold and the Parasite Genomics group led by Matt Berriman, who head efforts to resequence and reannotate the reference Plasmodium falciparum genome, 3D7.

Understanding the diversity and dynamics of Anopheles populations

A keystone of malaria control is to prevent transmission by the Anopheles vector. Hopes of eventually eliminating malaria rely greatly on this, but the failure of previous efforts to eradicate malaria has taught us that it is not easily accomplished, particularly because of the ability of Anopheles populations to develop resistance to insecticides as their usage increases. New technologies for large-scale sequencing provide unprecedented opportunities to overcome this problem by real-time monitoring of genome variation in Anopheles populations, and using this information to develop early warning systems for the emergence of insecticide resistance, and for other practical applications in vector control.

Working through MalariaGEN, we've established the Ag1000G project, which is using whole-genome sequencing to build a high-resolution view of genetic variation in natural populations of Anopheles gambiae, the principal vector of P. falciparum in Africa. At the same time, this multi-centre collaboration is building a catalogue of A. gambiae genome variation as a scientific resource for the malaria research community as a whole. A first step in the process has been to sequence samples from the major colonies used by the community to study parasite refractoriness and insecticide resistance.

Martin Donnelly, who works between the Liverpool School of Tropical Medicine and the Sanger Institute, is driving forward many of our Anopheles activities.

Statistical analysis of genome-wide association and short-read sequence data

Our research is underpinned by cutting-edge statistical and informatics solutions for the analysis, handling, and sharing of large-scale sequence and genotype data.

New high-throughput sequencing and large-scale genotyping technologies drive the need for novel statistical methods for genetic data analysis. This need is further underscored by the complexities of the population genetic structures we study: for example, the rich haplotypic diversity and low linkage disequilibrium (LD) of African populations pose unique challenges for GWA study design and analysis. Likewise, new statistical tools will be required to use short read sequence data to identify with confidence polymorphisms and structural variants, patterns of LD, and differences between Plasmodium populations. This is particularly challenging because of the low LD and AT rich nature of the parasite genome, as well as the presence of multiple parasite genomes in clinical samples.

Our epidemiological studies of human resistance and susceptibility to malaria include case-control and family trio designs, with sample sizes currently of approximately 30,000 individuals. For GWA analysis we have implemented analysis pipelines that convert chip intensities to genotype calls, through to ultimately testing for associations and positive selection, correcting for population artifacts, to discover putative variants for follow-up in the laboratory. This process has involved developing and applying methods to call genotypes (Illuminus), to understand the relationship of population structure and ethnicity within and across study sites, to determine strategies for the selection of tagging SNPs and to determine genotype in populations with low LD.

Resources

The genome browser in the MalariaGEN P. falciparum web application highlights potential challenges in accurately genotyping and SNP calling in particular regions of the genome.

The genome browser in the MalariaGEN P. falciparum web application highlights potential challenges in accurately genotyping and SNP calling in particular regions of the genome.

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Informatics solutions for analysis and sharing of large-scale sequence and genotype data

Our team is actively developing informatic technologies to manage and analyse the remarkable volume of data generated by our sequencing and genotyping projects, and to establish effective ways to share these complex epidemiological and genetic datasets across the malaria research community.

The Ag1000G web application was built using the Panoptes software framework.

The Ag1000G web application was built using the Panoptes software framework.

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In the past, we've produced an improved algorithm for genotype calling from the Illumina Bead Array platform (Illuminus) and detection of positive selection from haplotype information. We've also developed browser-based software packages for simplified presentation and browsing of linkage disequilibrium along chromosomes (Marker).

Our interest in developing tools to work with genetic and genomic data also led to the development of a number of additional tools including LookSeq (SNP-discovery and analysis in short-read sequence data), and ExplorerCat and MapSeq, early progenitors of the MalariaGEN P. falciparum web application. We also worked with the WorldWide Antimalarial Resistance Network (WWARN), a global network of malaria researchers building a web-based global antimalarial efficacy and resistance database to track resistance to malaria drugs, to develop the first release of their online data submission system.

Building on this substantial experience, we're collaborating with the Centre for Genomics and Global Health (CGGH) to develop a flexible software framework, Panoptes, which can be customised and rapidly deployed to create interactive web applications for browsing and querying genetic and genomic data. The first implementations of the framework are applications designed to share findings of two MalariaGEN projects: Ag1000G and Pf3k.

LookSeq

Screenshot of LookSeq, a browser-based read alignment viewer. LookSeq is a web-based application for alignment visualization, browsing and analysis of genome sequence data.

Screenshot of LookSeq, a browser-based read alignment viewer. LookSeq is a web-based application for alignment visualization, browsing and analysis of genome sequence data.

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LookSeq is a web-based application for alignment visualisation, browsing and analysis of genome sequence data.

LookSeq supports multiple sequencing technologies, alignment sources, and viewing modes; low or high-depth read pileups; and easy visualisation of putative single nucleotide and structural variation. The visible range, from whole chromosome to single base resolution, can be set manually or by scrolling or zooming the display with fast, on-the-fly rendering from the server-side alignment database. LookSeq uses a universal database for alignments of different sequencing technologies and algorithms. Sequence data from multiple sources can be viewed separately or aligned in a single display, facilitating direct comparison between datasets. LookSeq can also link to relevant external sites such as PubMed and other online analysis tools, via buttons or double-clicking on the displayed sequence annotation.

LookSeq requires no setup or installation, and is very intuitive to use.

  • LookSeq: a browser-based viewer for deep sequencing data.

    Manske HM and Kwiatkowski DP

    Genome research 2009;19;11;2125-32

Collaborations

Our research is highly collaborative. We work in partnership with clinical and epidemiological researchers in over 20 malaria-endemic countries through the Malaria Genomic Epidemiology Network (MalariaGEN), which brings together research groups with different projects and scientific objectives to carry out large-scale investigations that depend on samples, data and expertise from multiple investigators. There is also a close partnership with the Centre for Genomics and Global Health, whose mission is to translate genomic data into tools and actionable knowledge to combat major infectious diseases of the developing world.

Conferences

Genomic Epidemiology of Malaria (GEM)

Working with the Wellcome Trust Courses and Conferences, we host the annual Genomic Epidemiology of Malaria Conference at the Genome Campus in Hinxton. This meeting brings together diverse malaria scientists and clinicians working at the interface of genome science and technology, epidemiology and statistical and population genetics.

Pictures of past events are available to view.

Selected Publications

  • Genetic architecture of artemisinin-resistant Plasmodium falciparum.

    Miotto O, Amato R, Ashley EA, MacInnis B, Almagro-Garcia J, Amaratunga C, Lim P, Mead D, Oyola SO, Dhorda M, Imwong M, Woodrow C, Manske M, Stalker J, Drury E, Campino S, Amenga-Etego L, Thanh TN, Tran HT, Ringwald P, Bethell D, Nosten F, Phyo AP, Pukrittayakamee S, Chotivanich K, Chuor CM, Nguon C, Suon S, Sreng S, Newton PN, Mayxay M, Khanthavong M, Hongvanthong B, Htut Y, Han KT, Kyaw MP, Faiz MA, Fanello CI, Onyamboko M, Mokuolu OA, Jacob CG, Takala-Harrison S, Plowe CV, Day NP, Dondorp AM, Spencer CC, McVean G, Fairhurst RM, White NJ and Kwiatkowski DP

    Nature genetics 2015;47;3;226-34

  • Reappraisal of known malaria resistance loci in a large multicenter study.

    Malaria Genomic Epidemiology Network and Malaria Genomic Epidemiology Network

    Nature genetics 2014;46;11;1197-204

  • Monitoring parasite diversity for malaria elimination in sub-Saharan Africa.

    Ghansah A, Amenga-Etego L, Amambua-Ngwa A, Andagalu B, Apinjoh T, Bouyou-Akotet M, Cornelius V, Golassa L, Andrianaranjaka VH, Ishengoma D, Johnson K, Kamau E, Maïga-Ascofaré O, Mumba D, Tindana P, Tshefu-Kitoto A, Randrianarivelojosia M, William Y, Kwiatkowski DP and Djimde AA

    Science (New York, N.Y.) 2014;345;6202;1297-8

  • Adaptive introgression between Anopheles sibling species eliminates a major genomic island but not reproductive isolation.

    Clarkson CS, Weetman D, Essandoh J, Yawson AE, Maslen G, Manske M, Field SG, Webster M, Antão T, MacInnis B, Kwiatkowski D and Donnelly MJ

    Nature communications 2014;5;4248

  • Multiple populations of artemisinin-resistant Plasmodium falciparum in Cambodia.

    Miotto O, Almagro-Garcia J, Manske M, Macinnis B, Campino S, Rockett KA, Amaratunga C, Lim P, Suon S, Sreng S, Anderson JM, Duong S, Nguon C, Chuor CM, Saunders D, Se Y, Lon C, Fukuda MM, Amenga-Etego L, Hodgson AV, Asoala V, Imwong M, Takala-Harrison S, Nosten F, Su XZ, Ringwald P, Ariey F, Dolecek C, Hien TT, Boni MF, Thai CQ, Amambua-Ngwa A, Conway DJ, Djimdé AA, Doumbo OK, Zongo I, Ouedraogo JB, Alcock D, Drury E, Auburn S, Koch O, Sanders M, Hubbart C, Maslen G, Ruano-Rubio V, Jyothi D, Miles A, O'Brien J, Gamble C, Oyola SO, Rayner JC, Newbold CI, Berriman M, Spencer CC, McVean G, Day NP, White NJ, Bethell D, Dondorp AM, Plowe CV, Fairhurst RM and Kwiatkowski DP

    Nature genetics 2013;45;6;648-55

  • Analysis of Plasmodium falciparum diversity in natural infections by deep sequencing.

    Manske M, Miotto O, Campino S, Auburn S, Almagro-Garcia J, Maslen G, O'Brien J, Djimde A, Doumbo O, Zongo I, Ouedraogo JB, Michon P, Mueller I, Siba P, Nzila A, Borrmann S, Kiara SM, Marsh K, Jiang H, Su XZ, Amaratunga C, Fairhurst R, Socheat D, Nosten F, Imwong M, White NJ, Sanders M, Anastasi E, Alcock D, Drury E, Oyola S, Quail MA, Turner DJ, Ruano-Rubio V, Jyothi D, Amenga-Etego L, Hubbart C, Jeffreys A, Rowlands K, Sutherland C, Roper C, Mangano V, Modiano D, Tan JC, Ferdig MT, Amambua-Ngwa A, Conway DJ, Takala-Harrison S, Plowe CV, Rayner JC, Rockett KA, Clark TG, Newbold CI, Berriman M, MacInnis B and Kwiatkowski DP

    Nature 2012;487;7407;375-9

  • Characterization of within-host Plasmodium falciparum diversity using next-generation sequence data.

    Auburn S, Campino S, Miotto O, Djimde AA, Zongo I, Manske M, Maslen G, Mangano V, Alcock D, MacInnis B, Rockett KA, Clark TG, Doumbo OK, Ouédraogo JB and Kwiatkowski DP

    PloS one 2012;7;2;e32891

  • Optimizing Illumina next-generation sequencing library preparation for extremely AT-biased genomes.

    Oyola SO, Otto TD, Gu Y, Maslen G, Manske M, Campino S, Turner DJ, Macinnis B, Kwiatkowski DP, Swerdlow HP and Quail MA

    BMC genomics 2012;13;1

  • An effective method to purify Plasmodium falciparum DNA directly from clinical blood samples for whole genome high-throughput sequencing.

    Auburn S, Campino S, Clark TG, Djimde AA, Zongo I, Pinches R, Manske M, Mangano V, Alcock D, Anastasi E, Maslen G, Macinnis B, Rockett K, Modiano D, Newbold CI, Doumbo OK, Ouédraogo JB and Kwiatkowski DP

    PloS one 2011;6;7;e22213

  • Ethical issues in human genomics research in developing countries.

    de Vries J, Bull SJ, Doumbo O, Ibrahim M, Mercereau-Puijalon O, Kwiatkowski D and Parker M

    BMC medical ethics 2011;12;5

  • Population genetic analysis of Plasmodium falciparum parasites using a customized Illumina GoldenGate genotyping assay.

    Campino S, Auburn S, Kivinen K, Zongo I, Ouedraogo JB, Mangano V, Djimde A, Doumbo OK, Kiara SM, Nzila A, Borrmann S, Marsh K, Michon P, Mueller I, Siba P, Jiang H, Su XZ, Amaratunga C, Socheat D, Fairhurst RM, Imwong M, Anderson T, Nosten F, White NJ, Gwilliam R, Deloukas P, MacInnis B, Newbold CI, Rockett K, Clark TG and Kwiatkowski DP

    PloS one 2011;6;6;e20251

  • Methodological challenges of genome-wide association analysis in Africa.

    Teo YY, Small KS and Kwiatkowski DP

    Nature reviews. Genetics 2010;11;2;149-60

  • Ethical data release in genome-wide association studies in developing countries.

    Parker M, Bull SJ, de Vries J, Agbenyega T, Doumbo OK and Kwiatkowski DP

    PLoS medicine 2009;6;11;e1000143

  • LookSeq: a browser-based viewer for deep sequencing data.

    Manske HM and Kwiatkowski DP

    Genome research 2009;19;11;2125-32

  • SNP-o-matic.

    Manske HM and Kwiatkowski DP

    Bioinformatics (Oxford, England) 2009;25;18;2434-5

  • Genome-wide and fine-resolution association analysis of malaria in West Africa.

    Jallow M, Teo YY, Small KS, Rockett KA, Deloukas P, Clark TG, Kivinen K, Bojang KA, Conway DJ, Pinder M, Sirugo G, Sisay-Joof F, Usen S, Auburn S, Bumpstead SJ, Campino S, Coffey A, Dunham A, Fry AE, Green A, Gwilliam R, Hunt SE, Inouye M, Jeffreys AE, Mendy A, Palotie A, Potter S, Ragoussis J, Rogers J, Rowlands K, Somaskantharajah E, Whittaker P, Widden C, Donnelly P, Howie B, Marchini J, Morris A, SanJoaquin M, Achidi EA, Agbenyega T, Allen A, Amodu O, Corran P, Djimde A, Dolo A, Doumbo OK, Drakeley C, Dunstan S, Evans J, Farrar J, Fernando D, Hien TT, Horstmann RD, Ibrahim M, Karunaweera N, Kokwaro G, Koram KA, Lemnge M, Makani J, Marsh K, Michon P, Modiano D, Molyneux ME, Mueller I, Parker M, Peshu N, Plowe CV, Puijalon O, Reeder J, Reyburn H, Riley EM, Sakuntabhai A, Singhasivanon P, Sirima S, Tall A, Taylor TE, Thera M, Troye-Blomberg M, Williams TN, Wilson M, Kwiatkowski DP, Wellcome Trust Case Control Consortium and Malaria Genomic Epidemiology Network

    Nature genetics 2009;41;6;657-65

  • Tumor necrosis factor and lymphotoxin-alpha polymorphisms and severe malaria in African populations.

    Clark TG, Diakite M, Auburn S, Campino S, Fry AE, Green A, Richardson A, Small K, Teo YY, Wilson J, Jallow M, Sisay-Joof F, Pinder M, Griffiths MJ, Peshu N, Williams TN, Marsh K, Molyneux ME, Taylor TE, Rockett KA and Kwiatkowski DP

    The Journal of infectious diseases 2009;199;4;569-75

  • TLR9 polymorphisms in African populations: no association with severe malaria, but evidence of cis-variants acting on gene expression.

    Campino S, Forton J, Auburn S, Fry A, Diakite M, Richardson A, Hull J, Jallow M, Sisay-Joof F, Pinder M, Molyneux ME, Taylor TE, Rockett K, Clark TG and Kwiatkowski DP

    Malaria journal 2009;8;44

  • A global network for investigating the genomic epidemiology of malaria.

    Malaria Genomic Epidemiology Network

    Nature 2008;456;7223;732-7

  • Host genetic factors in resistance and susceptibility to malaria.

    Kwiatkowski DP and Luoni G

    Parassitologia 2006;48;4;450-67

  • Data sharing and intellectual property in a genomic epidemiology network: policies for large-scale research collaboration.

    Chokshi DA, Parker M and Kwiatkowski DP

    Bulletin of the World Health Organization 2006;84;5;382-7

  • How malaria has affected the human genome and what human genetics can teach us about malaria.

    Kwiatkowski DP

    American journal of human genetics 2005;77;2;171-92

Team

Team members

Cristina Ariani
Postdoctoral Fellow
Susana Campino
Senior Postdoctoral Scientist
Kalia Dede
kd9@sanger.ac.ukProgramme Administrator
Eleanor Drury
Laboratory Manager
Rachel Giacomantonio
Communications Manager
Will Hamilton
unknown
Mihir Kekre
Advanced Research Assistant
Cinzia Malangone
Senior Software Developer
Magnus Manske
Head of Informatics
Francois Meullenet
Research Administrator
Dawn Muddyman
Senior Project Manager
Samuel Oyola
so1@sanger.ac.ukStaff Scientist
James Stalker
Analysis Pipeline Manager
Arthur Talman
Postdoctoral Fellow

Cristina Ariani

- Postdoctoral Fellow

I am an evolutionary biologist with a strong interest in host-parasite interactions, genetics, genomics and ecology. I did my PhD at the University of Cambridge, in which I studied the genetic variation of traits that affect disease transmission in a major mosquito vector - Aedes aegypti. Previously I worked as a research assistant at the Institute of Zoology in London working on the conservation genetics of endangered species. I also hold a Master degree from the State University of Rio de Janeiro in Brazil, studying the ecology of lizards.

Research

I joined the Malaria Programme in November 2014 and shall be working on the interactions between the mosquito vector and the malarial parasite.

References

  • Environmental and Genetic Factors Determine Whether the Mosquito Aedes aegypti Lays Eggs Without a Blood Meal.

    Ariani CV, Smith SC, Osei-Poku J, Short K, Juneja P and Jiggins FM

    Department of Genetics, University of Cambridge, Cambridge, United Kingdom cristina.ariani@gmail.com.

    Some mosquito strains or species are able to lay eggs without taking a blood meal, a trait named autogeny. This may allow populations to persist through times or places where vertebrate hosts are scarce. Autogenous egg production is highly dependent on the environment in some species, but the ideal conditions for its expression in Aedes aegypti mosquitoes are unknown. We found that 3.2% of females in a population of Ae. aegypti from Kenya were autogenous. Autogeny was strongly influenced by temperature, with many more eggs laid at 28°C compared with 22°C. Good nutrition in larval stages and feeding on higher concentrations of sugar solution during the adult stage both result in more autogenous eggs being produced. The trait also has a genetic basis, as not all Ae. aegypti genotypes can lay autogenously. We conclude that Ae. aegypti requires a favorable environment and a suitable genotype to be able to lay eggs without a blood meal.

    The American journal of tropical medicine and hygiene 2015;92;4;715-21

  • Vector competence of Aedes aegypti mosquitoes for filarial nematodes is affected by age and nutrient limitation.

    Ariani CV, Juneja P, Smith S, Tinsley MC and Jiggins FM

    Department of Genetics, University of Cambridge, Downing Street, Cambridge CB24 6BG, United Kingdom. Electronic address: cristina.ariani@gmail.com.

    Mosquitoes are one of the most important vectors of human disease. The ability of mosquitoes to transmit disease is dependent on the age structure of the population, as mosquitoes must survive long enough for the parasites to complete their development and infect another human. Age could have additional effects due to mortality rates and vector competence changing as mosquitoes senesce, but these are comparatively poorly understood. We have investigated these factors using the mosquito Aedes aegypti and the filarial nematode Brugia malayi. Rather than observing any effects of immune senescence, we found that older mosquitoes were more resistant, but this only occurred if they had previously been maintained on a nutrient-poor diet of fructose. Constant blood feeding reversed this decline in vector competence, meaning that the number of parasites remained relatively unchanged as mosquitoes aged. Old females that had been maintained on fructose also experienced a sharp spike in mortality after an infected blood meal ("refeeding syndrome") and few survived long enough for the parasite to develop. Again, this effect was prevented by frequent blood meals. Our results indicate that old mosquitoes may be inefficient vectors due to low vector competence and high mortality, but that frequent blood meals can prevent these effects of age.

    Experimental gerontology 2015;61;47-53

  • Assembly of the genome of the disease vector Aedes aegypti onto a genetic linkage map allows mapping of genes affecting disease transmission.

    Juneja P, Osei-Poku J, Ho YS, Ariani CV, Palmer WJ, Pain A and Jiggins FM

    Department of Genetics, University of Cambridge, Cambridge, United Kingdom.

    The mosquito Aedes aegypti transmits some of the most important human arboviruses, including dengue, yellow fever and chikungunya viruses. It has a large genome containing many repetitive sequences, which has resulted in the genome being poorly assembled - there are 4,758 scaffolds, few of which have been assigned to a chromosome. To allow the mapping of genes affecting disease transmission, we have improved the genome assembly by scoring a large number of SNPs in recombinant progeny from a cross between two strains of Ae. aegypti, and used these to generate a genetic map. This revealed a high rate of misassemblies in the current genome, where, for example, sequences from different chromosomes were found on the same scaffold. Once these were corrected, we were able to assign 60% of the genome sequence to chromosomes and approximately order the scaffolds along the chromosome. We found that there are very large regions of suppressed recombination around the centromeres, which can extend to as much as 47% of the chromosome. To illustrate the utility of this new genome assembly, we mapped a gene that makes Ae. aegypti resistant to the human parasite Brugia malayi, and generated a list of candidate genes that could be affecting the trait.

    PLoS neglected tropical diseases 2014;8;1;e2652

  • Evolutionary history and identification of conservation units in the giant otter, Pteronura brasiliensis.

    Pickles RS, Groombridge JJ, Zambrana Rojas VD, Van Damme P, Gottelli D, Kundu S, Bodmer R, Ariani CV, Iyengar A and Jordan WC

    Institute of Zoology, Zoological Society of London, Regent's Park, London NW1 4RY, United Kingdom. robert.pickles@ioz.ac.uk

    The giant otter, Pteronura brasiliensis, occupies a range including the major drainage basins of South America, yet the degree of structure that exists within and among populations inhabiting these drainages is unknown. We sequenced portions of the mitochondrial DNA (mtDNA) cytochrome b (612bp) and control region (383 bp) genes in order to determine patterns of genetic variation within the species. We found high levels of mtDNA haplotype diversity (h = 0.93 overall) and support for subdivision into four distinct groups of populations, representing important centers of genetic diversity and useful units for prioritizing conservation within the giant otter. We tested these results against the predictions of three hypotheses of Amazonian diversification (Pleistocene Refugia, Paleogeography, and Hydrogeology). While the phylogeographic pattern conformed to the predictions of the Refugia Hypothesis, molecular dating using a relaxed clock revealed the phylogroups diverged from one another between 1.69 and 0.84 Ma, ruling out the influence of Late Pleistocene glacial refugia. However, the role of Plio-Pleistocene climate change could not be rejected. While the molecular dating also makes the influence of geological arches according to the Paleogeography Hypothesis extremely unlikely, the recent Pliocene formation of the Fitzcarrald Arch and its effect of subsequently altering drainage pattern could not be rejected. The data presented here support the interactions of both climatic and hydrological changes resulting from geological activity in the Plio-Pleistocene, in shaping the phylogeographic structure of the giant otter.

    Molecular phylogenetics and evolution 2011;61;3;616-27

  • Anurofauna of an Atlantic Rainforest fragment and its surroundings in Northern Rio de Janeiro State, Brazil.

    Almeida-Gomes M, Almeida-Santos M, Goyannes-Araújo P, Borges-Júnior VN, Vrcibradic D, Siqueira CC, Ariani CV, Dias AS, Souza VV, Pinto RR, Van Sluys M and Rocha CF

    Departamento de Ecologia, Universidade do Estado do Rio de Janeiro – UERJ, Rua São Francisco Xavier, 524, CEP 20550-011, Rio de Janeiro, RJ, Brazil. almeida.gomes@yahoo.com.br

    We carried out a study on the anurofaunal community from an Atlantic Forest fragment (Monte Verde mountains) and the surrounding area in Cambuci municipality, Rio de Janeiro State, Brazil, which constitutes one of the largest fragments remaining in the largely deforested landscape of the northern portion of the State. We combined three sampling methods: plot sampling, transects and pit-fall traps. We recorded twenty species of amphibians, of which only eleven were found within the forest fragment (and five of these also occurred in the surrounding matrix). Two of the species recorded in the present study (Crossodactylus sp. and Ischnocnema cf. parva) may represent undescribed taxa. Our records expand the distribution range of one species (Scinax trapicheiroi) to the north, and fill a geographic distribution gap for another one (Ischnocnema oea). The estimated overall density of frogs living in the leaf litter of the fragment (based on results of plot sampling) was 3.1 individuals/100 m², with Haddadus binotatus being the most abundant species (2.4 individuals/100 m²). Comparisons of our data with those of other studies suggest that anuran communities in forest fragments ca. 1,000 ha or smaller may be severely limited in their richness, and often include a large proportion of species tolerant to open areas, such as many hylids. Our results show the importance of increasing knowledge about the anurofaunal community of the northern portion of the State of Rio de Janeiro and preserve the forest remnants that still exist in the region.

    Brazilian journal of biology = Revista brasleira de biologia 2010;70;3 Suppl;871-7

  • A potential recovery of a population of the sand lizard Liolaemus lutzae Mertens, 1938 in an area within its range: a lizard endemic and threatened with extinction.

    Rocha CF, Siqueira CC and Ariani CV

    Departamento de Ecologia, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, RJ, Brazil. cfdrocha@uerj.br

    The endemic and threatened lizard Liolaemus lutzae has a relatively small geographic range restricted to only 200 km of along the coast of Rio de Janeiro State, Brazil, which are habitats under intensive anthropic disturbance. At the Barra da Tijuca beach, in Rio de Janeiro city an estimate of the population abundance made in 1991, compared to a previous estimate made in 1984, showed a considerable decrease (about 65%). Most of the decrease was attributed to anthropic disturbances that locally affected the beach vegetation, the species habitat. In this study we present estimates made in 2002 and in 2006 at the same area and compare them with the estimates of 1984 and 1991, using the same methodology in order to make comparable the data from different samplings years and to evaluate the present status of the local population. The estimated indexes of L. lutzae abundance in 2002 and in 2006 were higher than that of 1991. There was a significant increase in the mean number of recorded lizards in 2002 compared to 1991, but the mean number of lizards sighted in 2006 remained stable when compared with that of 2002. Our data based on the index of abundance recorded suggested that the number of L. lutzae at Barra da Tijuca beach recorded increased, which can be indicative of a potential recovery of the local population.

    Brazilian journal of biology = Revista brasleira de biologia 2009;69;1;185-7

  • Effects of a fire on a population of treefrogs (Scinax cf. alter, Lutz) in a restinga habitat in southern Brazil.

    Rocha CF, Ariani CV, Menezes VA and Vrcibradic D

    Departamento de Ecologia, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, Brazil. cfdrocha@uerj.br

    The area of the Dunas da Joaquina, in Santa Catarina island, contains one of the most important remnants of restinga habitat in Santa Catarina State, southern Brazil. In December 2003, a fire occurred in a portion of this area, affecting most of the vegetation, including the bromeliad community. In this study, the density of individuals and the diet composition of the bromelicolous treefrog Scinax cf. alter were compared between the area affected by the fire and an adjacent unburned area. One-hundred-and-fifty-eight ground bromeliads (Vriesea friburguensis) were dissected and searched for the presence of treefrogs among their leaves. We found 30 frogs in 29.5% (23/78) of the bromeliads from the unburned site, with a mean of 1.3 frogs per rosette, and 15 frogs in 12.5% (10/80) of the bromeliads from the burned site, with a mean of 1.6 frogs per rosette. Eight (27%) of the frogs from the unburned site and eleven (73%) of those from the burned site had empty stomachs. Frogs from the burned site also contained less prey per stomach than those from the unburned site. The data suggest that the fire has negatively affected the local population of Scinax cf. alter, though it is possible that the population can recover.

    Brazilian journal of biology = Revista brasleira de biologia 2008;68;3;539-43

  • Herpetofauna of an Atlantic rainforest area (Morro São João) in Rio de Janeiro State, Brazil.

    Almeida-Gomes M, Vrcibradic D, Siqueira CC, Kiefer MC, Klaion T, Almeida-Santos P, Nascimento D, Ariani CV, Borges-Junior VN, Freitas-Filho RF, van Sluys M and Rocha CF

    Departamento de Ecologia, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, RJ, 20550-011, Brazil. almeida.gomes@yahoo.com.br

    We studied the herpetofaunal community from the Atlantic forest of Morro São João, in Rio de Janeiro State, Brazil, and present data on species composition, richness, relative abundance and densities. We combined three sampling methods: plot sampling, visual encounter surveys and pit-fall traps. We recorded sixteen species of amphibians and nine of reptiles. The estimated densities (based on results of plot sampling) were 4.5 ind/100 m2 for amphibians and 0.8 ind/100 m2 for lizards, and the overall density (amphibians and lizards) was 5.3 ind/100 m2. For amphibians, Eleutherodactylus and Scinax were the most speciose genera with three species each, and Eleutherodactylus binotatus was the most abundant species (mean density of 3.0 frogs/100 m2). The reptile community of Morro São João was dominated by species of the families Gekkonidae and Gymnophtalmidae (Lacertilia) and Colubridae (Serpentes). The gymnophtalmid lizard Leposoma scincoides was the most abundant reptile species (mean density of 0.3 ind/100 m2). We compare densities obtained in our study data with those of other studied rainforest sites in various tropical regions of the world.

    Anais da Academia Brasileira de Ciências 2008;80;2;291-300

Susana Campino

- Senior Postdoctoral Scientist

Susana is a malariologist with expertise in host and parasite genomics. She holds a PhD in malaria genetics and immunology from Umea Biomedical University in Sweden, after which she completed a Marie Curie postdoctoral fellowship at the University of Oxford. Susana’s previous work involved developing experimental approaches to understand the role of genes in host susceptibility and parasite invasion, field-friendly methods for obtaining quality parasite gDNA for sequencing, and new genotyping assays for barcoding malaria infection. She has also worked with the Institute Pasteur in Paris and the Department of Immunogenetics at the Gulbenkian Institute of Science, Lisbon.

Research

Susana joined the Sanger Institute Malaria Programme in 2007, and played a key role in initiating the MalariaGEN Plasmodium falciparum Community Project which aims to describe global genetic diversity in malaria. Susana currently focuses on epidemiological and population genetic analyses, most recently examining invasion pathways using experimental parasite crosses. She also oversees the malaria laboratory, and works closely with collaborators in more than 15 countries on experimental and analytical aspects of their work

References

  • Using CF11 cellulose columns to inexpensively and effectively remove human DNA from Plasmodium falciparum-infected whole blood samples.

    Venkatesan M, Amaratunga C, Campino S, Auburn S, Koch O, Lim P, Uk S, Socheat D, Kwiatkowski DP, Fairhurst RM and Plowe CV

    Howard Hughes Medical Institute, University of Maryland School of Medicine, Baltimore, MD, USA.

    Background: Genome and transcriptome studies of Plasmodium nucleic acids obtained from parasitized whole blood are greatly improved by depletion of human DNA or enrichment of parasite DNA prior to next-generation sequencing and microarray hybridization. The most effective method currently used is a two-step procedure to deplete leukocytes: centrifugation using density gradient media followed by filtration through expensive, commercially available columns. This method is not easily implemented in field studies that collect hundreds of samples and simultaneously process samples for multiple laboratory analyses. Inexpensive syringes, hand-packed with CF11 cellulose powder, were recently shown to improve ex vivo cultivation of Plasmodium vivax obtained from parasitized whole blood. This study was undertaken to determine whether CF11 columns could be adapted to isolate Plasmodium falciparum DNA from parasitized whole blood and achieve current quantity and purity requirements for Illumina sequencing.

    Methods: The CF11 procedure was compared with the current two-step standard of leukocyte depletion using parasitized red blood cells cultured in vitro and parasitized blood obtained ex vivo from Cambodian patients with malaria. Procedural variations in centrifugation and column size were tested, along with a range of blood volumes and parasite densities.

    Results: CF11 filtration reliably produces 500 nanograms of DNA with less than 50% human DNA contamination, which is comparable to that obtained by the two-step method and falls within the current quality control requirements for Illumina sequencing. In addition, a centrifuge-free version of the CF11 filtration method to isolate P. falciparum DNA at remote and minimally equipped field sites in malaria-endemic areas was validated.

    Conclusions: CF11 filtration is a cost-effective, scalable, one-step approach to remove human DNA from P. falciparum-infected whole blood samples.

    Funded by: Howard Hughes Medical Institute; Medical Research Council: G0600718, G19/9; Wellcome Trust: 089275, 090532, 098051

    Malaria journal 2012;11;41

  • An effective method to purify Plasmodium falciparum DNA directly from clinical blood samples for whole genome high-throughput sequencing.

    Auburn S, Campino S, Clark TG, Djimde AA, Zongo I, Pinches R, Manske M, Mangano V, Alcock D, Anastasi E, Maslen G, Macinnis B, Rockett K, Modiano D, Newbold CI, Doumbo OK, Ouédraogo JB and Kwiatkowski DP

    Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, United Kingdom. sa3@sanger.ac.uk

    Highly parallel sequencing technologies permit cost-effective whole genome sequencing of hundreds of Plasmodium parasites. The ability to sequence clinical Plasmodium samples, extracted directly from patient blood without a culture step, presents a unique opportunity to sample the diversity of "natural" parasite populations in high resolution clinical and epidemiological studies. A major challenge to sequencing clinical Plasmodium samples is the abundance of human DNA, which may substantially reduce the yield of Plasmodium sequence. We tested a range of human white blood cell (WBC) depletion methods on P. falciparum-infected patient samples in search of a method displaying an optimal balance of WBC-removal efficacy, cost, simplicity, and applicability to low resource settings. In the first of a two-part study, combinations of three different WBC depletion methods were tested on 43 patient blood samples in Mali. A two-step combination of Lymphoprep plus Plasmodipur best fitted our requirements, although moderate variability was observed in human DNA quantity. This approach was further assessed in a larger sample of 76 patients from Burkina Faso. WBC-removal efficacy remained high (<30% human DNA in >70% samples) and lower variation was observed in human DNA quantities. In order to assess the Plasmodium sequence yield at different human DNA proportions, 59 samples with up to 60% human DNA contamination were sequenced on the Illumina Genome Analyzer platform. An average ~40-fold coverage of the genome was observed per lane for samples with ≤ 30% human DNA. Even in low resource settings, using a simple two-step combination of Lymphoprep plus Plasmodipur, over 70% of clinical sample preparations should exhibit sufficiently low human DNA quantities to enable ~40-fold sequence coverage of the P. falciparum genome using a single lane on the Illumina Genome Analyzer platform. This approach should greatly facilitate large-scale clinical and epidemiologic studies of P. falciparum.

    Funded by: Howard Hughes Medical Institute: 55005502; Medical Research Council: G0600718, G19/9; Wellcome Trust: 090532, 090770

    PloS one 2011;6;7;e22213

  • Drug-resistant genotypes and multi-clonality in Plasmodium falciparum analysed by direct genome sequencing from peripheral blood of malaria patients.

    Robinson T, Campino SG, Auburn S, Assefa SA, Polley SD, Manske M, MacInnis B, Rockett KA, Maslen GL, Sanders M, Quail MA, Chiodini PL, Kwiatkowski DP, Clark TG and Sutherland CJ

    Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdom.

    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 among PCR products derived from numerically dominant clones. However, minority clones are of clinical interest as they may harbour genes conferring drug resistance, leading to enhanced survival after treatment and the possibility of subsequent therapeutic failure. We deployed new generation sequencing to derive genome data for five non-propagated parasite isolates taken directly from 4 different patients treated for clinical malaria in a UK hospital. Analysis of depth of coverage and length of sequence intervals between paired reads identified both previously described and novel gene deletions and amplifications. Full-length sequence data was extracted for 6 loci considered to be under selection by antimalarial drugs, and both known and previously unknown amino acid substitutions were identified. Full mitochondrial genomes were extracted from the sequencing data for each isolate, and these are compared against a panel of polymorphic sites derived from published or unpublished but publicly available data. Finally, genome-wide analysis of clone multiplicity was performed, and the number of infecting parasite clones estimated for each isolate. Each patient harboured at least 3 clones of P. falciparum by this analysis, consistent with results obtained with conventional PCR analysis of polymorphic merozoite antigen loci. We conclude that genome sequencing of peripheral blood P. falciparum taken directly from malaria patients provides high quality data useful for drug resistance studies, genomic structural analyses and population genetics, and also robustly represents clonal multiplicity.

    Funded by: Medical Research Council: G0600718, G19/9; Wellcome Trust: 077012/Z/05/Z, 090532

    PloS one 2011;6;8;e23204

  • Population genetic analysis of Plasmodium falciparum parasites using a customized Illumina GoldenGate genotyping assay.

    Campino S, Auburn S, Kivinen K, Zongo I, Ouedraogo JB, Mangano V, Djimde A, Doumbo OK, Kiara SM, Nzila A, Borrmann S, Marsh K, Michon P, Mueller I, Siba P, Jiang H, Su XZ, Amaratunga C, Socheat D, Fairhurst RM, Imwong M, Anderson T, Nosten F, White NJ, Gwilliam R, Deloukas P, MacInnis B, Newbold CI, Rockett K, Clark TG and Kwiatkowski DP

    Wellcome Trust Sanger Institute, Hinxton, United Kingdom. sc11@sanger.ac.uk

    The diversity in the Plasmodium falciparum genome can be used to explore parasite population dynamics, with practical applications to malaria control. The ability to identify the geographic origin and trace the migratory patterns of parasites with clinically important phenotypes such as drug resistance is particularly relevant. With increasing single-nucleotide polymorphism (SNP) discovery from ongoing Plasmodium genome sequencing projects, a demand for high SNP and sample throughput genotyping platforms for large-scale population genetic studies is required. Low parasitaemias and multiple clone infections present a number of challenges to genotyping P. falciparum. We addressed some of these issues using a custom 384-SNP Illumina GoldenGate assay on P. falciparum DNA from laboratory clones (long-term cultured adapted parasite clones), short-term cultured parasite isolates and clinical (non-cultured isolates) samples from East and West Africa, Southeast Asia and Oceania. Eighty percent of the SNPs (n = 306) produced reliable genotype calls on samples containing as little as 2 ng of total genomic DNA and on whole genome amplified DNA. Analysis of artificial mixtures of laboratory clones demonstrated high genotype calling specificity and moderate sensitivity to call minor frequency alleles. Clear resolution of geographically distinct populations was demonstrated using Principal Components Analysis (PCA), and global patterns of population genetic diversity were consistent with previous reports. These results validate the utility of the platform in performing population genetic studies of P. falciparum.

    Funded by: Howard Hughes Medical Institute; Medical Research Council: G0600718, G19/9; NIAID NIH HHS: R37 AI048071; Wellcome Trust: 090532, 093956

    PloS one 2011;6;6;e20251

  • Genome-wide and fine-resolution association analysis of malaria in West Africa.

    Jallow M, Teo YY, Small KS, Rockett KA, Deloukas P, Clark TG, Kivinen K, Bojang KA, Conway DJ, Pinder M, Sirugo G, Sisay-Joof F, Usen S, Auburn S, Bumpstead SJ, Campino S, Coffey A, Dunham A, Fry AE, Green A, Gwilliam R, Hunt SE, Inouye M, Jeffreys AE, Mendy A, Palotie A, Potter S, Ragoussis J, Rogers J, Rowlands K, Somaskantharajah E, Whittaker P, Widden C, Donnelly P, Howie B, Marchini J, Morris A, SanJoaquin M, Achidi EA, Agbenyega T, Allen A, Amodu O, Corran P, Djimde A, Dolo A, Doumbo OK, Drakeley C, Dunstan S, Evans J, Farrar J, Fernando D, Hien TT, Horstmann RD, Ibrahim M, Karunaweera N, Kokwaro G, Koram KA, Lemnge M, Makani J, Marsh K, Michon P, Modiano D, Molyneux ME, Mueller I, Parker M, Peshu N, Plowe CV, Puijalon O, Reeder J, Reyburn H, Riley EM, Sakuntabhai A, Singhasivanon P, Sirima S, Tall A, Taylor TE, Thera M, Troye-Blomberg M, Williams TN, Wilson M, Kwiatkowski DP, Wellcome Trust Case Control Consortium and Malaria Genomic Epidemiology Network

    MRC Laboratories, Fajara, Banjul, Gambia.

    We report a genome-wide association (GWA) study of severe malaria in The Gambia. The initial GWA scan included 2,500 children genotyped on the Affymetrix 500K GeneChip, and a replication study included 3,400 children. We used this to examine the performance of GWA methods in Africa. We found considerable population stratification, and also that signals of association at known malaria resistance loci were greatly attenuated owing to weak linkage disequilibrium (LD). To investigate possible solutions to the problem of low LD, we focused on the HbS locus, sequencing this region of the genome in 62 Gambian individuals and then using these data to conduct multipoint imputation in the GWA samples. This increased the signal of association, from P = 4 × 10(-7) to P = 4 × 10(-14), with the peak of the signal located precisely at the HbS causal variant. Our findings provide proof of principle that fine-resolution multipoint imputation, based on population-specific sequencing data, can substantially boost authentic GWA signals and enable fine mapping of causal variants in African populations.

    Funded by: Chief Scientist Office: CZB/4/540, ETM/75; Howard Hughes Medical Institute; Medical Research Council: G0600230, G0600230(77610), G0600329, G0600718, G0800759, G19/9, G9828345, MC_U190081977, MC_U190081993; NIAID NIH HHS: U19 AI065683, U19 AI065683-04; Wellcome Trust: 061858, 064890, 076113, 076934, 077011, 077383, 077383/Z/05/Z, 081682, 089062, 090532

    Nature genetics 2009;41;6;657-65

  • TLR9 polymorphisms in African populations: no association with severe malaria, but evidence of cis-variants acting on gene expression.

    Campino S, Forton J, Auburn S, Fry A, Diakite M, Richardson A, Hull J, Jallow M, Sisay-Joof F, Pinder M, Molyneux ME, Taylor TE, Rockett K, Clark TG and Kwiatkowski DP

    Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK. sc11@sanger.ac.uk

    Background: During malaria infection the Toll-like receptor 9 (TLR9) is activated through induction with plasmodium DNA or another malaria motif not yet identified. Although TLR9 activation by malaria parasites is well reported, the implication to the susceptibility to severe malaria is not clear. The aim of this study was to assess the contribution of genetic variation at TLR9 to severe malaria.

    Methods: This study explores the contribution of TLR9 genetic variants to severe malaria using two approaches. First, an association study of four common single nucleotide polymorphisms was performed on both family- and population-based studies from Malawian and Gambian populations (n>6000 individual). Subsequently, it was assessed whether TLR9 expression is affected by cis-acting variants and if these variants could be mapped. For this work, an allele specific expression (ASE) assay on a panel of HapMap cell lines was carried out.

    Results: No convincing association was found with polymorphisms in TLR9 for malaria severity, in either Gambian or Malawian populations, using both case-control and family based study designs. Using an allele specific expression assay it was observed that TLR9 expression is affected by cis-acting variants, these results were replicated in a second experiment using biological replicates.

    Conclusion: By using the largest cohorts analysed to date, as well as a standardized phenotype definition and study design, no association of TLR9 genetic variants with severe malaria was found. This analysis considered all common variants in the region, but it is remains possible that there are rare variants with association signals. This report also shows that TLR9 expression is potentially modulated through cis-regulatory variants, which may lead to differential inflammatory responses to infection between individuals.

    Funded by: Medical Research Council: G0600230, G19/9; Wellcome Trust

    Malaria journal 2009;8;44

  • A global network for investigating the genomic epidemiology of malaria.

    Malaria Genomic Epidemiology Network

    The University of Buea, PO Box 63, Buea, South West Province, Cameroon.

    Large-scale studies of genomic variation could assist efforts to eliminate malaria. But there are scientific, ethical and practical challenges to carrying out such studies in developing countries, where the burden of disease is greatest. The Malaria Genomic Epidemiology Network (MalariaGEN) is now working to overcome these obstacles, using a consortial approach that brings together researchers from 21 countries.

    Funded by: Medical Research Council: G0200454, G0200454(62635), G0600230, G0600230(77610), G0600718, G19/9; Wellcome Trust: 076934, 077383, 077383/Z/05/Z

    Nature 2008;456;7223;732-7

  • Validating discovered Cis-acting regulatory genetic variants: application of an allele specific expression approach to HapMap populations.

    Campino S, Forton J, Raj S, Mohr B, Auburn S, Fry A, Mangano VD, Vandiedonck C, Richardson A, Rockett K, Clark TG and Kwiatkowski DP

    Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK. sc11@sanger.ac.uk

    Background: Localising regulatory variants that control gene expression is a challenge for genome research. Several studies have recently identified non-coding polymorphisms associated with inter-individual differences in gene expression. These approaches rely on the identification of signals of association against a background of variation due to other genetic and environmental factors. A complementary approach is to use an Allele-Specific Expression (ASE) assay, which is more robust to the effects of environmental variation and trans-acting genetic factors.

    Methodology/principal findings: Here we apply an ASE method which utilises heterozygosity within an individual to compare expression of the two alleles of a gene in a single cell. We used individuals from three HapMap population groups and analysed the allelic expression of genes with cis-regulatory regions previously identified using total gene expression studies. We were able to replicate the results in five of the six genes tested, and refined the cis- associated regions to a small number of variants. We also showed that by using multi-populations it is possible to refine the associated cis-effect DNA regions.

    Conclusions/significance: We discuss the efficacy and drawbacks of both total gene expression and ASE approaches in the discovery of cis-acting variants. We show that the ASE approach has significant advantages as it is a cleaner representation of cis-acting effects. We also discuss the implication of using different populations to map cis-acting regions and the importance of finding regulatory variants which contribute to human phenotypic variation.

    Funded by: Medical Research Council: G0600230, G19/9; Wellcome Trust

    PloS one 2008;3;12;e4105

  • Identification of common genetic variation that modulates alternative splicing.

    Hull J, Campino S, Rowlands K, Chan MS, Copley RR, Taylor MS, Rockett K, Elvidge G, Keating B, Knight J and Kwiatkowski D

    University Department of Paediatrics, John Radcliffe Hospital, Oxford, United Kingdom. jeremy.hull@paediatrics.ox.ac.uk

    Alternative splicing of genes is an efficient means of generating variation in protein function. Several disease states have been associated with rare genetic variants that affect splicing patterns. Conversely, splicing efficiency of some genes is known to vary between individuals without apparent ill effects. What is not clear is whether commonly observed phenotypic variation in splicing patterns, and hence potential variation in protein function, is to a significant extent determined by naturally occurring DNA sequence variation and in particular by single nucleotide polymorphisms (SNPs). In this study, we surveyed the splicing patterns of 250 exons in 22 individuals who had been previously genotyped by the International HapMap Project. We identified 70 simple cassette exon alternative splicing events in our experimental system; for six of these, we detected consistent differences in splicing pattern between individuals, with a highly significant association between splice phenotype and neighbouring SNPs. Remarkably, for five out of six of these events, the strongest correlation was found with the SNP closest to the intron-exon boundary, although the distance between these SNPs and the intron-exon boundary ranged from 2 bp to greater than 1,000 bp. Two of these SNPs were further investigated using a minigene splicing system, and in each case the SNPs were found to exert cis-acting effects on exon splicing efficiency in vitro. The functional consequences of these SNPs could not be predicted using bioinformatic algorithms. Our findings suggest that phenotypic variation in splicing patterns is determined by the presence of SNPs within flanking introns or exons. Effects on splicing may represent an important mechanism by which SNPs influence gene function.

    Funded by: Medical Research Council: G0600230, G19/9; Wellcome Trust: 074318

    PLoS genetics 2007;3;6;e99

  • Unique genetic variation revealed by a microsatellite polymorphism survey in ten wild-derived inbred strains.

    Campino S, Behrschmidt C, Bagot S, Guénet JL, Cazenave PA, Holmberg D and Penha-Gonçalves C

    Instituto Gulbenkian de Ciência, Rua da Quinta Grande, 6, Oeiras, Portugal.

    Here we report on a genome polymorphism survey using 254 microsatellite markers in ten recently wild-derived inbred strains. Allele size analysis showed that the rate of polymorphism of these wild-derived mouse strains when compared with any of the common laboratory strains is on average 79.8%. We found 632 wild-derived alleles that were not present in the common laboratory strains, representing a 61% increase over the genetic variation observed in the laboratory strains. We also found that on average 14.5% of the microsatellite alleles of any given wild-derived inbred strain were unique. Our results indicate that the recently wild-derived mouse strains represent repositories of unique naturally occurring genetic variability and may prove invaluable for the study of complex phenotypes and in the construction of new mouse models of human disease.

    Genomics 2002;79;5;618-20

Kalia Dede

kd9@sanger.ac.uk Programme Administrator

Kalia graduated the University of Patras (Greece) with a B.Sc. in Biology. In 2008 she received the Leventis Foundation Distinguished Scholar Award and joined Prof. Fraguedakis-Tsolis and Prof. Chondropoulos laboratory to pursue an M.Sc. in Ecology, Management and Conservation of Natural Environment. During her M.Sc. Kalia studied the micromammals of the genus Mus in two different habitats in Cyprus, using mtDNA and geometric morphometric analyses.

Research

After graduating, Kalia moved to the UK where she joined the Wellcome Trust Sanger Institute as Programme Administrator in 2014. Working hand in hand with the Research Administrator, she provides high-level administrative and organisational support to the Sanger Institute Malaria Programme and its Faculty, driving forward the aims and objectives of the Programme.

Eleanor Drury

- Laboratory Manager

Eleanor graduated from Newcastle University in 2000 with a degree in Applied Biology. She began her scientific career working for Cambridge University in association with the Juvenile Diabetes Foundation and the Wellcome Trust, before moving into industry and running the RNA team at Pharmagene. Eleanor joined the Sanger Institute in 2004 and worked with the Medical Sequencing team, before joining the Malaria Programme in 2010.

Research

As an Advanced Research Assistant, Eleanor works on the Plasmodium Genome Variation project, which aims to describe the genetic diversity between malaria parasites from across the globe using high throughput genome sequencing. Her work involves sample reception, quantification and quality control, as well as laboratory support including parasite culture, protocol development and troubleshooting.

References

  • The GENCODE exome: sequencing the complete human exome.

    Coffey AJ, Kokocinski F, Calafato MS, Scott CE, Palta P, Drury E, Joyce CJ, Leproust EM, Harrow J, Hunt S, Lehesjoki AE, Turner DJ, Hubbard TJ and Palotie A

    Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Cambridge, UK.

    Sequencing the coding regions, the exome, of the human genome is one of the major current strategies to identify low frequency and rare variants associated with human disease traits. So far, the most widely used commercial exome capture reagents have mainly targeted the consensus coding sequence (CCDS) database. We report the design of an extended set of targets for capturing the complete human exome, based on annotation from the GENCODE consortium. The extended set covers an additional 5594 genes and 10.3 Mb compared with the current CCDS-based sets. The additional regions include potential disease genes previously inaccessible to exome resequencing studies, such as 43 genes linked to ion channel activity and 70 genes linked to protein kinase activity. In total, the new GENCODE exome set developed here covers 47.9 Mb and performed well in sequence capture experiments. In the sample set used in this study, we identified over 5000 SNP variants more in the GENCODE exome target (24%) than in the CCDS-based exome sequencing.

    Funded by: NHGRI NIH HHS: 5U54HG004555; Wellcome Trust: 077198, WT062023, WT077198, WT089062

    European journal of human genetics : EJHG 2011;19;7;827-31

  • An evaluation of different target enrichment methods in pooled sequencing designs for complex disease association studies.

    Day-Williams AG, McLay K, Drury E, Edkins S, Coffey AJ, Palotie A and Zeggini E

    Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridgeshire, United Kingdom.

    Pooled sequencing can be a cost-effective approach to disease variant discovery, but its applicability in association studies remains unclear. We compare sequence enrichment methods coupled to next-generation sequencing in non-indexed pools of 1, 2, 10, 20 and 50 individuals and assess their ability to discover variants and to estimate their allele frequencies. We find that pooled resequencing is most usefully applied as a variant discovery tool due to limitations in estimating allele frequency with high enough accuracy for association studies, and that in-solution hybrid-capture performs best among the enrichment methods examined regardless of pool size.

    Funded by: Wellcome Trust: WT088885/Z/09/Z

    PloS one 2011;6;11;e26279

  • Clustered coding variants in the glutamate receptor complexes of individuals with schizophrenia and bipolar disorder.

    Frank RA, McRae AF, Pocklington AJ, van de Lagemaat LN, Navarro P, Croning MD, Komiyama NH, Bradley SJ, Challiss RA, Armstrong JD, Finn RD, Malloy MP, MacLean AW, Harris SE, Starr JM, Bhaskar SS, Howard EK, Hunt SE, Coffey AJ, Ranganath V, Deloukas P, Rogers J, Muir WJ, Deary IJ, Blackwood DH, Visscher PM and Grant SG

    Wellcome Trust Sanger Institute, Genome Campus, Hinxton, Cambridgeshire, United Kingdom.

    Current models of schizophrenia and bipolar disorder implicate multiple genes, however their biological relationships remain elusive. To test the genetic role of glutamate receptors and their interacting scaffold proteins, the exons of ten glutamatergic 'hub' genes in 1304 individuals were re-sequenced in case and control samples. No significant difference in the overall number of non-synonymous single nucleotide polymorphisms (nsSNPs) was observed between cases and controls. However, cluster analysis of nsSNPs identified two exons encoding the cysteine-rich domain and first transmembrane helix of GRM1 as a risk locus with five mutations highly enriched within these domains. A new splice variant lacking the transmembrane GPCR domain of GRM1 was discovered in the human brain and the GRM1 mutation cluster could perturb the regulation of this variant. The predicted effect on individuals harbouring multiple mutations distributed in their ten hub genes was also examined. Diseased individuals possessed an increased load of deleteriousness from multiple concurrent rare and common coding variants. Together, these data suggest a disease model in which the interplay of compound genetic coding variants, distributed among glutamate receptors and their interacting proteins, contribute to the pathogenesis of schizophrenia and bipolar disorders.

    Funded by: Chief Scientist Office: CZB/4/505, ETM/55; Medical Research Council: MC_U127592696; Wellcome Trust

    PloS one 2011;6;4;e19011

  • Genome-wide association study of CNVs in 16,000 cases of eight common diseases and 3,000 shared controls.

    Wellcome Trust Case Control Consortium, Craddock N, Hurles ME, Cardin N, Pearson RD, Plagnol V, Robson S, Vukcevic D, Barnes C, Conrad DF, Giannoulatou E, Holmes C, Marchini JL, Stirrups K, Tobin MD, Wain LV, Yau C, Aerts J, Ahmad T, Andrews TD, Arbury H, Attwood A, Auton A, Ball SG, Balmforth AJ, Barrett JC, Barroso I, Barton A, Bennett AJ, Bhaskar S, Blaszczyk K, Bowes J, Brand OJ, Braund PS, Bredin F, Breen G, Brown MJ, Bruce IN, Bull J, Burren OS, Burton J, Byrnes J, Caesar S, Clee CM, Coffey AJ, Connell JM, Cooper JD, Dominiczak AF, Downes K, Drummond HE, Dudakia D, Dunham A, Ebbs B, Eccles D, Edkins S, Edwards C, Elliot A, Emery P, Evans DM, Evans G, Eyre S, Farmer A, Ferrier IN, Feuk L, Fitzgerald T, Flynn E, Forbes A, Forty L, Franklyn JA, Freathy RM, Gibbs P, Gilbert P, Gokumen O, Gordon-Smith K, Gray E, Green E, Groves CJ, Grozeva D, Gwilliam R, Hall A, Hammond N, Hardy M, Harrison P, Hassanali N, Hebaishi H, Hines S, Hinks A, Hitman GA, Hocking L, Howard E, Howard P, Howson JM, Hughes D, Hunt S, Isaacs JD, Jain M, Jewell DP, Johnson T, Jolley JD, Jones IR, Jones LA, Kirov G, Langford CF, Lango-Allen H, Lathrop GM, Lee J, Lee KL, Lees C, Lewis K, Lindgren CM, Maisuria-Armer M, Maller J, Mansfield J, Martin P, Massey DC, McArdle WL, McGuffin P, McLay KE, Mentzer A, Mimmack ML, Morgan AE, Morris AP, Mowat C, Myers S, Newman W, Nimmo ER, O'Donovan MC, Onipinla A, Onyiah I, Ovington NR, Owen MJ, Palin K, Parnell K, Pernet D, Perry JR, Phillips A, Pinto D, Prescott NJ, Prokopenko I, Quail MA, Rafelt S, Rayner NW, Redon R, Reid DM, Renwick, Ring SM, Robertson N, Russell E, St Clair D, Sambrook JG, Sanderson JD, Schuilenburg H, Scott CE, Scott R, Seal S, Shaw-Hawkins S, Shields BM, Simmonds MJ, Smyth DJ, Somaskantharajah E, Spanova K, Steer S, Stephens J, Stevens HE, Stone MA, Su Z, Symmons DP, Thompson JR, Thomson W, Travers ME, Turnbull C, Valsesia A, Walker M, Walker NM, Wallace C, Warren-Perry M, Watkins NA, Webster J, Weedon MN, Wilson AG, Woodburn M, Wordsworth BP, Young AH, Zeggini E, Carter NP, Frayling TM, Lee C, McVean G, Munroe PB, Palotie A, Sawcer SJ, Scherer SW, Strachan DP, Tyler-Smith C, Brown MA, Burton PR, Caulfield MJ, Compston A, Farrall M, Gough SC, Hall AS, Hattersley AT, Hill AV, Mathew CG, Pembrey M, Satsangi J, Stratton MR, Worthington J, Deloukas P, Duncanson A, Kwiatkowski DP, McCarthy MI, Ouwehand W, Parkes M, Rahman N, Todd JA, Samani NJ and Donnelly P

    Copy number variants (CNVs) account for a major proportion of human genetic polymorphism and have been predicted to have an important role in genetic susceptibility to common disease. To address this we undertook a large, direct genome-wide study of association between CNVs and eight common human diseases. Using a purpose-designed array we typed approximately 19,000 individuals into distinct copy-number classes at 3,432 polymorphic CNVs, including an estimated approximately 50% of all common CNVs larger than 500 base pairs. We identified several biological artefacts that lead to false-positive associations, including systematic CNV differences between DNAs derived from blood and cell lines. Association testing and follow-up replication analyses confirmed three loci where CNVs were associated with disease-IRGM for Crohn's disease, HLA for Crohn's disease, rheumatoid arthritis and type 1 diabetes, and TSPAN8 for type 2 diabetes-although in each case the locus had previously been identified in single nucleotide polymorphism (SNP)-based studies, reflecting our observation that most common CNVs that are well-typed on our array are well tagged by SNPs and so have been indirectly explored through SNP studies. We conclude that common CNVs that can be typed on existing platforms are unlikely to contribute greatly to the genetic basis of common human diseases.

    Funded by: Arthritis Research UK: 17552; British Heart Foundation: RG/09/012/28096; Chief Scientist Office: CZB/4/540, ETM/137, ETM/75; Medical Research Council: G0000934, G0400874, G0500115, G0501942, G0600329, G0600705, G0700491, G0701003, G0701420, G0701810, G0701810(85517), G0800383, G0800509, G0800759, G0801418B, G19/9, G90/106, G9521010, G9817803B, MC_UP_A390_1107; Wellcome Trust: 061858, 083948, 089989, 090532

    Nature 2010;464;7289;713-20

  • Target-enrichment strategies for next-generation sequencing.

    Mamanova L, Coffey AJ, Scott CE, Kozarewa I, Turner EH, Kumar A, Howard E, Shendure J and Turner DJ

    The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, UK.

    We have not yet reached a point at which routine sequencing of large numbers of whole eukaryotic genomes is feasible, and so it is often necessary to select genomic regions of interest and to enrich these regions before sequencing. There are several enrichment approaches, each with unique advantages and disadvantages. Here we describe our experiences with the leading target-enrichment technologies, the optimizations that we have performed and typical results that can be obtained using each. We also provide detailed protocols for each technology so that end users can find the best compromise between sensitivity, specificity and uniformity for their particular project.

    Funded by: NHGRI NIH HHS: 5R21HG004749, R21 HG004749; NHLBI NIH HHS: 5R01HL094976, R01 HL094976; Wellcome Trust: WT079643

    Nature methods 2010;7;2;111-8

  • Esophageal atresia, hypoplasia of zygomatic complex, microcephaly, cup-shaped ears, congenital heart defect, and mental retardation--new MCA/MR syndrome in two affected sibs and a mildly affected mother?

    Wieczorek D, Shaw-Smith C, Kohlhase J, Schmitt W, Buiting K, Coffey A, Howard E, Hehr U and Gillessen-Kaesbach G

    Institut für Humangenetik, Universitätsklinikum Essen, Germany, and Department of Medical Genetics, Addenbrooke's Hospital, Cambridge, UK. dagmar.wieczorek@uni-due.de

    The previously undescribed combination of esophageal atresia, hypoplasia of the zygomatic complex, microcephaly, cup-shaped ears, congenital heart defect, and mental retardation was diagnosed in two siblings of different sexes, with the brother being more severely affected. The mother presented with zygomatic arch hypoplasia of the right side only. We discuss major differential diagnoses: Goldenhar, Feingold, CHARGE, and Treacher Collins syndromes show a few overlapping clinical features, but these diagnoses are unlikely as the clinical findings are unusual for Goldenhar syndrome and mutational screening of the MYCN, the CHD7, and the TCOF1 genes did not reveal any abnormalities. Autosomal recessive oto-facial syndrome, hypomandibular faciocranial dysostosis, and Ozkan syndromes were clinically excluded. A microdeletion 22q11.2 was excluded by FISH analysis, a microdeletion 2p23-p24 by microsatellite analyses, a subtelomeric chromosomal aberration by MLPA, and a small genomic deletion/duplication by CGH array. As X-inactivation studies did not show skewed X-inactivation in the mother, we consider X-chromosomal recessive inheritance of this condition less likely. We discuss autosomal dominant inheritance with variable expressivity or mosaicism in the mother as the likely genetic mechanism in this new multiple congenital anomaly/mental retardation (MCA/MR) syndrome.

    American journal of medical genetics. Part A 2007;143A;11;1135-42

Rachel Giacomantonio

- Communications Manager

Rachel holds an HonBSc from the University of Toronto, where she spent her summers studying par homologues in M. smegmatis. After graduating, she held various positions in communications and programme management with the World Agroforestry Centre (Nairobi, Kenya) and Dalhousie University (Halifax, Canada). Prior to joining the team in 2012, Rachel worked in a similar capacity with the Worldwide Antimalarial Resistance Network (WWARN).

Research

Rachel is the Communications Manager for the Genomics and Global Health Programme at the University of Oxford, supporting both MalariaGEN and the MRC Centre for Genomics and Global Health (CGGH). Rachel’s focus is on ensuring that the scientific, collaborative and educational activities of the Programme are appropriately communicated in order to support the Programme’s strategic objectives. Operationally, her role is varied and involves coordinating activities across numerous channels including web and direct communications. She supports product launch and development plans for digital resources, maintains the CGGH and MalariaGEN websites, and manages social media activities.

Will Hamilton

- unknown

Will Hamilton is part of the University of Cambridge MB-PhD programme, combining a research PhD with training in clinical medicine. He started his PhD in 2011 at the Mahidol Oxford Tropical Medicine Research Unit (MORU) in Bangkok, Thailand, before moving to Sanger.

Will's research background is mainly in laboratory work, investigating innate defence against retroviruses such as HIV in the Mothes Laboratory at Yale, and protein expression in Trypanosoma brucei, the parasite responsible for African sleeping sickness, in the Field Laboratory in Cambridge. His clinical interests include infectious disease, tropical medicine and global public health.

Research

Will investigates how the Plasmodium falciparum genome changes (mutates) over time. Genetic mutations are the driving force behind evolutionary change, and this diversity is an important factor in how P. falciparum develops antimalarial drug resistance and evades the human immune system. Whole genome sequencing provides a uniquely overarching view of parasite evolution and identifies highly dynamic regions of the genome that are of evolutionary and pathophysiological interest. Will is also investigating how DNA repair processes impact on Plasmodium genome evolution, using experimental genetic approaches.

Mihir Kekre

- Advanced Research Assistant

Mihir graduated with a Bachelor’s degree in Biotechnology from Manipal University, India in 2010. Following this, he completed his Master’s from King’s College London in 2011, majoring in Biopharmaceuticals and Drug Discovery. He joined the Malaria Programme as a research assistant in the summer of 2012.

Research

Currently an advanced research assistant, Mihir’s role primarily encompasses developing novel strategies for leucodepletion and processing of Plasmodium-infected clinical samples. A major part of this effort involves designing, testing and validating simplistic, low-cost and high yielding methods that can be both efficiently and effortlessly carried out at resource-limited endemic field sites.

He also assists in management of the MalariaGEN pipeline that involves clinical sample reception, quantification, improving DNA handling and streamlining various aspects of process quality control. His other work includes culturing Plasmodium falciparum for in-vitro studies and maintenance of Containment Level 2 & 3 laboratories.

Cinzia Malangone

- Senior Software Developer

Cinzia graduated with a Masters degree in Computer Science from the University of Turin in 2003. She worked for several years in the Insurance and Retail sectors, before starting her biological career working on a cardiovascular applications project at the University of Turin.

Upon joining the Wellcome Trust Sanger Institute in 2010, Cinzia worked with the Production Software Development team on the development of Sequencescape, a LIMs based sample management system for tracking samples through the Illumina sequencing pipeline.

Research

Cinzia joined the Malaria Programme in April 2011 and works on the design and development of web applications to explore catalogues of genetic variation, the sequencing analysis pipeline, and on ExplorerCat, a web-based application for publishing genotyping data.

Magnus Manske

- Head of Informatics

In 2006, Magnus graduated magna cum laude from the University of Cologne with a PhD in Biochemistry. Shortly after completing his PhD, he joined the Sanger Institute Malaria Programme as a Senior Computer Programmer, later becoming a Senior Staff Scientist; he is currently the Head of Informatics. Magnus has also worked voluntarily for many years as an author and programmer for Wikipedia. He is the original author of MediaWiki, the software that powers Wikipedia and many other wiki-based sites.

Research

Magnus works on the analysis of sequencing and genotyping data for MalariaGEN projects such as the P. falciparum Community Project, and also writes interactive data visualization applications for the web.

http://www.linkedin.com/profile/view?id=58317594

References

  • Analysis of Plasmodium falciparum diversity in natural infections by deep sequencing.

    Manske M, Miotto O, Campino S, Auburn S, Almagro-Garcia J, Maslen G, O'Brien J, Djimde A, Doumbo O, Zongo I, Ouedraogo JB, Michon P, Mueller I, Siba P, Nzila A, Borrmann S, Kiara SM, Marsh K, Jiang H, Su XZ, Amaratunga C, Fairhurst R, Socheat D, Nosten F, Imwong M, White NJ, Sanders M, Anastasi E, Alcock D, Drury E, Oyola S, Quail MA, Turner DJ, Ruano-Rubio V, Jyothi D, Amenga-Etego L, Hubbart C, Jeffreys A, Rowlands K, Sutherland C, Roper C, Mangano V, Modiano D, Tan JC, Ferdig MT, Amambua-Ngwa A, Conway DJ, Takala-Harrison S, Plowe CV, Rayner JC, Rockett KA, Clark TG, Newbold CI, Berriman M, MacInnis B and Kwiatkowski DP

    Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, UK.

    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 malaria, either directly or after short-term culture. Analysis of 86,158 exonic single nucleotide polymorphisms that passed genotyping quality control in 227 samples from Africa, Asia and Oceania provides genome-wide estimates of allele frequency distribution, population structure and linkage disequilibrium. By comparing the genetic diversity of individual infections with that of the local parasite population, we derive a metric of within-host diversity that is related to the level of inbreeding in the population. An open-access web application has been established for the exploration of regional differences in allele frequency and of highly differentiated loci in the P. falciparum genome.

    Funded by: Howard Hughes Medical Institute: 55005502; Medical Research Council: G0600718, G19/9; Wellcome Trust: 075491/Z/04, 077012/Z/05/Z, 082370, 089275, 090532, 090532/Z/09/Z, 090770, 090770/Z/09/Z, 092654, 093956, 098051

    Nature 2012;487;7407;375-9

  • Optimizing Illumina next-generation sequencing library preparation for extremely AT-biased genomes.

    Oyola SO, Otto TD, Gu Y, Maslen G, Manske M, Campino S, Turner DJ, Macinnis B, Kwiatkowski DP, Swerdlow HP and Quail MA

    Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK. so1@sanger.ac.uk

    Background: 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 currently available NGS platforms. The genomes of some important pathogenic organisms like Plasmodium falciparum (high AT content) and Mycobacterium tuberculosis (high GC content) display extremes of base composition. The standard library preparation procedures that employ PCR amplification have been shown to cause uneven read coverage particularly across AT and GC rich regions, leading to problems in genome assembly and variation analyses. Alternative library-preparation approaches that omit PCR amplification require large quantities of starting material and hence are not suitable for small amounts of DNA/RNA such as those from clinical isolates. We have developed and optimized library-preparation procedures suitable for low quantity starting material and tolerant to extremely high AT content sequences.

    Results: We have used our optimized conditions in parallel with standard methods to prepare Illumina sequencing libraries from a non-clinical and a clinical isolate (containing ~53% host contamination). By analyzing and comparing the quality of sequence data generated, we show that our optimized conditions that involve a PCR additive (TMAC), produces amplified libraries with improved coverage of extremely AT-rich regions and reduced bias toward GC neutral templates.

    Conclusion: We have developed a robust and optimized Next-Generation Sequencing library amplification method suitable for extremely AT-rich genomes. The new amplification conditions significantly reduce bias and retain the complexity of either extremes of base composition. This development will greatly benefit sequencing clinical samples that often require amplification due to low mass of DNA starting material.

    Funded by: Medical Research Council: G0600718, G19/9; Wellcome Trust: 079355/Z/06/Z, 090532

    BMC genomics 2012;13;1

  • An effective method to purify Plasmodium falciparum DNA directly from clinical blood samples for whole genome high-throughput sequencing.

    Auburn S, Campino S, Clark TG, Djimde AA, Zongo I, Pinches R, Manske M, Mangano V, Alcock D, Anastasi E, Maslen G, Macinnis B, Rockett K, Modiano D, Newbold CI, Doumbo OK, Ouédraogo JB and Kwiatkowski DP

    Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, United Kingdom. sa3@sanger.ac.uk

    Highly parallel sequencing technologies permit cost-effective whole genome sequencing of hundreds of Plasmodium parasites. The ability to sequence clinical Plasmodium samples, extracted directly from patient blood without a culture step, presents a unique opportunity to sample the diversity of "natural" parasite populations in high resolution clinical and epidemiological studies. A major challenge to sequencing clinical Plasmodium samples is the abundance of human DNA, which may substantially reduce the yield of Plasmodium sequence. We tested a range of human white blood cell (WBC) depletion methods on P. falciparum-infected patient samples in search of a method displaying an optimal balance of WBC-removal efficacy, cost, simplicity, and applicability to low resource settings. In the first of a two-part study, combinations of three different WBC depletion methods were tested on 43 patient blood samples in Mali. A two-step combination of Lymphoprep plus Plasmodipur best fitted our requirements, although moderate variability was observed in human DNA quantity. This approach was further assessed in a larger sample of 76 patients from Burkina Faso. WBC-removal efficacy remained high (<30% human DNA in >70% samples) and lower variation was observed in human DNA quantities. In order to assess the Plasmodium sequence yield at different human DNA proportions, 59 samples with up to 60% human DNA contamination were sequenced on the Illumina Genome Analyzer platform. An average ~40-fold coverage of the genome was observed per lane for samples with ≤ 30% human DNA. Even in low resource settings, using a simple two-step combination of Lymphoprep plus Plasmodipur, over 70% of clinical sample preparations should exhibit sufficiently low human DNA quantities to enable ~40-fold sequence coverage of the P. falciparum genome using a single lane on the Illumina Genome Analyzer platform. This approach should greatly facilitate large-scale clinical and epidemiologic studies of P. falciparum.

    Funded by: Howard Hughes Medical Institute: 55005502; Medical Research Council: G0600718, G19/9; Wellcome Trust: 090532, 090770

    PloS one 2011;6;7;e22213

  • An optimized microarray platform for assaying genomic variation in Plasmodium falciparum field populations.

    Tan JC, Miller BA, Tan A, Patel JJ, Cheeseman IH, Anderson TJ, Manske M, Maslen G, Kwiatkowski DP and Ferdig MT

    The Eck Institute for Global Health, University of Notre Dame, 100 Galvin Life Sciences, Notre Dame, IN 46556, USA.

    We present an optimized probe design for copy number variation (CNV) and SNP genotyping in the Plasmodium falciparum genome. We demonstrate that variable length and isothermal probes are superior to static length probes. We show that sample preparation and hybridization conditions mitigate the effects of host DNA contamination in field samples. The microarray and workflow presented can be used to identify CNVs and SNPs with 95% accuracy in a single hybridization, in field samples containing up to 92% human DNA contamination.

    Funded by: Medical Research Council: G19/9; NCRR NIH HHS: RR013556; NIAID NIH HHS: AI072517, AI075145; Wellcome Trust: 090532

    Genome biology 2011;12;4;R35

  • Drug-resistant genotypes and multi-clonality in Plasmodium falciparum analysed by direct genome sequencing from peripheral blood of malaria patients.

    Robinson T, Campino SG, Auburn S, Assefa SA, Polley SD, Manske M, MacInnis B, Rockett KA, Maslen GL, Sanders M, Quail MA, Chiodini PL, Kwiatkowski DP, Clark TG and Sutherland CJ

    Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdom.

    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 among PCR products derived from numerically dominant clones. However, minority clones are of clinical interest as they may harbour genes conferring drug resistance, leading to enhanced survival after treatment and the possibility of subsequent therapeutic failure. We deployed new generation sequencing to derive genome data for five non-propagated parasite isolates taken directly from 4 different patients treated for clinical malaria in a UK hospital. Analysis of depth of coverage and length of sequence intervals between paired reads identified both previously described and novel gene deletions and amplifications. Full-length sequence data was extracted for 6 loci considered to be under selection by antimalarial drugs, and both known and previously unknown amino acid substitutions were identified. Full mitochondrial genomes were extracted from the sequencing data for each isolate, and these are compared against a panel of polymorphic sites derived from published or unpublished but publicly available data. Finally, genome-wide analysis of clone multiplicity was performed, and the number of infecting parasite clones estimated for each isolate. Each patient harboured at least 3 clones of P. falciparum by this analysis, consistent with results obtained with conventional PCR analysis of polymorphic merozoite antigen loci. We conclude that genome sequencing of peripheral blood P. falciparum taken directly from malaria patients provides high quality data useful for drug resistance studies, genomic structural analyses and population genetics, and also robustly represents clonal multiplicity.

    Funded by: Medical Research Council: G0600718, G19/9; Wellcome Trust: 077012/Z/05/Z, 090532

    PloS one 2011;6;8;e23204

  • Ten simple rules for editing Wikipedia.

    Logan DW, Sandal M, Gardner PP, Manske M and Bateman A

    PLoS computational biology 2010;6;9

  • SnoopCGH: software for visualizing comparative genomic hybridization data.

    Almagro-Garcia J, Manske M, Carret C, Campino S, Auburn S, Macinnis BL, Maslen G, Pain A, Newbold CI, Kwiatkowski DP and Clark TG

    Wellcome Trust Sanger Institute, Hinxton, The Weatherall Institute of Molecular Medicine and Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK. jg10@sanger.ac.uk

    Unlabelled: Array-based comparative genomic hybridization (CGH) technology is used to discover and validate genomic structural variation, including copy number variants, insertions, deletions and other structural variants (SVs). The visualization and summarization of the array CGH data outputs, potentially across many samples, is an important process in the identification and analysis of SVs. We have developed a software tool for SV analysis using data from array CGH technologies, which is also amenable to short-read sequence data.

    Availability and implementation: SnoopCGH is written in java and is available from http://snoopcgh.sourceforge.net/

    Funded by: Medical Research Council: G0600718, G19/9; Wellcome Trust

    Bioinformatics (Oxford, England) 2009;25;20;2732-3

  • Genome-wide and fine-resolution association analysis of malaria in West Africa.

    Jallow M, Teo YY, Small KS, Rockett KA, Deloukas P, Clark TG, Kivinen K, Bojang KA, Conway DJ, Pinder M, Sirugo G, Sisay-Joof F, Usen S, Auburn S, Bumpstead SJ, Campino S, Coffey A, Dunham A, Fry AE, Green A, Gwilliam R, Hunt SE, Inouye M, Jeffreys AE, Mendy A, Palotie A, Potter S, Ragoussis J, Rogers J, Rowlands K, Somaskantharajah E, Whittaker P, Widden C, Donnelly P, Howie B, Marchini J, Morris A, SanJoaquin M, Achidi EA, Agbenyega T, Allen A, Amodu O, Corran P, Djimde A, Dolo A, Doumbo OK, Drakeley C, Dunstan S, Evans J, Farrar J, Fernando D, Hien TT, Horstmann RD, Ibrahim M, Karunaweera N, Kokwaro G, Koram KA, Lemnge M, Makani J, Marsh K, Michon P, Modiano D, Molyneux ME, Mueller I, Parker M, Peshu N, Plowe CV, Puijalon O, Reeder J, Reyburn H, Riley EM, Sakuntabhai A, Singhasivanon P, Sirima S, Tall A, Taylor TE, Thera M, Troye-Blomberg M, Williams TN, Wilson M, Kwiatkowski DP, Wellcome Trust Case Control Consortium and Malaria Genomic Epidemiology Network

    MRC Laboratories, Fajara, Banjul, Gambia.

    We report a genome-wide association (GWA) study of severe malaria in The Gambia. The initial GWA scan included 2,500 children genotyped on the Affymetrix 500K GeneChip, and a replication study included 3,400 children. We used this to examine the performance of GWA methods in Africa. We found considerable population stratification, and also that signals of association at known malaria resistance loci were greatly attenuated owing to weak linkage disequilibrium (LD). To investigate possible solutions to the problem of low LD, we focused on the HbS locus, sequencing this region of the genome in 62 Gambian individuals and then using these data to conduct multipoint imputation in the GWA samples. This increased the signal of association, from P = 4 × 10(-7) to P = 4 × 10(-14), with the peak of the signal located precisely at the HbS causal variant. Our findings provide proof of principle that fine-resolution multipoint imputation, based on population-specific sequencing data, can substantially boost authentic GWA signals and enable fine mapping of causal variants in African populations.

    Funded by: Chief Scientist Office: CZB/4/540, ETM/75; Howard Hughes Medical Institute; Medical Research Council: G0600230, G0600230(77610), G0600329, G0600718, G0800759, G19/9, G9828345, MC_U190081977, MC_U190081993; NIAID NIH HHS: U19 AI065683, U19 AI065683-04; Wellcome Trust: 061858, 064890, 076113, 076934, 077011, 077383, 077383/Z/05/Z, 081682, 089062, 090532

    Nature genetics 2009;41;6;657-65

  • A global network for investigating the genomic epidemiology of malaria.

    Malaria Genomic Epidemiology Network

    The University of Buea, PO Box 63, Buea, South West Province, Cameroon.

    Large-scale studies of genomic variation could assist efforts to eliminate malaria. But there are scientific, ethical and practical challenges to carrying out such studies in developing countries, where the burden of disease is greatest. The Malaria Genomic Epidemiology Network (MalariaGEN) is now working to overcome these obstacles, using a consortial approach that brings together researchers from 21 countries.

    Funded by: Medical Research Council: G0200454, G0200454(62635), G0600230, G0600230(77610), G0600718, G19/9; Wellcome Trust: 076934, 077383, 077383/Z/05/Z

    Nature 2008;456;7223;732-7

  • The RNA WikiProject: community annotation of RNA families.

    Daub J, Gardner PP, Tate J, Ramsköld D, Manske M, Scott WG, Weinberg Z, Griffiths-Jones S and Bateman A

    The online encyclopedia Wikipedia has become one of the most important online references in the world and has a substantial and growing scientific content. A search of Google with many RNA-related keywords identifies a Wikipedia article as the top hit. We believe that the RNA community has an important and timely opportunity to maximize the content and quality of RNA information in Wikipedia. To this end, we have formed the RNA WikiProject (http://en.wikipedia.org/wiki/Wikipedia:WikiProject_RNA) as part of the larger Molecular and Cellular Biology WikiProject. We have created over 600 new Wikipedia articles describing families of noncoding RNAs based on the Rfam database, and invite the community to update, edit, and correct these articles. The Rfam database now redistributes this Wikipedia content as the primary textual annotation of its RNA families. Users can, therefore, for the first time, directly edit the content of one of the major RNA databases. We believe that this Wikipedia/Rfam link acts as a functioning model for incorporating community annotation into molecular biology databases.

    Funded by: Howard Hughes Medical Institute; Wellcome Trust: 077044

    RNA (New York, N.Y.) 2008;14;12;2462-4

Francois Meullenet

- Research Administrator

Francois completed a PhD and a post-doctoral placement at the University of Nottingham, using molecular and biophysical methods to understand the molecular basis of myotonic dystrophy and to develop early stage-drug screening assays. He went on to work in grants administration and management at Breast Cancer Campaign, The Royal Society, and Arthritis Research UK.

Research

Francois joined the Sanger Institute in 2014, providing high-level administrative and strategic support to the Malaria Programme. As the Research Administrator, he is the interface between the Malaria Programme and Institute’s Management Operations, supporting many aspects of the Programme, including recruitment, partnerships, and scientific and financial monitoring.

Dawn Muddyman

- Senior Project Manager

I'm a Prince2 qualified senior project manager with a background in epidemiology and public health (PhD & MPhil, University of Cambridge). Before joining the Malaria Programme in 2013 I managed the Wellcome Trust UK10K Project, which sequenced the genomes of 10,000 people to determine the role of rare genetic variants in health and disease. I've also managed projects examining the sustainability of informatics resources in the research community, and have interned at the Department for HIV/AIDS at the World Health Organization (WHO) in Geneva.

Research

I'm responsible for managing flagship projects on parasite genome variation within the Sanger Institute Malaria Programme, including the Pf3k project and the Recon project; developing genetic reconnaissance technologies to support malaria elimination in the Mekong region.

References

  • Managing clinically significant findings in research: the UK10K example.

    Kaye J, Hurles M, Griffin H, Grewal J, Bobrow M, Timpson N, Smee C, Bolton P, Durbin R, Dyke S, Fitzpatrick D, Kennedy K, Kent A, Muddyman D, Muntoni F, Raymond LF, Semple R, Spector T and UK 10K

    Nuffield Department of Population Health, HeLEX - Centre for Health, Law and Emerging Technologies, University of Oxford, Oxford, UK.

    Recent advances in sequencing technology allow data on the human genome to be generated more quickly and in greater detail than ever before. Such detail includes findings that may be of significance to the health of the research participant involved. Although research studies generally do not feed back information on clinically significant findings (CSFs) to participants, this stance is increasingly being questioned. There may be difficulties and risks in feeding clinically significant information back to research participants, however, the UK10K consortium sought to address these by creating a detailed management pathway. This was not intended to create any obligation upon the researchers to feed back any CSFs they discovered. Instead, it provides a mechanism to ensure that any such findings can be passed on to the participant where appropriate. This paper describes this mechanism and the specific criteria, which must be fulfilled in order for a finding and participant to qualify for feedback. This mechanism could be used by future research consortia, and may also assist in the development of sound principles for dealing with CSFs.

    Funded by: Department of Health: NF-SI-0510-10268; Medical Research Council: MC_UU_12012/5/B, MC_UU_12013/3; Wellcome Trust: 091310, 092731, 096599, 098498, 100140, 102215, WT091310, WT096599/2/11/Z

    European journal of human genetics : EJHG 2014;22;9;1100-4

  • Implementing a successful data-management framework: the UK10K managed access model.

    Muddyman D, Smee C, Griffin H and Kaye J

    The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK.

    This paper outlines the history behind open access principles and describes the development of a managed access data-sharing process for the UK10K Project, currently Britain's largest genomic sequencing consortium (2010 to 2013). Funded by the Wellcome Trust, the purpose of UK10K was two-fold: to investigate how low-frequency and rare genetic variants contribute to human disease, and to provide an enduring data resource for future research into human genetics. In this paper, we discuss the challenge of reconciling data-sharing principles with the practicalities of delivering a sequencing project of UK10K's scope and magnitude. We describe the development of a sustainable, easy-to-use managed access system that allowed rapid access to UK10K data, while protecting the interests of participants and data generators alike. Specifically, we focus in depth on the three key issues that emerge in the data pipeline: study recruitment, data release and data access.

    Funded by: Wellcome Trust: 092731

    Genome medicine 2013;5;11;100

Samuel Oyola

so1@sanger.ac.uk Staff Scientist

Samuel holds a PhD in Molecular and Cellular Biology from the University of Cambridge. Before joining the Kwiatkowski group, Samuel did his Postdoctoral research on Leishmania functional genomics and the host-parasite interactions in the lab of Professor Debbie Smith at the University of York.

Research

Samuel supports the MalariaGEN P. falciparum Community Project and is leading the malaria sequencing research and development team, which is focused on developing new tools that facilitate high-throughput sequencing of malaria clinical samples and genome analysis. Samuel is keen on translating high-throughput sequencing technology into basic healthcare applications.

References

  • Optimized whole-genome amplification strategy for extremely AT-biased template.

    Oyola SO, Manske M, Campino S, Claessens A, Hamilton WL, Kekre M, Drury E, Mead D, Gu Y, Miles A, MacInnis B, Newbold C, Berriman M and Kwiatkowski DP

    Wellcome Trust Sanger Institute, Hinxton, UK so1@sanger.ac.uk.

    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 amplification (WGA) of low-yield samples from P. falciparum prior to short-read sequencing. We have developed WGA conditions that incorporate tetramethylammonium chloride for improved amplification and coverage of AT-rich regions of the genome. We show that this method reduces amplification bias and chimera formation. Our data show that this method is suitable for as low as 10 pg input DNA, and offers the possibility of sequencing the parasite genome from small blood samples.

    Funded by: Medical Research Council: G0600718; Wellcome Trust: 090532, 090532/Z/09/Z, 090770, 090770/Z/09/Z, 098051

    DNA research : an international journal for rapid publication of reports on genes and genomes 2014;21;6;661-71

  • A genome wide association study of Plasmodium falciparum susceptibility to 22 antimalarial drugs in Kenya.

    Wendler JP, Okombo J, Amato R, Miotto O, Kiara SM, Mwai L, Pole L, O'Brien J, Manske M, Alcock D, Drury E, Sanders M, Oyola SO, Malangone C, Jyothi D, Miles A, Rockett KA, MacInnis BL, Marsh K, Bejon P, Nzila A and Kwiatkowski DP

    Medical Research Council (MRC) Centre for Genomics and Global Health, University of Oxford, Oxford, United Kingdom; Wellcome Trust Sanger Institute, Hinxton, Cambridge, United Kingdom; Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdom.

    Background: 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.

    Methods and principal findings: Parasites isolated from children with acute febrile malaria were adapted to culture, and sensitivity was determined by in vitro growth in the presence of anti-malarial drugs. Parasites were genotyped using whole genome sequencing techniques. Associations between 6250 single nucleotide polymorphisms (SNPs) and resistance to individual anti-malarial agents were determined, with false discovery rate adjustment for multiple hypothesis testing. We identified expected associations in the pfcrt region with chloroquine (CQ) activity, and other novel loci associated with amodiaquine, quinazoline, and quinine activities. Signals for CQ and primaquine (PQ) overlap in and around pfcrt, and interestingly the phenotypes are inversely related for these two drugs. We catalog the variation in dhfr, dhps, mdr1, nhe, and crt, including novel SNPs, and confirm the presence of a dhfr-164L quadruple mutant in coastal Kenya. Mutations implicated in sulfadoxine-pyrimethamine resistance are at or near fixation in this sample set.

    Conclusions/significance: Sequence-based GWA studies are powerful tools for phenotypic association tests. Using this approach on falciparum parasites from coastal Kenya we identified known and previously unreported genes associated with phenotypic resistance to anti-malarial drugs, and observe in high-resolution haplotype visualizations a possible signature of an inverse selective relationship between CQ and PQ.

    Funded by: Medical Research Council: G0600718, G1002624; Wellcome Trust: 090532, 090532/Z/09/Z, 090770, 090770/Z/09/Z, 092654, 098051

    PloS one 2014;9;5;e96486

  • Multiple populations of artemisinin-resistant Plasmodium falciparum in Cambodia.

    Miotto O, Almagro-Garcia J, Manske M, Macinnis B, Campino S, Rockett KA, Amaratunga C, Lim P, Suon S, Sreng S, Anderson JM, Duong S, Nguon C, Chuor CM, Saunders D, Se Y, Lon C, Fukuda MM, Amenga-Etego L, Hodgson AV, Asoala V, Imwong M, Takala-Harrison S, Nosten F, Su XZ, Ringwald P, Ariey F, Dolecek C, Hien TT, Boni MF, Thai CQ, Amambua-Ngwa A, Conway DJ, Djimdé AA, Doumbo OK, Zongo I, Ouedraogo JB, Alcock D, Drury E, Auburn S, Koch O, Sanders M, Hubbart C, Maslen G, Ruano-Rubio V, Jyothi D, Miles A, O'Brien J, Gamble C, Oyola SO, Rayner JC, Newbold CI, Berriman M, Spencer CC, McVean G, Day NP, White NJ, Bethell D, Dondorp AM, Plowe CV, Fairhurst RM and Kwiatkowski DP

    Medical Research Council MRC Centre for Genomics and Global Health, University of Oxford, Oxford, UK.

    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 subpopulations with extremely high levels of genetic differentiation. Of particular interest are three subpopulations, all associated with clinical resistance to artemisinin, which have skewed allele frequency spectra and high levels of haplotype homozygosity, indicative of founder effects and recent population expansion. We provide a catalog of SNPs that show high levels of differentiation in the artemisinin-resistant subpopulations, including codon variants in transporter proteins and DNA mismatch repair proteins. These data provide a population-level genetic framework for investigating the biological origins of artemisinin resistance and for defining molecular markers to assist in its elimination.

    Funded by: Howard Hughes Medical Institute: 55005502; Medical Research Council: 1252410, G0600718, G19/9, MC_U190081987; Wellcome Trust: 082370, 089275, 089276, 090532, 090532/Z/09/Z, 090770, 090770/Z/09/Z, 093956, 098051, G0600718

    Nature genetics 2013;45;6;648-55

  • Efficient depletion of host DNA contamination in malaria clinical sequencing.

    Oyola SO, Gu Y, Manske M, Otto TD, O'Brien J, Alcock D, Macinnis B, Berriman M, Newbold CI, Kwiatkowski DP, Swerdlow HP and Quail MA

    Wellcome Trust Sanger Institute, Hinxton, Cambridge, United Kingdom. Samuel.oyola@sanger.ac.uk

    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 microbial pathogens. Success of these applications is hugely dependent on efficient generation of clean genetic material that is free from host DNA contamination for rapid preparation of sequencing libraries. The presence of large amounts of host DNA severely affects the efficiency of characterizing pathogens using WGS and is therefore a serious impediment to clinical and epidemiological sequencing for health care and public health applications. We have developed a simple enzymatic treatment method that takes advantage of the methylation of human DNA to selectively deplete host contamination from clinical samples prior to sequencing. Using malaria clinical samples with over 80% human host DNA contamination, we show that the enzymatic treatment enriches Plasmodium falciparum DNA up to ∼9-fold and generates high-quality, nonbiased sequence reads covering >98% of 86,158 catalogued typeable single-nucleotide polymorphism loci.

    Funded by: Medical Research Council: G19/9; Wellcome Trust: 079355/Z/06/Z, 090532

    Journal of clinical microbiology 2013;51;3;745-51

  • A method for selectively enriching microbial DNA from contaminating vertebrate host DNA.

    Feehery GR, Yigit E, Oyola SO, Langhorst BW, Schmidt VT, Stewart FJ, Dimalanta ET, Amaral-Zettler LA, Davis T, Quail MA and Pradhan S

    New England Biolabs Inc., Ipswich, Massachusetts, United States of America.

    DNA samples derived from vertebrate skin, bodily cavities and body fluids contain both host and microbial DNA; the latter often present as a minor component. Consequently, DNA sequencing of a microbiome sample frequently yields reads originating from the microbe(s) of interest, but with a vast excess of host genome-derived reads. In this study, we used a methyl-CpG binding domain (MBD) to separate methylated host DNA from microbial DNA based on differences in CpG methylation density. MBD fused to the Fc region of a human antibody (MBD-Fc) binds strongly to protein A paramagnetic beads, forming an effective one-step enrichment complex that was used to remove human or fish host DNA from bacterial and protistan DNA for subsequent sequencing and analysis. We report enrichment of DNA samples from human saliva, human blood, a mock malaria-infected blood sample and a black molly fish. When reads were mapped to reference genomes, sequence reads aligning to host genomes decreased 50-fold, while bacterial and Plasmodium DNA sequences reads increased 8-11.5-fold. The Shannon-Wiener diversity index was calculated for 149 bacterial species in saliva before and after enrichment. Unenriched saliva had an index of 4.72, while the enriched sample had an index of 4.80. The similarity of these indices demonstrates that bacterial species diversity and relative phylotype abundance remain conserved in enriched samples. Enrichment using the MBD-Fc method holds promise for targeted microbiome sequence analysis across a broad range of sample types.

    Funded by: Wellcome Trust: 079355/Z/06/Z, 098051

    PloS one 2013;8;10;e76096

  • Analysis of Plasmodium falciparum diversity in natural infections by deep sequencing.

    Manske M, Miotto O, Campino S, Auburn S, Almagro-Garcia J, Maslen G, O'Brien J, Djimde A, Doumbo O, Zongo I, Ouedraogo JB, Michon P, Mueller I, Siba P, Nzila A, Borrmann S, Kiara SM, Marsh K, Jiang H, Su XZ, Amaratunga C, Fairhurst R, Socheat D, Nosten F, Imwong M, White NJ, Sanders M, Anastasi E, Alcock D, Drury E, Oyola S, Quail MA, Turner DJ, Ruano-Rubio V, Jyothi D, Amenga-Etego L, Hubbart C, Jeffreys A, Rowlands K, Sutherland C, Roper C, Mangano V, Modiano D, Tan JC, Ferdig MT, Amambua-Ngwa A, Conway DJ, Takala-Harrison S, Plowe CV, Rayner JC, Rockett KA, Clark TG, Newbold CI, Berriman M, MacInnis B and Kwiatkowski DP

    Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, UK.

    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 malaria, either directly or after short-term culture. Analysis of 86,158 exonic single nucleotide polymorphisms that passed genotyping quality control in 227 samples from Africa, Asia and Oceania provides genome-wide estimates of allele frequency distribution, population structure and linkage disequilibrium. By comparing the genetic diversity of individual infections with that of the local parasite population, we derive a metric of within-host diversity that is related to the level of inbreeding in the population. An open-access web application has been established for the exploration of regional differences in allele frequency and of highly differentiated loci in the P. falciparum genome.

    Funded by: Howard Hughes Medical Institute: 55005502; Medical Research Council: G0600718, G19/9; Wellcome Trust: 075491/Z/04, 077012/Z/05/Z, 082370, 089275, 090532, 090532/Z/09/Z, 090770, 090770/Z/09/Z, 092654, 093956, 098051

    Nature 2012;487;7407;375-9

  • Optimal enzymes for amplifying sequencing libraries.

    Quail MA, Otto TD, Gu Y, Harris SR, Skelly TF, McQuillan JA, Swerdlow HP and Oyola SO

    Nature methods 2012;9;1;10-1

  • Functional analysis of Leishmania cyclopropane fatty acid synthetase.

    Oyola SO, Evans KJ, Smith TK, Smith BA, Hilley JD, Mottram JC, Kaye PM and Smith DF

    Centre for Immunology and Infection, Department of Biology/Hull York Medical School, University of York, York, United Kingdom.

    The single gene encoding cyclopropane fatty acid synthetase (CFAS) is present in Leishmania infantum, L. mexicana and L. braziliensis but absent from L. major, a causative agent of cutaneous leishmaniasis. In L. infantum, usually causative agent of visceral leishmaniasis, the CFAS gene is transcribed in both insect (extracellular) and host (intracellular) stages of the parasite life cycle. Tagged CFAS protein is stably detected in intracellular L. infantum but only during the early log phase of extracellular growth, when it shows partial localisation to the endoplasmic reticulum. Lipid analyses of L. infantum wild type, CFAS null and complemented parasites detect a low abundance CFAS-dependent C19Δ fatty acid, characteristic of a cyclopropanated species, in wild type and add-back cells. Sub-cellular fractionation studies locate the C19Δ fatty acid to both ER and plasma membrane-enriched fractions. This fatty acid is not detectable in wild type L. major, although expression of the L. infantum CFAS gene in L. major generates cyclopropanated fatty acids, indicating that the substrate for this modification is present in L. major, despite the absence of the modifying enzyme. Loss of the L. infantum CFAS gene does not affect extracellular parasite growth, phagocytosis or early survival in macrophages. However, while endocytosis is also unaffected in the extracellular CFAS nulls, membrane transporter activity is defective and the null parasites are more resistant to oxidative stress. Following infection in vivo, L. infantum CFAS nulls exhibit lower parasite burdens in both the liver and spleen of susceptible hosts but it has not been possible to complement this phenotype, suggesting that loss of C19Δ fatty acid may lead to irreversible changes in cell physiology that cannot be rescued by re-expression. Aberrant cyclopropanation in L. major decreases parasite virulence but does not influence parasite tissue tropism.

    Funded by: Medical Research Council: G1000230; Wellcome Trust: 067441, 076355, 085349, 086658

    PloS one 2012;7;12;e51300

  • Optimizing Illumina next-generation sequencing library preparation for extremely AT-biased genomes.

    Oyola SO, Otto TD, Gu Y, Maslen G, Manske M, Campino S, Turner DJ, Macinnis B, Kwiatkowski DP, Swerdlow HP and Quail MA

    Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK. so1@sanger.ac.uk

    Background: 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 currently available NGS platforms. The genomes of some important pathogenic organisms like Plasmodium falciparum (high AT content) and Mycobacterium tuberculosis (high GC content) display extremes of base composition. The standard library preparation procedures that employ PCR amplification have been shown to cause uneven read coverage particularly across AT and GC rich regions, leading to problems in genome assembly and variation analyses. Alternative library-preparation approaches that omit PCR amplification require large quantities of starting material and hence are not suitable for small amounts of DNA/RNA such as those from clinical isolates. We have developed and optimized library-preparation procedures suitable for low quantity starting material and tolerant to extremely high AT content sequences.

    Results: We have used our optimized conditions in parallel with standard methods to prepare Illumina sequencing libraries from a non-clinical and a clinical isolate (containing ~53% host contamination). By analyzing and comparing the quality of sequence data generated, we show that our optimized conditions that involve a PCR additive (TMAC), produces amplified libraries with improved coverage of extremely AT-rich regions and reduced bias toward GC neutral templates.

    Conclusion: We have developed a robust and optimized Next-Generation Sequencing library amplification method suitable for extremely AT-rich genomes. The new amplification conditions significantly reduce bias and retain the complexity of either extremes of base composition. This development will greatly benefit sequencing clinical samples that often require amplification due to low mass of DNA starting material.

    Funded by: Medical Research Council: G0600718, G19/9; Wellcome Trust: 079355/Z/06/Z, 090532

    BMC genomics 2012;13;1

  • Comparative genomic analysis of three Leishmania species that cause diverse human disease.

    Peacock CS, Seeger K, Harris D, Murphy L, Ruiz JC, Quail MA, Peters N, Adlem E, Tivey A, Aslett M, Kerhornou A, Ivens A, Fraser A, Rajandream MA, Carver T, Norbertczak H, Chillingworth T, Hance Z, Jagels K, Moule S, Ormond D, Rutter S, Squares R, Whitehead S, Rabbinowitsch E, Arrowsmith C, White B, Thurston S, Bringaud F, Baldauf SL, Faulconbridge A, Jeffares D, Depledge DP, Oyola SO, Hilley JD, Brito LO, Tosi LR, Barrell B, Cruz AK, Mottram JC, Smith DF and Berriman M

    Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridgeshire CB10 1SA, UK. csp@sanger.ac.uk

    Leishmania parasites cause a broad spectrum of clinical disease. Here we report the sequencing of the genomes of two species of Leishmania: Leishmania infantum and Leishmania braziliensis. The comparison of these sequences with the published genome of Leishmania major reveals marked conservation of synteny and identifies only approximately 200 genes with a differential distribution between the three species. L. braziliensis, contrary to Leishmania species examined so far, possesses components of a putative RNA-mediated interference pathway, telomere-associated transposable elements and spliced leader-associated SLACS retrotransposons. We show that pseudogene formation and gene loss are the principal forces shaping the different genomes. Genes that are differentially distributed between the species encode proteins implicated in host-pathogen interactions and parasite survival in the macrophage.

    Funded by: Medical Research Council: G0000508; Wellcome Trust: 076355, 085775

    Nature genetics 2007;39;7;839-47

James Stalker

- Analysis Pipeline Manager

Jim graduated from the University of Cambridge with a degree in Natural Sciences (Genetics) in 1995. After a couple of years post-graduate research on lepidoptera he moved into industry as a computer programmer. In 2000, Jim joined the Sanger Centre as a web developer, and has since held various roles at the Institute including building the original Ensembl web team, running an ad-hoc bioinformatics support team, and informatics team leader on the 1000Genomes and UK10K projects.

Research

Jim joined the Kwiatkowski malaria group in 2012 as Analysis Pipelines Manager. He's in charge of the team responsible for tracking and analysing the sequence data generated by the group, and releasing processed data back to the group and the wider community.

Arthur Talman

- Postdoctoral Fellow

Throughout my studies I have been interested in host-pathogen interactions and mechanisms of malaria transmission. In particular, I participated in field studies of apicomplexan parasite transmission in rural Africa and Southeast Asia. For my PhD at Imperial College London, I investigated malaria sexual reproduction and transmission to Anopheline mosquitoes from a molecular perspective. I recently completed my post-doctoral training by conducting research into dissemination of bacterial and viral pathogens at the Yale School of Medicine’s Microbial Pathogenesis department. I joined Mara Lawniczak group in February 2015.

Research

My current research focuses on understanding the strategies and patterns observed during malaria parasite sexual reproduction, which occurs during their transmission from human host to mosquito vector. Specifically male and female parasites are picked up during mosquito feeding and mate in the blood meal. The aim of my research is to understand how distinct parasite populations interact during this process.

References

  • An essential role of the basal body protein SAS-6 in Plasmodium male gamete development and malaria transmission.

    Marques SR, Ramakrishnan C, Carzaniga R, Blagborough AM, Delves MJ, Talman AM and Sinden RE

    Department of Life Sciences, Imperial College of London, London, SW7 2AZ, UK.

    Gametocytes are the sole Plasmodium parasite stages that infect mosquitoes; therefore development of functional gametes is required for malaria transmission. Flagellum assembly of the Plasmodium male gamete differs from that of most other eukaryotes in that it is intracytoplasmic but retains a key conserved feature: axonemes assemble from basal bodies. The centriole/basal body protein SAS-6 normally regulates assembly and duplication of these organelles and its depletion causes severe flagellar/ciliary abnormalities in a diverse array of eukaryotes. Since basal body and flagellum assembly are intimately coupled to male gamete development in Plasmodium, we hypothesized that SAS-6 disruption may cause gametogenesis defects and perturb transmission. We show that Plasmodium berghei sas6 knockouts display severely abnormal male gametogenesis presenting reduced basal body numbers, axonemal assembly defects and abnormal nuclear allocation. The defects in gametogenesis reduce fertilization and render Pbsas6 knockouts less infectious to mosquitoes. Additionally, we show that lack of Pbsas6 blocks transmission from mosquito to vertebrate host, revealing an additional yet undefined role in ookinete to sporulating oocysts transition. These findings underscore the vulnerability of the basal body/SAS-6 to malaria transmission blocking interventions.

    Funded by: Wellcome Trust

    Cellular microbiology 2015;17;2;191-206

  • Actin network disassembly powers dissemination of Listeria monocytogenes.

    Talman AM, Chong R, Chia J, Svitkina T and Agaisse H

    Department of Microbial Pathogenesis, Boyer Center for Molecular Medicine, Yale School of Medicine, New Haven, CT 06536, USA.

    Several bacterial pathogens hijack the actin assembly machinery and display intracellular motility in the cytosol of infected cells. At the cell cortex, intracellular motility leads to bacterial dissemination through formation of plasma membrane protrusions that resolve into vacuoles in adjacent cells. Here, we uncover a crucial role for actin network disassembly in dissemination of Listeria monocytogenes. We found that defects in the disassembly machinery decreased the rate of actin tail turnover but did not affect the velocity of the bacteria in the cytosol. By contrast, defects in the disassembly machinery had a dramatic impact on bacterial dissemination. Our results suggest a model of L. monocytogenes dissemination in which the disassembly machinery, through local recycling of the actin network in protrusions, fuels continuous actin assembly at the bacterial pole and concurrently exhausts cytoskeleton components from the network distal to the bacterium, which enables membrane apposition and resolution of protrusions into vacuoles.

    Funded by: NIAID NIH HHS: R01 AI073904, R01-AI073904, R21-AI094228; NIGMS NIH HHS: R01 GM 095977, R01 GM095977

    Journal of cell science 2014;127;Pt 1;240-9

  • Proteomic analysis of the Plasmodium male gamete reveals the key role for glycolysis in flagellar motility.

    Talman AM, Prieto JH, Marques S, Ubaida-Mohien C, Lawniczak M, Wass MN, Xu T, Frank R, Ecker A, Stanway RS, Krishna S, Sternberg MJ, Christophides GK, Graham DR, Dinglasan RR, Yates JR and Sinden RE

    Division of Cell and Molecular Biology, Imperial College, London, UK. arthur.talman@yale.edu.

    Background: Gametogenesis and fertilization play crucial roles in malaria transmission. While male gametes are thought to be amongst the simplest eukaryotic cells and are proven targets of transmission blocking immunity, little is known about their molecular organization. For example, the pathway of energy metabolism that power motility, a feature that facilitates gamete encounter and fertilization, is unknown.

    Methods: Plasmodium berghei microgametes were purified and analysed by whole-cell proteomic analysis for the first time. Data are available via ProteomeXchange with identifier PXD001163.

    Results: 615 proteins were recovered, they included all male gamete proteins described thus far. Amongst them were the 11 enzymes of the glycolytic pathway. The hexose transporter was localized to the gamete plasma membrane and it was shown that microgamete motility can be suppressed effectively by inhibitors of this transporter and of the glycolytic pathway.

    Conclusions: This study describes the first whole-cell proteomic analysis of the malaria male gamete. It identifies glycolysis as the likely exclusive source of energy for flagellar beat, and provides new insights in original features of Plasmodium flagellar organization.

    Funded by: Biotechnology and Biological Sciences Research Council: BB/F020481/1; Medical Research Council: G1100339; NHLBI NIH HHS: N01-HV-00240; NIAID NIH HHS: 5R21 AI72615-2; NIGMS NIH HHS: P 41 GM103533; PHS HHS: HHSN268201000032C; Wellcome Trust

    Malaria journal 2014;13;315

  • PbGEST mediates malaria transmission to both mosquito and vertebrate host.

    Talman AM, Lacroix C, Marques SR, Blagborough AM, Carzaniga R, Ménard R and Sinden RE

    Department of Life Sciences, Imperial College London, SW72AZ London, UK. amtalman@gmail.com

    The malaria life cycle relies on the successful transfer of the parasite between its human and mosquito hosts. We identified a Plasmodium berghei secreted protein (PBANKA_131270) that plays distinct roles in both the mammal-to-mosquito and the mosquito-to-mammal transitions. This protein, here named gamete egress and sporozoite traversal (GEST), plays an important role in the egress of male and female gametes from the vertebrate red blood cell. Interestingly, GEST is also required following the bite of the infected mosquito, for sporozoite progression through the skin. We found PbGEST to be secreted shortly after activation of the intraerythrocytic gametocyte, and during sporozoite migration. These findings indicate that a single malaria protein may have pleiotropic roles in different parasites stages mediating transmission between its insect and mammalian hosts.

    Funded by: Biotechnology and Biological Sciences Research Council; Wellcome Trust

    Molecular microbiology 2011;82;2;462-74

  • The flagellum in malarial parasites.

    Sinden RE, Talman A, Marques SR, Wass MN and Sternberg MJ

    The Malaria Centre, The Department of Life Sciences, Imperial College London, London SW7 2AZ, United Kingdom. r.sinden@imperial.ac.uk

    The malarial parasites assemble flagella exclusively during the formation of the male gamete in the midgut of the female mosquito vector. The observation of gamete formation ex vivo reported by Laveran (Laveran MA: De la nature parasitaire des accidents de l'impaludisme. Comptes Rendues De La Societe de Biologie. Paris 1881, 93:627-630) was seminal to the discovery of the parasite itself. Following ingestion of malaria-infected blood by the mosquito, microgamete formation from the terminally arrested gametocytes is exceptionally rapid, completing three mitotic divisions in just a few minutes, and is precisely regulated. This review attempts to draw together the diverse original observations with subsequent electron microscopic studies, and recent work on the signalling pathways regulating sexual development, together with transcriptomic and proteomic studies that are paving the way to new understandings of the molecular mechanisms involved and the potential they offer for effective interventions to block the transmission of the parasites in natural communities.

    Current opinion in microbiology 2010;13;4;491-500

  • The Armadillo repeat protein PF16 is essential for flagellar structure and function in Plasmodium male gametes.

    Straschil U, Talman AM, Ferguson DJ, Bunting KA, Xu Z, Bailes E, Sinden RE, Holder AA, Smith EF, Coates JC and Rita Tewari

    Institute of Genetics, School of Biology, University of Nottingham, Nottingham, United Kingdom.

    Malaria, caused by the apicomplexan parasite Plasmodium, threatens 40% of the world's population. Transmission between vertebrate and insect hosts depends on the sexual stages of the life-cycle. The male gamete of Plasmodium parasite is the only developmental stage that possesses a flagellum. Very little is known about the identity or function of proteins in the parasite's flagellar biology. Here, we characterise a Plasmodium PF16 homologue using reverse genetics in the mouse malaria parasite Plasmodium berghei. PF16 is a conserved Armadillo-repeat protein that regulates flagellar structure and motility in organisms as diverse as green algae and mice. We show that P. berghei PF16 is expressed in the male gamete flagellum, where it plays a crucial role maintaining the correct microtubule structure in the central apparatus of the axoneme as studied by electron microscopy. Disruption of the PF16 gene results in abnormal flagellar movement and reduced fertility, but does not lead to complete sterility, unlike pf16 mutations in other organisms. Using homology modelling, bioinformatics analysis and complementation studies in Chlamydomonas, we show that some regions of the PF16 protein are highly conserved across all eukaryotes, whereas other regions may have species-specific functions. PF16 is the first ARM-repeat protein characterised in the malaria parasite genus Plasmodium and this study opens up a novel model for analysis of Plasmodium flagellar biology that may provide unique insights into an ancient organelle and suggest novel intervention strategies to control the malaria parasite.

    Funded by: Medical Research Council: G0900109, MC_U117532067, U117532067; Wellcome Trust

    PloS one 2010;5;9;e12901

  • A Plasmodium falciparum strain expressing GFP throughout the parasite's life-cycle.

    Talman AM, Blagborough AM and Sinden RE

    Division of Cell and Molecular Biology, Imperial College London, London, United Kingdom.

    The human malaria parasite Plasmodium falciparum is responsible for the majority of malaria-related deaths. Tools allowing the study of the basic biology of P. falciparum throughout the life cycle are critical to the development of new strategies to target the parasite within both human and mosquito hosts. We here present 3D7HT-GFP, a strain of P. falciparum constitutively expressing the Green Fluorescent Protein (GFP) throughout the life cycle, which has retained its capacity to complete sporogonic development. The GFP expressing cassette was inserted in the Pf47 locus. Using this transgenic strain, parasite tracking and population dynamics studies in mosquito stages and exo-erythrocytic schizogony is greatly facilitated. The development of 3D7HT-GFP will permit a deeper understanding of the biology of parasite-host vector interactions, and facilitate the development of high-throughput malaria transmission assays and thus aid development of new intervention strategies against both parasite and mosquito.

    PloS one 2010;5;2;e9156

  • Heritability of the human infectious reservoir of malaria parasites.

    Lawaly YR, Sakuntabhai A, Marrama L, Konate L, Phimpraphi W, Sokhna C, Tall A, Sarr FD, Peerapittayamongkol C, Louicharoen C, Schneider BS, Levescot A, Talman A, Casademont I, Menard D, Trape JF, Rogier C, Kaewkunwal J, Sura T, Nuchprayoon I, Ariey F, Baril L, Singhasivanon P, Mercereau-Puijalon O and Paul R

    Institut Pasteur de Dakar, Laboratoire d'Entomologie Médicale, Dakar, Senegal.

    Background: Studies on human genetic factors associated with malaria have hitherto concentrated on their role in susceptibility to and protection from disease. In contrast, virtually no attention has been paid to the role of human genetics in eliciting the production of parasite transmission stages, the gametocytes, and thus enhancing the spread of disease.

    Methods and findings: We analysed four longitudinal family-based cohort studies from Senegal and Thailand followed for 2-8 years and evaluated the relative impact of the human genetic and non-genetic factors on gametocyte production in infections of Plasmodium falciparum or P. vivax. Prevalence and density of gametocyte carriage were evaluated in asymptomatic and symptomatic infections by examination of Giemsa-stained blood smears and/or RT-PCR (for falciparum in one site). A significant human genetic contribution was found to be associated with gametocyte prevalence in asymptomatic P. falciparum infections. By contrast, there was no heritability associated with the production of gametocytes for P. falciparum or P. vivax symptomatic infections. Sickle cell mutation, HbS, was associated with increased gametocyte prevalence but its contribution was small.

    Conclusions: The existence of a significant human genetic contribution to gametocyte prevalence in asymptomatic infections suggests that candidate gene and genome wide association approaches may be usefully applied to explore the underlying human genetics. Prospective epidemiological studies will provide an opportunity to generate novel and perhaps more epidemiologically pertinent gametocyte data with which similar analyses can be performed and the role of human genetics in parasite transmission ascertained.

    PloS one 2010;5;6;e11358

  • Attraction between sexes: male-female gametocyte behaviour within a Leucocytozoon toddi (Haemosporida).

    Barraclough RK, Duval L, Talman AM, Ariey F and Robert V

    Groupe de Recherche sur le Paludisme, Institut Pasteur de Madagascar, BP 1274, Antananarivo 101, Madagascar. r.k.barraclough@massey.ac.nz

    Understanding the breeding systems of Plasmodium, and the closely related Haemoproteus and Leucocytozoon (Apicomplexa: Haemosporida), is fundamental to virulence and transmission research. We report an unusual binding behaviour between gametocytes of Leucocytozoon toddi. This aggregative behaviour was notably characterised by a disparity in the likelihood of clustering by female and male gametocytes. Thus, indicating a possible difference in the 'stickiness' of gametocytes per sex. Overall, 12% of gametocytes in this high-parasitaemia infection (0.269 gametocytes per 100 red blood cells (RBCs)) were incorporated into aggregations involving substantial contact. The gametocyte sexual combinations within aggregations varied significantly from expected according to the background 0.49 sex ratio within this sample, with female-female contacts occurring more and male-male contacts occurring less frequently than expected. A second L. toddi (identical for 709 bp of the cyt b mitochondrial gene) with lower parasitemia (0.035 gametocytes per 100 RBCs) showed no significant binding. Interestingly, the ratios of male gametocytes in both of these parasites were greater than expected under sex-ratio theory and similar to the 50% observed in species with syzygy breeding strategies. We discuss the ramifications of this observation in terms of sex-ratio theory and breeding strategies and provide speculative explanations for this unusual gametocyte behaviour.

    Parasitology research 2008;102;6;1321-7

  • Influence of chemotherapy on the Plasmodium gametocyte sex ratio of mice and humans.

    Talman AM, Paul RE, Sokhna CS, Domarle O, Ariey F, Trape JF and Robert V

    Groupe de Recherche sur le Paludisme, Institut Pasteur de Madagascar, Antananarivo, Madagascar. atalman@pasteur.mg

    Plasmodium species, the etiologic agents of malaria, are obligatory sexual organisms. Gametocytes, the precursors of gametes, are responsible for parasite transmission from human to mosquito. The sex ratio of gametocytes has been shown to have consequences for the success of this shift from vertebrate host to insect vector. We attempted to document the effect of chemotherapy on the sex ratio of two different Plasmodium species: Plasmodium falciparum in children from endemic area with uncomplicated malaria treated with chloroquine (CQ) or sulfadoxine-pyrimethamine (SP), and P. vinckei petteri in mice treated with CQ or untreated. The studies involved 53 patients without gametocytes at day 0 (13 CQ and 40 SP) followed for 14 days, and 15 mice (10 CQ and 5 controls) followed for five days. During the course of infection, a positive correlation was observed between the time of the length of infection and the proportion of male gametocytes in both Plasmodium species. No effects of treatment (CQ versus SP for P. falciparum or CQ versus controls for P. vinckei petteri) on the gametocyte sex ratio were found for either Plasmodium species. This indicates that parasites do not respond to chemotherapy by altering their sex allocation strategy, even though, in the case of P. falciparum, they apparently increase their overall investment in sexual stages. This suggests that malaria parasite species respond to different environmental cues for their sex differentiation and sex determination.

    The American journal of tropical medicine and hygiene 2004;71;6;739-44

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