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

Roberto Amato
Research Associate in Statistical Genomics
Cristina Ariani
Postdoctoral Fellow
Sandra Bernaldo de Quiros Fernandez
sb40@sanger.ac.ukResearch Assistant
Susana Campino
Senior Postdoctoral Scientist
Kalia Dede
kd9@sanger.ac.ukProgramme Administrator
Eleanor Drury
Laboratory Manager
Rachel Giacomantonio
Communications Manager
Will Hamilton
unknown
Christopher Jacob
Staff Scientist
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

Roberto Amato

- Research Associate in Statistical Genomics

I got a PhD in Computational Biology and Bioinformatics from the University of Naples "Federico II" and I also hold a BSc and an MSc in Computer Science. Initially based at the Wellcome Trust Centre for Human Genetics in Oxford, I then joined the Wellcome Trust Sanger Institute’s Malaria Programme where I support several global collaborations including the MalariaGEN P. falciparum Community Project and Pf3k.

Research

I'm involved in the analysis of natural genetic variation in the Plasmodium parasites that cause malaria. My primary focus is on developing new statistical methods to understand the evolution of these parasites at a population level, in order to shed light on the underlying genetics of antimalarial drug resistance.

References

  • 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

    1] Wellcome Trust Sanger Institute, Hinxton, UK. [2] Medical Research Council (MRC) Centre for Genomics and Global Health, University of Oxford, Oxford, UK. [3] Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand.

    We report a large multicenter genome-wide association study of Plasmodium falciparum resistance to artemisinin, the frontline antimalarial drug. Across 15 locations in Southeast Asia, we identified at least 20 mutations in kelch13 (PF3D7_1343700) affecting the encoded propeller and BTB/POZ domains, which were associated with a slow parasite clearance rate after treatment with artemisinin derivatives. Nonsynonymous polymorphisms in fd (ferredoxin), arps10 (apicoplast ribosomal protein S10), mdr2 (multidrug resistance protein 2) and crt (chloroquine resistance transporter) also showed strong associations with artemisinin resistance. Analysis of the fine structure of the parasite population showed that the fd, arps10, mdr2 and crt polymorphisms are markers of a genetic background on which kelch13 mutations are particularly likely to arise and that they correlate with the contemporary geographical boundaries and population frequencies of artemisinin resistance. These findings indicate that the risk of new resistance-causing mutations emerging is determined by specific predisposing genetic factors in the underlying parasite population.

    Funded by: Howard Hughes Medical Institute; Intramural NIH HHS; Medical Research Council: G0600718; NIAID NIH HHS: R01 AI101713; Wellcome Trust: 090532/Z/09/Z, 090770, 090770/Z/09/Z, 098051

    Nature genetics 2015;47;3;226-34

  • Spread of artemisinin resistance in Plasmodium falciparum malaria.

    Ashley EA, Dhorda M, Fairhurst RM, Amaratunga C, Lim P, Suon S, Sreng S, Anderson JM, Mao S, Sam B, Sopha C, Chuor CM, Nguon C, Sovannaroth S, Pukrittayakamee S, Jittamala P, Chotivanich K, Chutasmit K, Suchatsoonthorn C, Runcharoen R, Hien TT, Thuy-Nhien NT, Thanh NV, Phu NH, Htut Y, Han KT, Aye KH, Mokuolu OA, Olaosebikan RR, Folaranmi OO, Mayxay M, Khanthavong M, Hongvanthong B, Newton PN, Onyamboko MA, Fanello CI, Tshefu AK, Mishra N, Valecha N, Phyo AP, Nosten F, Yi P, Tripura R, Borrmann S, Bashraheil M, Peshu J, Faiz MA, Ghose A, Hossain MA, Samad R, Rahman MR, Hasan MM, Islam A, Miotto O, Amato R, MacInnis B, Stalker J, Kwiatkowski DP, Bozdech Z, Jeeyapant A, Cheah PY, Sakulthaew T, Chalk J, Intharabut B, Silamut K, Lee SJ, Vihokhern B, Kunasol C, Imwong M, Tarning J, Taylor WJ, Yeung S, Woodrow CJ, Flegg JA, Das D, Smith J, Venkatesan M, Plowe CV, Stepniewska K, Guerin PJ, Dondorp AM, Day NP, White NJ and Tracking Resistance to Artemisinin Collaboration (TRAC)

    The authors' affiliations are listed in the Appendix.

    Background: Artemisinin resistance in Plasmodium falciparum has emerged in Southeast Asia and now poses a threat to the control and elimination of malaria. Mapping the geographic extent of resistance is essential for planning containment and elimination strategies.

    Methods: Between May 2011 and April 2013, we enrolled 1241 adults and children with acute, uncomplicated falciparum malaria in an open-label trial at 15 sites in 10 countries (7 in Asia and 3 in Africa). Patients received artesunate, administered orally at a daily dose of either 2 mg per kilogram of body weight per day or 4 mg per kilogram, for 3 days, followed by a standard 3-day course of artemisinin-based combination therapy. Parasite counts in peripheral-blood samples were measured every 6 hours, and the parasite clearance half-lives were determined.

    Results: The median parasite clearance half-lives ranged from 1.9 hours in the Democratic Republic of Congo to 7.0 hours at the Thailand-Cambodia border. Slowly clearing infections (parasite clearance half-life >5 hours), strongly associated with single point mutations in the "propeller" region of the P. falciparum kelch protein gene on chromosome 13 (kelch13), were detected throughout mainland Southeast Asia from southern Vietnam to central Myanmar. The incidence of pretreatment and post-treatment gametocytemia was higher among patients with slow parasite clearance, suggesting greater potential for transmission. In western Cambodia, where artemisinin-based combination therapies are failing, the 6-day course of antimalarial therapy was associated with a cure rate of 97.7% (95% confidence interval, 90.9 to 99.4) at 42 days.

    Conclusions: Artemisinin resistance to P. falciparum, which is now prevalent across mainland Southeast Asia, is associated with mutations in kelch13. Prolonged courses of artemisinin-based combination therapies are currently efficacious in areas where standard 3-day treatments are failing. (Funded by the U.K. Department of International Development and others; ClinicalTrials.gov number, NCT01350856.).

    Funded by: Intramural NIH HHS; Wellcome Trust: 077166, 090532, 090770, 093956

    The New England journal of medicine 2014;371;5;411-23

  • 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

  • A network-based approach to dissect the cilia/centrosome complex interactome.

    Amato R, Morleo M, Giaquinto L, di Bernardo D and Franco B

    Telethon Institute of Genetics and Medicine (TIGEM), Naples, Italy. franco@tigem.it.

    Background: Cilia are microtubule-based organelles protruding from almost all mammalian cells which, when dysfunctional, result in genetic disorders called "ciliopathies". High-throughput studies have revealed that cilia are composed of thousands of proteins. However, despite many efforts, much remains to be determined regarding the biological functions of this increasingly important complex organelle.

    Results: We have derived an online tool, from a systematic network-based approach to dissect the cilia/centrosome complex interactome (CCCI). The tool integrates all current available data into a model which provides an "interaction" perspective on ciliary function. We generated a network of interactions between human proteins organized into functionally relevant "communities", which can be defined as groups of genes that are both highly inter-connected and strongly co-expressed. We then combined sequence and co-expression data in order to identify the transcription factors responsible for regulating genes within their respective communities. Our analyses have discovered communities significantly specialized for delegating specific biological functions such as mRNA processing, protein translation, folding and degradation processes that had never been associated with ciliary proteins until now.

    Conclusions: CCCI will allow us to clarify the roles of previously unknown ciliary functions, elucidate the molecular mechanisms underlying ciliary-associated phenotypes, and apply our knowledge of the functional roles of relatively uncharacterized molecular entities to disease phenotypes and new clinical applications.

    BMC genomics 2014;15;658

  • Simulating gene-gene and gene-environment interactions in complex diseases: Gene-Environment iNteraction Simulator 2.

    Pinelli M, Scala G, Amato R, Cocozza S and Miele G

    Gruppo Interdipartimentale di Bioinformatica e Biologia Computazionale, Università di Napoli "Federico II" - Università di Salerno, Italy. michele.pinelli@unina.it

    Background: The analysis of complex diseases is an important problem in human genetics. Because multifactoriality is expected to play a pivotal role, many studies are currently focused on collecting information on the genetic and environmental factors that potentially influence these diseases. However, there is still a lack of efficient and thoroughly tested statistical models that can be used to identify implicated features and their interactions. Simulations using large biologically realistic data sets with known gene-gene and gene-environment interactions that influence the risk of a complex disease are a convenient and useful way to assess the performance of statistical methods.

    Results: The Gene-Environment iNteraction Simulator 2 (GENS2) simulates interactions among two genetic and one environmental factor and also allows for epistatic interactions. GENS2 is based on data with realistic patterns of linkage disequilibrium, and imposes no limitations either on the number of individuals to be simulated or on number of non-predisposing genetic/environmental factors to be considered. The GENS2 tool is able to simulate gene-environment and gene-gene interactions. To make the Simulator more intuitive, the input parameters are expressed as standard epidemiological quantities. GENS2 is written in Python language and takes advantage of operators and modules provided by the simuPOP simulation environment. It can be used through a graphical or a command-line interface and is freely available from http://sourceforge.net/projects/gensim. The software is released under the GNU General Public License version 3.0.

    Conclusions: Data produced by GENS2 can be used as a benchmark for evaluating statistical tools designed for the identification of gene-gene and gene-environment interactions.

    BMC bioinformatics 2012;13;132

  • Signs of selective pressure on genetic variants affecting human height.

    Amato R, Miele G, Monticelli A and Cocozza S

    Gruppo Interdipartimentale di Bioinformatica e Biologia Computazionale, Università di Napoli Federico II-Università di Salerno, Naples, Italy. roamato@na.infn.it

    Many decades of scientific investigation have proved the role of selective pressure in Homo Sapiens at least at the level of individual genes or loci. Nevertheless, there are examples of polygenic traits that are bound to be under selection, but studies devoted to apply population genetics methods to unveil such occurrence are still lacking. Stature provides a relevant example of well-studied polygenic trait for which is now available a genome-wide association study which has identified the genes involved in this trait, and which is known to be under selection. We studied the behavior of F(ST) in a simulated toy model to detect population differentiation on a generic polygenic phenotype under selection. The simulations showed that the set of alleles involved in the trait has a higher mean F(ST) value than those undergoing genetic drift only. In view of this we looked for an increase in the mean F(ST) value of the 180 variants associated to human height. For this set of alleles we found F(ST) to be significantly higher than the genomic background (p = 0.0356). On the basis of a statistical analysis we excluded that the increase was just due to the presence of outliers. We also proved as marginal the role played by local adaptation phenomena, even on different phenotypes in linkage disequilibrium with genetic variants involved in height. The increase of F(ST) for the set of alleles involved in a polygenic trait seems to provide an example of symmetry breaking phenomenon concerning the population differentiation. The splitting in the allele frequencies would be driven by the initial conditions in the population dynamics which are stochastically modified by events like drift, bottlenecks, etc, and other stochastic events like the born of new mutations.

    PloS one 2011;6;11;e27588

  • A novel approach to simulate gene-environment interactions in complex diseases.

    Amato R, Pinelli M, D'Andrea D, Miele G, Nicodemi M, Raiconi G and Cocozza S

    Gruppo Interdipartimentale di Bioinformatica e Biologia Computazionale, Università di Napoli "Federico II"-Università di Salerno, Italy. roamato@na.infn.it

    Background: Complex diseases are multifactorial traits caused by both genetic and environmental factors. They represent the major part of human diseases and include those with largest prevalence and mortality (cancer, heart disease, obesity, etc.). Despite a large amount of information that has been collected about both genetic and environmental risk factors, there are few examples of studies on their interactions in epidemiological literature. One reason can be the incomplete knowledge of the power of statistical methods designed to search for risk factors and their interactions in these data sets. An improvement in this direction would lead to a better understanding and description of gene-environment interactions. To this aim, a possible strategy is to challenge the different statistical methods against data sets where the underlying phenomenon is completely known and fully controllable, for example simulated ones.

    Results: We present a mathematical approach that models gene-environment interactions. By this method it is possible to generate simulated populations having gene-environment interactions of any form, involving any number of genetic and environmental factors and also allowing non-linear interactions as epistasis. In particular, we implemented a simple version of this model in a Gene-Environment iNteraction Simulator (GENS), a tool designed to simulate case-control data sets where a one gene-one environment interaction influences the disease risk. The main aim has been to allow the input of population characteristics by using standard epidemiological measures and to implement constraints to make the simulator behaviour biologically meaningful.

    Conclusions: By the multi-logistic model implemented in GENS it is possible to simulate case-control samples of complex disease where gene-environment interactions influence the disease risk. The user has full control of the main characteristics of the simulated population and a Monte Carlo process allows random variability. A knowledge-based approach reduces the complexity of the mathematical model by using reasonable biological constraints and makes the simulation more understandable in biological terms. Simulated data sets can be used for the assessment of novel statistical methods or for the evaluation of the statistical power when designing a study.

    BMC bioinformatics 2010;11;8

  • Schizophrenia and vitamin D related genes could have been subject to latitude-driven adaptation.

    Amato R, Pinelli M, Monticelli A, Miele G and Cocozza S

    Gruppo Interdipartimentale di Bioinformatica e Biologia Computazionale, Università di Napoli "Federico II"-Università di Salerno, Naples, Italy. roamato@na.infn.it

    Background: Many natural phenomena are directly or indirectly related to latitude. Living at different latitudes, indeed, has its consequences with being exposed to different climates, diets, light/dark cycles, etc. In humans, one of the best known examples of genetic traits following a latitudinal gradient is skin pigmentation. Nevertheless, also several diseases show latitudinal clinals such as hypertension, cancer, dismetabolic conditions, schizophrenia, Parkinson's disease and many more.

    Results: We investigated, for the first time on a wide genomic scale, the latitude-driven adaptation phenomena. In particular, we selected a set of genes showing signs of latitude-dependent population differentiation. The biological characterization of these genes showed enrichment for neural-related processes. In light of this, we investigated whether genes associated to neuropsychiatric diseases were enriched by Latitude-Related Genes (LRGs). We found a strong enrichment of LRGs in the set of genes associated to schizophrenia. In an attempt to try to explain this possible link between latitude and schizophrenia, we investigated their associations with vitamin D. We found in a set of vitamin D related genes a significant enrichment of both LRGs and of genes involved in schizophrenia.

    Conclusions: Our results suggest a latitude-driven adaptation for both schizophrenia and vitamin D related genes. In addition we confirm, at a molecular level, the link between schizophrenia and vitamin D. Finally, we discuss a model in which schizophrenia is, at least partly, a maladaptive by-product of latitude dependent adaptive changes in vitamin D metabolism.

    BMC evolutionary biology 2010;10;351

  • Genome-wide scan for signatures of human population differentiation and their relationship with natural selection, functional pathways and diseases.

    Amato R, Pinelli M, Monticelli A, Marino D, Miele G and Cocozza S

    Gruppo Interdipartimentale di Bioinformatica e Biologia Computazionale, Università di Napoli Federico II-Università di Salerno, Naples, Italy. roamato@na.infn.it

    Genetic differences both between individuals and populations are studied for their evolutionary relevance and for their potential medical applications. Most of the genetic differentiation among populations are caused by random drift that should affect all loci across the genome in a similar manner. When a locus shows extraordinary high or low levels of population differentiation, this may be interpreted as evidence for natural selection. The most used measure of population differentiation was devised by Wright and is known as fixation index, or F(ST). We performed a genome-wide estimation of F(ST) on about 4 millions of SNPs from HapMap project data. We demonstrated a heterogeneous distribution of F(ST) values between autosomes and heterochromosomes. When we compared the F(ST) values obtained in this study with another evolutionary measure obtained by comparative interspecific approach, we found that genes under positive selection appeared to show low levels of population differentiation. We applied a gene set approach, widely used for microarray data analysis, to detect functional pathways under selection. We found that one pathway related to antigen processing and presentation showed low levels of F(ST), while several pathways related to cell signalling, growth and morphogenesis showed high F(ST) values. Finally, we detected a signature of selection within genes associated with human complex diseases. These results can help to identify which process occurred during human evolution and adaptation to different environments. They also support the hypothesis that common diseases could have a genetic background shaped by human evolution.

    PloS one 2009;4;11;e7927

  • A multi-step approach to time series analysis and gene expression clustering.

    Amato R, Ciaramella A, Deniskina N, Del Mondo C, di Bernardo D, Donalek C, Longo G, Mangano G, Miele G, Raiconi G, Staiano A and Tagliaferri R

    Dipartimento di Scienze Fisiche, University of Naples Federico II, Naples, Italy.

    Motivation: The huge growth in gene expression data calls for the implementation of automatic tools for data processing and interpretation.

    Results: We present a new and comprehensive machine learning data mining framework consisting in a non-linear PCA neural network for feature extraction, and probabilistic principal surfaces combined with an agglomerative approach based on Negentropy aimed at clustering gene microarray data. The method, which provides a user-friendly visualization interface, can work on noisy data with missing points and represents an automatic procedure to get, with no a priori assumptions, the number of clusters present in the data. Cell-cycle dataset and a detailed analysis confirm the biological nature of the most significant clusters.

    Availability: The software described here is a subpackage part of the ASTRONEURAL package and is available upon request from the corresponding author.

    Supplementary information: Supplementary data are available at Bioinformatics online.

    Funded by: Telethon: TGM06S01, TGM06Z06

    Bioinformatics (Oxford, England) 2006;22;5;589-96

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

  • Exome and transcriptome sequencing of Aedes aegypti identifies a locus that confers resistance to Brugia malayi and alters the immune response.

    Juneja P, Ariani CV, Ho YS, Akorli J, Palmer WJ, Pain A and Jiggins FM

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

    Many mosquito species are naturally polymorphic for their abilities to transmit parasites, a feature which is of great interest for controlling vector-borne disease. Aedes aegypti, the primary vector of dengue and yellow fever and a laboratory model for studying lymphatic filariasis, is genetically variable for its capacity to harbor the filarial nematode Brugia malayi. The genome of Ae. aegypti is large and repetitive, making genome resequencing difficult and expensive. We designed exome captures to target protein-coding regions of the genome, and used association mapping in a wild Kenyan population to identify a single, dominant, sex-linked locus underlying resistance. This falls in a region of the genome where a resistance locus was previously mapped in a line established in 1936, suggesting that this polymorphism has been maintained in the wild for the at least 80 years. We then crossed resistant and susceptible mosquitoes to place both alleles of the gene into a common genetic background, and used RNA-seq to measure the effect of this locus on gene expression. We found evidence for Toll, IMD, and JAK-STAT pathway activity in response to early stages of B. malayi infection when the parasites are beginning to die in the resistant genotype. We also found that resistant mosquitoes express anti-microbial peptides at the time of parasite-killing, and that this expression is suppressed in susceptible mosquitoes. Together, we have found that a single resistance locus leads to a higher immune response in resistant mosquitoes, and we identify genes in this region that may be responsible for this trait.

    Funded by: Medical Research Council: G0900747; Wellcome Trust: 090532/Z/09/Z

    PLoS pathogens 2015;11;3;e1004765

  • 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

Sandra Bernaldo de Quiros Fernandez

sb40@sanger.ac.uk Research Assistant

After finishing my Bachelor's Degree in Biology at the University of Oviedo my interest in conducting research led me in a collaboration with the Laboratory of Tissue Engineering as an undergraduate Research Student. After that I obtained a Master's Degree in Oncology and Cancer Biology. Later I joined Carlos-Suarez-Nieto team to pursue my PhD in head and neck Cancer. While finishing my PhD,I continued my work abroad, and I joined the Mouse genomics Team at the Wellcome Trust Sanger Institute as a Visiting PhD student.

Research

I am involved in the Plasmodium Genome Variation project which aims to describe the genetic diversity between malaria parasites from across the globe using high-throughput genome sequencing. I also support other laboratory activities in the Kwiatkowski group at Sanger including sample reception, quantification and quality, parasite culture, protocol development and troubleshooting.

References

  • Identification of somatic VHL gene mutations in sporadic head and neck paragangliomas in association with activation of the HIF-1α/miR-210 signaling pathway.

    Merlo A, de Quirós SB, de Santa-María IS, Pitiot AS, Balbín M, Astudillo A, Scola B, Arístegui M, Quer M, Suarez C and Chiara MD

    Hospital Universitario Central de Asturias. Centro General, Laboratory Instituto Universitario de Oncología del Principado de Asturias, no. 2, 1 planta Centro, C/Celestino Villamil s/n, E-33006 Oviedo, Asturias, Spain. mdchiara.uo@uniovi.es.

    Context: Head and neck paragangliomas (HNPGLs) arise from parasympathetic paraganglias and 35% to 45% are hereditary caused by mutations in succinate dehydrogenase (SDH) genes. The connection between SDH and tumor development is unclear. The most accepted hypothesis proposes a central role for the pseudohypoxic (pHx) pathway activated by hypoxia-inducible factor (HIF). Paradoxically, we showed that activation of HIF in HNPGLs is restricted to a subset of HNPGLs lacking SDH mutations. These tumors overexpress HIF-1α protein and target genes and the HIF-inducible microRNA miR-210 (pHx-HNPGLs).

    Objective: The present study aimed at unraveling the SDH-independent mechanisms involved in the activation of HIF in HNPGLs.

    Design: The VHL gene was analyzed in 53 tumors by gene sequencing, multiplex-ligation-dependent probe amplification, and quantitative PCR. The miR-210, HIF-1α, and CA9 levels were used as markers of the pHx gene signature. Meta-analysis of the transcriptome of pHx-HNPGLs was performed using the Oncomine platform. Assays in cells lacking functional pVHL and HIF-1α were performed to analyze the role of pVHL/HIF-1α on miR-210 expression.

    Results: We identified, for the first time, somatic VHL mutations in HNPGLs. These were found in 2 of 4 pHx-HNPGLs with concomitant loss of heterozygosity in one of them; but not in non-pHx-HNPGLs. Meta-analysis of the transcriptome of pHx-HNPGLs revealed that these tumors are highly related to clear cell renal cell carcinoma. Cell-based assays showed that loss of pVHL lead to upregulation of miR-210 mainly via HIF-1α activation.

    Conclusions: VHL, involved in tumorigenesis of PGLs and clear cell renal cell carcinomas, may be an important player in the pathogenesis of sporadic HNPGLs via activation of an HIF-1α/miR-210 pHx pathway.

    The Journal of clinical endocrinology and metabolism 2013;98;10;E1661-6

  • Identification of TRPC6 as a possible candidate target gene within an amplicon at 11q21-q22.2 for migratory capacity in head and neck squamous cell carcinomas.

    Bernaldo de Quirós S, Merlo A, Secades P, Zambrano I, de Santa María IS, Ugidos N, Jantus-Lewintre E, Sirera R, Suarez C and Chiara MD

    Servicio de Otorrinolaringología, Hospital Universitario Central de Asturias, Instituto Universitario de Oncología del Principado de Asturias, Universidad de Oviedo, Oviedo, Spain.

    Background: Cytogenetic and gene expression analyses in head and neck squamous cell carcinomas (HNSCC) have allowed identification of genomic aberrations that may contribute to cancer pathophysiology. Nevertheless, the molecular consequences of numerous genetic alterations still remain unclear.

    Methods: To identify novel genes implicated in HNSCC pathogenesis, we analyzed the genomic alterations present in five HNSCC-derived cell lines by array CGH, and compared high level focal gene amplifications with gene expression levels to identify genes whose expression is directly impacted by these genetic events. Next, we knocked down TRPC6, one of the most highly amplified and over-expressed genes, to characterize the biological roles of TRPC6 in carcinogenesis. Finally, real time PCR was performed to determine TRPC6 gene dosage and mRNA levels in normal mucosa and human HNSCC tissues.

    Results: The data showed that the HNSCC-derived cell lines carry most of the recurrent genomic abnormalities previously described in primary tumors. High-level genomic amplifications were found at four chromosomal sites (11q21-q22.2, 18p11.31-p11.21, 19p13.2-p13.13, and 21q11) with associated gene expression changes in selective candidate genes suggesting that they may play an important role in the malignant behavior of HNSCC. One of the most dramatic alterations of gene transcription involved the TRPC6 gene (located at 11q21-q22.2) which has been recently implicated in tumour invasiveness. siRNA-induced knockdown of TRPC6 expression in HNSCC-derived cells dramatically inhibited HNSCC-cell invasion but did not significantly alter cell proliferation. Importantly, amplification and concomitant overexpression of TRPC6 was also found in HNSCC tumour samples.

    Conclusions: Altogether, these data show that TRPC6 is likely to be a target for 11q21-22.2 amplification that confers enhanced invasive behavior to HNSCC cells. Therefore, TRPC6 may be a promising therapeutic target in the treatment of HNSCC.

    BMC cancer 2013;13;116

  • Identification of a signaling axis HIF-1α/microRNA-210/ISCU independent of SDH mutation that defines a subgroup of head and neck paragangliomas.

    Merlo A, de Quiros SB, Secades P, Zambrano I, Balbín M, Astudillo A, Scola B, Arístegui M, Suarez C and Chiara MD

    Hospital Universitario Central de Asturias, Centro General, C/Celestino Villamil s/n, E-33006 Oviedo, Asturias, Spain.

    Background: Head and neck paragangliomas (HNPGLs) are rare tumors associated with the parasympathetic nervous system. Most are sporadic, but about one third result from germline mutations in succinate dehydrogenase (SDH) genes (SDHB, SDHC, SDHD, SDHA, or SDHAF2). Although a molecular connection between SDH dysfunction and tumor development is still unclear, the most accepted hypothesis proposes a central role of the pseudohypoxic pathway. SDH dysfunction induces abnormal stabilization of the hypoxia-inducible factors (HIFs) that regulate target genes involved in proliferation, apoptosis, angiogenesis, and metabolism. The involvement of these pathways in the development of sporadic HNPGLs is presently unknown.

    Objective: To get some insights into the hypoxic/pseudohypoxic molecular basis of HNPGLs, we attempted to define the gene, microRNA (miRNA), and HIF-1α expression patterns that distinguish tumors from normal paraganglia tissue.

    Design: Genome microarray and TaqMan low-density arrays were used to analyze gene and miRNA expression, respectively, in 17 HNPGL tumor tissues and three normal human carotid bodies. Twelve HNPGLs were used for validation of data. HIF-1α, SDHB, and iron-sulfur cluster scaffold protein (ISCU) protein expression was analyzed by immunohistochemistry.

    Results: We found activation of a canonical HIF-1α-related gene expression signaling only in a subset of HNPGLs from patients that did not harbor germline or somatic SDH mutations. The pseudohypoxic signature consisted in the overexpression of both HIF-1α-target genes and the HIF-1α-inducible miRNA, miR-210, and down-regulation of the miR-210 target gene, ISCU1/2. A decreased level of the iron-sulfur-containing protein SDHB was found by immunohistochemical analysis performed in two of these tumors.

    Conclusions: Collectively, this study unveiled a putative signaling axis of HIF-1α/miRNA-210/ISCU in a subset of HNPGLs that could have an impact on SDHB protein stability by a mechanism independent of SDH mutations, thus providing a foundation to better understand the functional interplay between HIF, miR-210, and mitochondria and its relevance in the pathogenesis of HNPGLs.

    The Journal of clinical endocrinology and metabolism 2012;97;11;E2194-200

  • MAX mutations cause hereditary and sporadic pheochromocytoma and paraganglioma.

    Burnichon N, Cascón A, Schiavi F, Morales NP, Comino-Méndez I, Abermil N, Inglada-Pérez L, de Cubas AA, Amar L, Barontini M, de Quirós SB, Bertherat J, Bignon YJ, Blok MJ, Bobisse S, Borrego S, Castellano M, Chanson P, Chiara MD, Corssmit EP, Giacchè M, de Krijger RR, Ercolino T, Girerd X, Gómez-García EB, Gómez-Graña A, Guilhem I, Hes FJ, Honrado E, Korpershoek E, Lenders JW, Letón R, Mensenkamp AR, Merlo A, Mori L, Murat A, Pierre P, Plouin PF, Prodanov T, Quesada-Charneco M, Qin N, Rapizzi E, Raymond V, Reisch N, Roncador G, Ruiz-Ferrer M, Schillo F, Stegmann AP, Suarez C, Taschin E, Timmers HJ, Tops CM, Urioste M, Beuschlein F, Pacak K, Mannelli M, Dahia PL, Opocher G, Eisenhofer G, Gimenez-Roqueplo AP and Robledo M

    Assistance Publique-Hôpitaux de Paris, Hôpital Européen Georges Pompidou, Paris, France.

    Purpose: Pheochromocytomas (PCC) and paragangliomas (PGL) are genetically heterogeneous neural crest-derived neoplasms. Recently we identified germline mutations in a new tumor suppressor susceptibility gene, MAX (MYC-associated factor X), which predisposes carriers to PCC. How MAX mutations contribute to PCC/PGL and associated phenotypes remain unclear. This study aimed to examine the prevalence and associated phenotypic features of germline and somatic MAX mutations in PCC/PGL.

    Design: We sequenced MAX in 1,694 patients with PCC or PGL (without mutations in other major susceptibility genes) from 17 independent referral centers. We screened for large deletions/duplications in 1,535 patients using a multiplex PCR-based method. Somatic mutations were searched for in tumors from an additional 245 patients. The frequency and type of MAX mutation was assessed overall and by clinical characteristics.

    Results: Sixteen MAX pathogenic mutations were identified in 23 index patients. All had adrenal tumors, including 13 bilateral or multiple PCCs within the same gland (P < 0.001), 15.8% developed additional tumors at thoracoabdominal sites, and 37% had familial antecedents. Age at diagnosis was lower (P = 0.001) in MAX mutation carriers compared with nonmutated cases. Two patients (10.5%) developed metastatic disease. A mutation affecting MAX was found in five tumors, four of them confirmed as somatic (1.65%). MAX tumors were characterized by substantial increases in normetanephrine, associated with normal or minor increases in metanephrine.

    Conclusions: Germline mutations in MAX are responsible for 1.12% of PCC/PGL in patients without evidence of other known mutations and should be considered in the genetic work-up of these patients.

    Clinical cancer research : an official journal of the American Association for Cancer Research 2012;18;10;2828-37

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, G19/9, G90/106, G9521010, 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.

Christopher Jacob

- Staff Scientist

I graduated from the Graduate School at the University of Maryland School of Medicine with a PhD in Molecular Medicine with a concentration in Genome Biology. My thesis research was focused on the population genetics of malaria parasites in Southeast Asia highlighting how migration and selection influence drug resistance. My PhD followed a BS in Biology from the College of Mount Saint Joseph in Cincinnati, Ohio.

Research

My research focus is directed towards the use of genomics for understanding and solving clinical questions relating to the effective treatment of malaria. My work will include genotyping and data analysis of parasites from ongoing studies collected as part of the reconnaissance project monitor parasite populations and drug resistance in Southeast Asia.

References

  • Hemoglobin C Trait Provides Protection From Clinical Falciparum Malaria in Malian Children.

    Travassos MA, Coulibaly D, Laurens MB, Dembélé A, Tolo Y, Koné AK, Traoré K, Niangaly A, Guindo A, Wu Y, Berry AA, Jacob CG, Takala-Harrison S, Adams M, Shrestha B, Mu AZ, Kouriba B, Lyke KE, Diallo DA, Doumbo OK, Plowe CV and Thera MA

    Center for Malaria Research, Institute for Global Health, University of Maryland School of Medicine Howard Hughes Medical Institute, Baltimore, Maryland.

    Background:  Hemoglobin C trait, like hemoglobin S trait, protects against severe malaria in children, but it is unclear whether hemoglobin C trait also protects against uncomplicated malaria. We hypothesized that Malian children with hemoglobin C trait would have a lower risk of clinical malaria than children with hemoglobin AA.

    Methods:  Three hundred children aged 0-6 years were enrolled in a cohort study of malaria incidence in Bandiagara, Mali, with continuous passive and monthly active follow-up from June 2009 to June 2010.

    Results:  Compared to hemoglobin AA children (n = 242), hemoglobin AC children (n = 39) had a longer time to first clinical malaria episode (hazard ratio [HR], 0.19; P = .001; 364 median malaria-free days vs 181 days), fewer episodes of clinical malaria, and a lower cumulative parasite burden. Similarly, hemoglobin AS children (n = 14) had a longer time to first clinical malaria episode than hemoglobin AA children (HR, 0.15; P = .015; 364 median malaria-free days vs 181 days), but experienced the most asymptomatic malaria infections of any group.

    Conclusions:  Both hemoglobin C and S traits exerted a protective effect against clinical malaria episodes, but appeared to do so by mechanisms that differentially affect the response to infecting malaria parasites.

    Funded by: FIC NIH HHS: D43 TW001589

    The Journal of infectious diseases 2015

  • A Single Mutation in K13 Predominates in Southern China and Is Associated With Delayed Clearance of Plasmodium falciparum Following Artemisinin Treatment.

    Huang F, Takala-Harrison S, Jacob CG, Liu H, Sun X, Yang H, Nyunt MM, Adams M, Zhou S, Xia Z, Ringwald P, Bustos MD, Tang L and Plowe CV

    National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, World Health Organization Collaborating Centre for Malaria, Schistosomiasis and Filariasis, Key Laboratory of Parasite and Vector Biology, Ministry of Health, Shanghai, PR China Center for Malaria Research, Institute for Global Health, University of Maryland School of Medicine, Baltimore.

    Background:  Artemisinin resistance in Plasmodium falciparum has emerged in Southeast Asia and poses a threat to malaria control and elimination. Mutations in a P. falciparum gene encoding a kelch protein on chromosome 13 have been associated with delayed parasite clearance following artemisinin treatment elsewhere in the region, but not yet in China.

    Methods:  Therapeutic efficacy studies of artesunate and dihydroartemisinin-piperaquine were conducted from 2009 to 2012 in the Yunnan Province of China near the border with Myanmar. K13 mutations were genotyped by capillary sequencing of DNA extracted from dried blood spots collected in these clinical trials and in routine surveillance. Associations between K13 mutations and delayed parasite clearance were tested using regression models.

    Results:  Parasite clearance half-lives were prolonged after artemisinin treatment, with 44% of infections having half-lives >5 hours (n = 109). Fourteen mutations in K13 were observed, with an overall prevalence of 47.7% (n = 329). A single mutation, F446I, predominated, with a prevalence of 36.5%. Infections with F446I were significantly associated with parasitemia on day 3 following artemisinin treatment and with longer clearance half-lives.

    Conclusions:  Plasmodium falciparum infections in southern China displayed markedly delayed clearance following artemisinin treatment. F446I was the predominant K13 mutation and was associated with delayed parasite clearance.

    Funded by: NIAID NIH HHS: R01 AI101713

    The Journal of infectious diseases 2015

  • Independent emergence of artemisinin resistance mutations among Plasmodium falciparum in Southeast Asia.

    Takala-Harrison S, Jacob CG, Arze C, Cummings MP, Silva JC, Dondorp AM, Fukuda MM, Hien TT, Mayxay M, Noedl H, Nosten F, Kyaw MP, Nhien NT, Imwong M, Bethell D, Se Y, Lon C, Tyner SD, Saunders DL, Ariey F, Mercereau-Puijalon O, Menard D, Newton PN, Khanthavong M, Hongvanthong B, Starzengruber P, Fuehrer HP, Swoboda P, Khan WA, Phyo AP, Nyunt MM, Nyunt MH, Brown TS, Adams M, Pepin CS, Bailey J, Tan JC, Ferdig MT, Clark TG, Miotto O, MacInnis B, Kwiatkowski DP, White NJ, Ringwald P and Plowe CV

    Howard Hughes Medical Institute/Center for Vaccine Development.

    Background: The emergence of artemisinin-resistant Plasmodium falciparum in Southeast Asia threatens malaria treatment efficacy. Mutations in a kelch protein encoded on P. falciparum chromosome 13 (K13) have been associated with resistance in vitro and in field samples from Cambodia.

    Methods: P. falciparum infections from artesunate efficacy trials in Bangladesh, Cambodia, Laos, Myanmar, and Vietnam were genotyped at 33 716 genome-wide single-nucleotide polymorphisms (SNPs). Linear mixed models were used to test associations between parasite genotypes and parasite clearance half-lives following artesunate treatment. K13 mutations were tested for association with artemisinin resistance, and extended haplotypes on chromosome 13 were examined to determine whether mutations arose focally and spread or whether they emerged independently.

    Results: The presence of nonreference K13 alleles was associated with prolonged parasite clearance half-life (P = 1.97 × 10(-12)). Parasites with a mutation in any of the K13 kelch domains displayed longer parasite clearance half-lives than parasites with wild-type alleles. Haplotype analysis revealed both population-specific emergence of mutations and independent emergence of the same mutation in different geographic areas.

    Conclusions: K13 appears to be a major determinant of artemisinin resistance throughout Southeast Asia. While we found some evidence of spreading resistance, there was no evidence of resistance moving westward from Cambodia into Myanmar.

    Funded by: Howard Hughes Medical Institute; Medical Research Council: G0600718; NIAID NIH HHS: R01 AI101713, R01AI101713, U19 AI110820, U19AI10820; NIGMS NIH HHS: R25 GM055036; Wellcome Trust: 090532, 090532/Z/09/Z, 090770, 090770/Z/09/Z, 093956, 098051

    The Journal of infectious diseases 2015;211;5;670-9

  • 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

    1] Wellcome Trust Sanger Institute, Hinxton, UK. [2] Medical Research Council (MRC) Centre for Genomics and Global Health, University of Oxford, Oxford, UK. [3] Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand.

    We report a large multicenter genome-wide association study of Plasmodium falciparum resistance to artemisinin, the frontline antimalarial drug. Across 15 locations in Southeast Asia, we identified at least 20 mutations in kelch13 (PF3D7_1343700) affecting the encoded propeller and BTB/POZ domains, which were associated with a slow parasite clearance rate after treatment with artemisinin derivatives. Nonsynonymous polymorphisms in fd (ferredoxin), arps10 (apicoplast ribosomal protein S10), mdr2 (multidrug resistance protein 2) and crt (chloroquine resistance transporter) also showed strong associations with artemisinin resistance. Analysis of the fine structure of the parasite population showed that the fd, arps10, mdr2 and crt polymorphisms are markers of a genetic background on which kelch13 mutations are particularly likely to arise and that they correlate with the contemporary geographical boundaries and population frequencies of artemisinin resistance. These findings indicate that the risk of new resistance-causing mutations emerging is determined by specific predisposing genetic factors in the underlying parasite population.

    Funded by: Howard Hughes Medical Institute; Intramural NIH HHS; Medical Research Council: G0600718; NIAID NIH HHS: R01 AI101713; Wellcome Trust: 090532/Z/09/Z, 090770, 090770/Z/09/Z, 098051

    Nature genetics 2015;47;3;226-34

  • Plasmodium falciparum field isolates from areas of repeated emergence of drug resistant malaria show no evidence of hypermutator phenotype.

    Brown TS, Jacob CG, Silva JC, Takala-Harrison S, Djimdé A, Dondorp AM, Fukuda M, Noedl H, Nyunt MM, Kyaw MP, Mayxay M, Hien TT, Plowe CV and Cummings MP

    Johns Hopkins University School of Medicine, Baltimore, MD, USA; Howard Hughes Medical Institute/Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, MD, USA.

    Multiple transcontinental waves of drug resistance in Plasmodium falciparum have originated in Southeast Asia before spreading westward, first into the rest of Asia and then to sub-Saharan Africa. In vitro studies have suggested that hypermutator P. falciparum parasites may exist in Southeast Asia and that an increased rate of acquisition of new mutations in these parasites may explain the repeated emergence of drug resistance in Southeast Asia. This study is the first to test the hypermutator hypothesis using field isolates. Using genome-wide SNP data from human P. falciparum infections in Southeast Asia and West Africa and a test for relative rate differences we found no evidence of increased relative substitution rates in P. falciparum isolates from Southeast Asia. Instead, we found significantly increased substitution rates in Mali and Bangladesh populations relative to those in populations from Southeast Asia. Additionally we found no association between increased relative substitution rates and parasite clearance following treatment with artemisinin derivatives.

    Funded by: Howard Hughes Medical Institute; NIAID NIH HHS: R01 AI101713, R01AI101713, R03AI101680, U19 AI110820, U19AI10820

    Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases 2015;30;318-22

  • A microarray platform and novel SNP calling algorithm to evaluate Plasmodium falciparum field samples of low DNA quantity.

    Jacob CG, Tan JC, Miller BA, Tan A, Takala-Harrison S, Ferdig MT and Plowe CV

    Malaria Group, Howard Hughes Medical Institute / Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, MD 21201, USA. cplowe@medicine.umaryland.edu.

    Background: Analysis of single nucleotide polymorphisms (SNPs) derived from whole-genome studies allows for rapid evaluation of genome-wide diversity, and genomic epidemiology studies of Plasmodium falciparum provide insights into parasite population structure, gene flow, drug resistance and vaccine development. In areas with adequate cold chain facilities, large volumes of leukocyte-depleted patient blood can be frozen for use in parasite genomic analyses. In more remote endemic areas smaller volumes of infected blood are taken by finger prick, and dried and stored on filter paper. These dried blood spots do not generally yield enough concentrated parasite DNA for whole-genome sequencing.

    Results: A DNA microarray was designed for use on field samples to type a genome-wide set of SNPs which prior sequencing had shown to be variable in Africa, Southeast Asia, and Papua New Guinea. An algorithm was designed to call SNPs in samples with low parasite DNA. With this new algorithm SNP-calling accuracy of 98% was measured by hybridizing purified DNA from malaria lab strains and comparing calls with SNPs called from full genome sequences. An average accuracy of >98% was likewise obtained for DNA extracted from malaria field samples collected in studies in Southeast Asia, with an average call rate of > 82%.

    Conclusion: This new high-density microarray provided high quality SNP calls from a wide range of parasite DNA quantities, and represents a robust tool for genome-wide analysis of malaria parasites in diverse settings.

    Funded by: Howard Hughes Medical Institute; NIAID NIH HHS: R01 AI101713, R01AI10171302

    BMC genomics 2014;15;719

  • A novel clade of unique eukaryotic ribonucleotide reductase R2 subunits is exclusive to apicomplexan parasites.

    Munro JB, Jacob CG and Silva JC

    Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD, 21201, USA.

    Apicomplexa are protist parasites of tremendous medical and economic importance, causing millions of deaths and billions of dollars in losses each year. Apicomplexan-related diseases may be controlled via inhibition of essential enzymes. Ribonucleotide reductase (RNR) provides the only de novo means of synthesizing deoxyribonucleotides, essential precursors for DNA replication and repair. RNR has long been the target of antibacterial and antiviral therapeutics. However, targeting this ubiquitous protein in eukaryotic pathogens may be problematic unless these proteins differ significantly from that of their respective host. The typical eukaryotic RNR enzymes belong to class Ia, and the holoenzyme consists minimally of two R1 and two R2 subunits (α₂β₂). We generated a comparative, annotated, structure-based, multiple-sequence alignment of R2 subunits, identified a clade of R2 subunits unique to Apicomplexa, and determined its phylogenetic position. Our analyses revealed that the apicomplexan-specific sequences share characteristics with both class I R2 and R2lox proteins. The putative radical-harboring residue, essential for the reduction reaction by class Ia R2-containing holoenzymes, was not conserved within this group. Phylogenetic analyses suggest that class Ia subunits are not monophyletic and consistently placed the apicomplexan-specific clade sister to the remaining class Ia eukaryote R2 subunits. Our research suggests that the novel apicomplexan R2 subunit may be a promising candidate for chemotherapeutic-induced inhibition as it differs greatly from known eukaryotic host RNRs and may be specifically targeted.

    Journal of molecular evolution 2013;77;3;92-106

  • Genetic loci associated with delayed clearance of Plasmodium falciparum following artemisinin treatment in Southeast Asia.

    Takala-Harrison S, Clark TG, Jacob CG, Cummings MP, Miotto O, Dondorp AM, Fukuda MM, Nosten F, Noedl H, Imwong M, Bethell D, Se Y, Lon C, Tyner SD, Saunders DL, Socheat D, Ariey F, Phyo AP, Starzengruber P, Fuehrer HP, Swoboda P, Stepniewska K, Flegg J, Arze C, Cerqueira GC, Silva JC, Ricklefs SM, Porcella SF, Stephens RM, Adams M, Kenefic LJ, Campino S, Auburn S, MacInnis B, Kwiatkowski DP, Su XZ, White NJ, Ringwald P and Plowe CV

    Howard Hughes Medical Institute/Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, MD 21201, USA.

    The recent emergence of artemisinin-resistant Plasmodium falciparum malaria in western Cambodia could threaten prospects for malaria elimination. Identification of the genetic basis of resistance would provide tools for molecular surveillance, aiding efforts to contain resistance. Clinical trials of artesunate efficacy were conducted in Bangladesh, in northwestern Thailand near the Myanmar border, and at two sites in western Cambodia. Parasites collected from trial participants were genotyped at 8,079 single nucleotide polymorphisms (SNPs) using a P. falciparum-specific SNP array. Parasite genotypes were examined for signatures of recent positive selection and association with parasite clearance phenotypes to identify regions of the genome associated with artemisinin resistance. Four SNPs on chromosomes 10 (one), 13 (two), and 14 (one) were significantly associated with delayed parasite clearance. The two SNPs on chromosome 13 are in a region of the genome that appears to be under strong recent positive selection in Cambodia. The SNPs on chromosomes 10 and 13 lie in or near genes involved in postreplication repair, a DNA damage-tolerance pathway. Replication and validation studies are needed to refine the location of loci responsible for artemisinin resistance and to understand the mechanism behind it; however, two SNPs on chromosomes 10 and 13 may be useful markers of delayed parasite clearance in surveillance for artemisinin resistance in Southeast Asia.

    Funded by: Howard Hughes Medical Institute; Intramural NIH HHS; Medical Research Council: G0600718, G19/9; NCRR NIH HHS: K12RR023250; PHS HHS: HHSN261200800001E; Wellcome Trust: 077012/Z/05/Z, 089275, 090532, 090770, 090770/Z/09/Z, 093956, 098051

    Proceedings of the National Academy of Sciences of the United States of America 2013;110;1;240-5

  • Reduced susceptibility of Plasmodium falciparum to artesunate in southern Myanmar.

    Kyaw MP, Nyunt MH, Chit K, Aye MM, Aye KH, Aye MM, Lindegardh N, Tarning J, Imwong M, Jacob CG, Rasmussen C, Perin J, Ringwald P and Nyunt MM

    Department of Medical Research (Lower Myanmar), Yangon, The Republic of the Union of Myanmar.

    Background: Plasmodium falciparum resistance to artemisinins, the first line treatment for malaria worldwide, has been reported in western Cambodia. Resistance is characterized by significantly delayed clearance of parasites following artemisinin treatment. Artemisinin resistance has not previously been reported in Myanmar, which has the highest falciparum malaria burden among Southeast Asian countries.

    Methods: A non-randomized, single-arm, open-label clinical trial of artesunate monotherapy (4 mg/kg daily for seven days) was conducted in adults with acute blood-smear positive P. falciparum malaria in Kawthaung, southern Myanmar. Parasite density was measured every 12 hours until two consecutive negative smears were obtained. Participants were followed weekly at the study clinic for three additional weeks. Co-primary endpoints included parasite clearance time (the time required for complete clearance of initial parasitemia), parasite clearance half-life (the time required for parasitemia to decrease by 50% based on the linear portion of the parasite clearance slope), and detectable parasitemia 72 hours after commencement of artesunate treatment. Drug pharmacokinetics were measured to rule out delayed clearance due to suboptimal drug levels.

    Results: The median (range) parasite clearance half-life and time were 4.8 (2.1-9.7) and 60 (24-96) hours, respectively. The frequency distributions of parasite clearance half-life and time were bimodal, with very slow parasite clearance characteristic of the slowest-clearing Cambodian parasites (half-life longer than 6.2 hours) in approximately 1/3 of infections. Fourteen of 52 participants (26.9%) had a measurable parasitemia 72 hours after initiating artesunate treatment. Parasite clearance was not associated with drug pharmacokinetics.

    Conclusions: A subset of P. falciparum infections in southern Myanmar displayed markedly delayed clearance following artemisinin treatment, suggesting either emergence of artemisinin resistance in southern Myanmar or spread to this location from its site of origin in western Cambodia. Resistance containment efforts are underway in Myanmar.

    Trial registration: Australian New Zealand Clinical Trials Registry ACTRN12610000896077.

    Funded by: Howard Hughes Medical Institute; Wellcome Trust: 089275

    PloS one 2013;8;3;e57689

  • Successful human infection with P. falciparum using three aseptic Anopheles stephensi mosquitoes: a new model for controlled human malaria infection.

    Laurens MB, Billingsley P, Richman A, Eappen AG, Adams M, Li T, Chakravarty S, Gunasekera A, Jacob CG, Sim BK, Edelman R, Plowe CV, Hoffman SL and Lyke KE

    Howard Hughes Medical Institute/Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, Maryland, United States of America.

    Unlabelled: Controlled human malaria infection (CHMI) is a powerful method for assessing the efficacy of anti-malaria vaccines and drugs targeting pre-erythrocytic and erythrocytic stages of the parasite. CHMI has heretofore required the bites of 5 Plasmodium falciparum (Pf) sporozoite (SPZ)-infected mosquitoes to reliably induce Pf malaria. We reported that CHMI using the bites of 3 PfSPZ-infected mosquitoes reared aseptically in compliance with current good manufacturing practices (cGMP) was successful in 6 participants. Here, we report results from a subsequent CHMI study using 3 PfSPZ-infected mosquitoes reared aseptically to validate the initial clinical trial. We also compare results of safety, tolerability, and transmission dynamics in participants undergoing CHMI using 3 PfSPZ-infected mosquitoes reared aseptically to published studies of CHMI using 5 mosquitoes. Nineteen adults aged 18-40 years were bitten by 3 Anopheles stephensi mosquitoes infected with the chloroquine-sensitive NF54 strain of Pf. All 19 participants developed malaria (100%); 12 of 19 (63%) on Day 11. The mean pre-patent period was 258.3 hours (range 210.5-333.8). The geometric mean parasitemia at first diagnosis by microscopy was 9.5 parasites/µL (range 2-44). Quantitative polymerase chain reaction (qPCR) detected parasites an average of 79.8 hours (range 43.8-116.7) before microscopy. The mosquitoes had a geometric mean of 37,894 PfSPZ/mosquito (range 3,500-152,200). Exposure to the bites of 3 aseptically-raised, PfSPZ-infected mosquitoes is a safe, effective procedure for CHMI in malaria-naïve adults. The aseptic model should be considered as a new standard for CHMI trials in non-endemic areas. Microscopy is the gold standard used for the diagnosis of Pf malaria after CHMI, but qPCR identifies parasites earlier. If qPCR continues to be shown to be highly specific, and can be made to be practical, rapid, and standardized, it should be considered as an alternative for diagnosis.

    Trial registration: ClinicalTrials.gov NCT00744133 NCT00744133.

    Funded by: Howard Hughes Medical Institute; NIAID NIH HHS: HHSN272200800057C, N01-AI-80001, R01 AI-067954, R44AI055229, R44AI058375; PHS HHS: HHSN272200800057C

    PloS one 2013;8;7;e68969

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: Medical Research Council: MC_UU_12013/3; Wellcome Trust: 091310, 092731, 096599, 098498, 100140, 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: 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.

Group leader

Dominic's photo Professor Dominic Kwiatkowski
Dominic's profile

Joint projects

Matt's photo Dr Matt Berriman
Matt's profile

Oliver's photo Dr Oliver Billker
Oliver's group

Julian's photo Dr Julian Rayner
Julian's group

Mara's photo Dr Mara Lawniczak
Mara's profile

* quick link - http://q.sanger.ac.uk/mal-kwia