title: Whole genome sequencing of Plasmodium falciparum from dried blood spots using selective whole genome amplification.
abstract: BACKGROUND
  Translating genomic technologies into healthcare applications for the malaria parasite Plasmodium falciparum has been limited by the technical and logistical difficulties of obtaining high quality clinical samples from the field. Sampling by dried blood spot (DBS) finger-pricks can be performed safely and efficiently with minimal resource and storage requirements compared with venous blood (VB). Here, the use of selective whole genome amplification (sWGA) to sequence the P. falciparum genome from clinical DBS samples was evaluated, and the results compared with current methods that use leucodepleted VB.
  
  
  METHODS
  Parasite DNA with high (>95%) human DNA contamination was selectively amplified by Phi29 polymerase using short oligonucleotide probes of 8-12 mers as primers. These primers were selected on the basis of their differential frequency of binding the desired (P. falciparum DNA) and contaminating (human) genomes.
  
  
  RESULTS
  Using sWGA method, clinical samples from 156 malaria patients, including 120 paired samples for head-to-head comparison of DBS and leucodepleted VB were sequenced. Greater than 18-fold enrichment of P. falciparum DNA was achieved from DBS extracts. The parasitaemia threshold to achieve >5× coverage for 50% of the genome was 0.03% (40 parasites per 200 white blood cells). Over 99% SNP concordance between VB and DBS samples was achieved after excluding missing calls.
  
  
  CONCLUSIONS
  The sWGA methods described here provide a reliable and scalable way of generating P. falciparum genome sequence data from DBS samples. The current data indicate that it will be possible to get good quality sequence on most if not all drug resistance loci from the majority of symptomatic malaria patients. This technique overcomes a major limiting factor in P. falciparum genome sequencing from field samples, and paves the way for large-scale epidemiological applications.
authors.affiliation: Wellcome Trust Sanger Institute, Hinxton, CB10 1SA, UK. so1@sanger.ac.uk.|International Livestock Research Institute, Box 30709, Nairobi, Kenya. so1@sanger.ac.uk.
authors.forename: Samuel O
authors.initials: SO
authors.lastname: Oyola
authors.affiliation: Wellcome Trust Sanger Institute, Hinxton, CB10 1SA, UK. cristina.ariani@sanger.ac.uk.
authors.forename: Cristina V
authors.initials: CV
authors.lastname: Ariani
authors.affiliation: Wellcome Trust Sanger Institute, Hinxton, CB10 1SA, UK.|Addenbrooke's Hospital, University of Cambridge School of Clinical Medicine, Hills Rd, Cambridge, CB2 0SP, UK.
authors.forename: William L
authors.initials: WL
authors.lastname: Hamilton
authors.affiliation: Wellcome Trust Sanger Institute, Hinxton, CB10 1SA, UK.
authors.forename: Mihir
authors.initials: M
authors.lastname: Kekre
authors.affiliation: Navrongo Health Research Centre, Post Office Box 114, Navrongo, Ghana.
authors.forename: Lucas N
authors.initials: LN
authors.lastname: Amenga-Etego
authors.affiliation: Noguchi Memorial Institute for Medical Research, University of Ghana, P. O. Box LG 581, Legon, Accra, Ghana.
authors.forename: Anita
authors.initials: A
authors.lastname: Ghansah
authors.affiliation: Wellcome Trust Sanger Institute, Hinxton, CB10 1SA, UK.
authors.forename: Gavin G
authors.initials: GG
authors.lastname: Rutledge
authors.affiliation: Broad Institute, 415 Main St, Cambridge, MA, 02142, USA.
authors.forename: Seth
authors.initials: S
authors.lastname: Redmond
authors.affiliation: Wellcome Trust Sanger Institute, Hinxton, CB10 1SA, UK.
authors.forename: Magnus
authors.initials: M
authors.lastname: Manske
authors.affiliation: Wellcome Trust Sanger Institute, Hinxton, CB10 1SA, UK.
authors.forename: Dushyanth
authors.initials: D
authors.lastname: Jyothi
authors.affiliation: Wellcome Trust Sanger Institute, Hinxton, CB10 1SA, UK.
authors.forename: Chris G
authors.initials: CG
authors.lastname: Jacob
authors.affiliation: Wellcome Trust Sanger Institute, Hinxton, CB10 1SA, UK.
authors.forename: Thomas D
authors.initials: TD
authors.lastname: Otto
authors.affiliation: MRC Centre for Genomics and Global Health, University of Oxford, Oxford, OX3 7BN, UK.|Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, OX3 7BN, UK.
authors.forename: Kirk
authors.initials: K
authors.lastname: Rockett
authors.affiliation: Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, OX3 9DS, UK.
authors.forename: Chris I
authors.initials: CI
authors.lastname: Newbold
authors.affiliation: Wellcome Trust Sanger Institute, Hinxton, CB10 1SA, UK.
authors.forename: Matthew
authors.initials: M
authors.lastname: Berriman
authors.affiliation: Wellcome Trust Sanger Institute, Hinxton, CB10 1SA, UK.|MRC Centre for Genomics and Global Health, University of Oxford, Oxford, OX3 7BN, UK.|Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, OX3 7BN, UK.
authors.forename: Dominic P
authors.initials: DP
authors.lastname: Kwiatkowski
mesh_terms: D001769:Blood / Q000469:parasitology*; D017931:DNA Primers / Q000235:genetics; D016054:DNA, Protozoan / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D003890:Desiccation; D018503:Genome, Protozoan; D006801:Humans; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D010963:Plasmodium falciparum / Q000235:genetics* / Q000302:isolation & purification; D017422:Sequence Analysis, DNA; D013048:Specimen Handling / Q000379:methods*
chemical_list: D017931:DNA Primers; D016054:DNA, Protozoan
keywords: Dried blood spot; Field samples; Malaria; Selective whole genome amplification; Whole genome sequencing