title: Drug-resistant genotypes and multi-clonality in Plasmodium falciparum analysed by direct genome sequencing from peripheral blood of malaria patients.
abstract: 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sequencing data for each isolate, and these are compared against a panel of polymorphic sites derived from published or unpublished but publicly available data.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.
authors.affiliation: Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdom.
authors.forename: Timothy
authors.initials: T
authors.lastname: Robinson
authors.forename: Susana G
authors.initials: SG
authors.lastname: Campino
authors.forename: Sarah
authors.initials: S
authors.lastname: Auburn
authors.forename: Samuel A
authors.initials: SA
authors.lastname: Assefa
authors.forename: Spencer D
authors.initials: SD
authors.lastname: Polley
authors.forename: Magnus
authors.initials: M
authors.lastname: Manske
authors.forename: Bronwyn
authors.initials: B
authors.lastname: MacInnis
authors.forename: Kirk A
authors.initials: KA
authors.lastname: Rockett
authors.forename: Gareth L
authors.initials: GL
authors.lastname: Maslen
authors.forename: Mandy
authors.initials: M
authors.lastname: Sanders
authors.forename: Michael A
authors.initials: MA
authors.lastname: Quail
authors.forename: Peter L
authors.initials: PL
authors.lastname: Chiodini
authors.forename: Dominic P
authors.initials: DP
authors.lastname: Kwiatkowski
authors.forename: Taane G
authors.initials: TG
authors.lastname: Clark
authors.forename: Colin J
authors.initials: CJ
authors.lastname: Sutherland
mesh_terms: D000328:Adult; D000953:Antigens, Protozoan / Q000235:genetics; D001483:Base Sequence; D002875:Chromosomes; D002999:Clone Cells; D004351:Drug Resistance / Q000235:genetics*; D018628:Gene Dosage / Q000235:genetics; D017125:Genes, Protozoan / Q000235:genetics; D056426:Genetic Loci / Q000235:genetics; D016678:Genome / Q000235:genetics*; D054629:Genome, Mitochondrial / Q000235:genetics; D006239:Haplotypes / Q000235:genetics; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008288:Malaria / Q000097:blood* / Q000469:parasitology*; D008875:Middle Aged; D008969:Molecular Sequence Data; D010963:Plasmodium falciparum / Q000235:genetics*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D015800:Protozoan Proteins / Q000235:genetics; D017422:Sequence Analysis, DNA / Q000379:methods*; D017384:Sequence Deletion / Q000235:genetics
chemical_list: D000953:Antigens, Protozoan; D015800:Protozoan Proteins; C065974:merozoite surface protein 2, Plasmodium