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sequencing data for each isolate, and these are compared against a panel of polymorphic sites derived from published or unpublished but publicly available data.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.

Figure 1. Identifying amplifications as areas of high MPS coverage.

Length of sequence intervals between paired reads (nt) and coverage (read frequency) are plotted against chromosome position and %GC content for two loci in isolates OX001 and OX006. pfgh1 displays high coverage consistent with amplification in OX006, but not OX001. pfef2 displays high coverage consistent with amplification in OX001, but not OX006. Loci of interest (red circles) are shown within100 km of genomic context. Red colouring within the read pile-ups signify polymorphic sites within a read in which a non-reference allele is present (i.e. SNP).

Figure 2. Deletions in pfrbp2 homologues a and b appear as areas with inflated sequence intervals in four isolates.

Two isoforms of RBP2 are encoded by adjacent genes on chromosome 13, arranged head to head and transcribed in opposite directions. 60 kb around these genes are depicted, for four isolates. Loci of interest (red arrows) have either a ∼600 bp deletion in the carboxy-terminal serine-rich domain of homologue b (red elipses), or a ∼2–300 bp deletion in the low-complexity protein domain immediately upstream in both genes (blue elipses). Y-axis depicts sequence interval between paired reads. X-axis depicts nucleotide coordinates along the chromosome, as in Figure 1.

Figure 3. Evidence of a major deletion at the right end of chromosome 3 in isolate OX005.

Paired reads across the whole of chromosome 3 are presented in pile-up view for two isolates, OX005 and OX006 (upper panel). Y-axis depicts sequence interval between paired reads, and X-axis gives chromosome coordinates as in Figures 1 and 2. A detailed view of ∼70 kb around the clag3.2 and clag3.1 loci is also shown for 4 isolates (lower panel). The locus between PFC0110w and PFC0120w is a degenerate var gene lacking a full-length ORF in 3D7 and other parasite sequences in the available databases.