Abstract
BackgroundAmplicon deep sequencing permits sensitive detection of minority clones and improves discriminatory power for genotyping multi-clone Plasmodium falciparum infections. New amplicon sequencing and data analysis protocols are needed for genotyping in epidemiological studies and drug efficacy trials of P. falciparum.MethodsTargeted sequencing of molecular marker csp and novel marker cpmp was conducted in duplicate on mixtures of parasite culture strains and 37 field samples. A protocol allowing to multiplex up to 384 samples in a single sequencing run was applied. Software “HaplotypR” was developed for data analysis.ResultsCpmp was highly diverse (He = 0.96) in contrast to csp (He = 0.57). Minority clones were robustly detected if their frequency was >1%. False haplotype calls owing to sequencing errors were observed below that threshold.ConclusionsTo reliably detect haplotypes at very low frequencies, experiments are best performed in duplicate and should aim for coverage of >10′000 reads/amplicon. When compared to length polymorphic marker msp2, highly multiplexed amplicon sequencing displayed greater sensitivity in detecting minority clones.
Highlights
Amplicon deep sequencing permits sensitive detection of minority clones and improves discriminatory power for genotyping multi-clone Plasmodium falciparum infections
Marker selection A protocol for deep sequencing and data analysis was developed for two molecular markers, namely the P. falciparum csp gene (PF3D7_0304600) and gene PF3D7_0104100, annotated in the malaria sequence database PlasmoDB as “conserved Plasmodium membrane protein”
The newly validated marker cpmp was identified by calculating heterozygosity in 200 bp windows of 3′411 genomic P. falciparum sequences from 23 countries available from the MalariaGEN dataset [17]
Summary
Amplicon deep sequencing permits sensitive detection of minority clones and improves discriminatory power for genotyping multi-clone Plasmodium falciparum infections. Identification of new infections is crucial in clinical trials of antimalarial drugs, where persisting clones need to be distinguished from new clones in post-treatment samples from patients with recurrent parasitaemia [2, 3]. For such diverse applications, genotyping methods based on length polymorphic markers had been applied for decades, by targeting microsatellite markers or genes encoding parasite surface antigens such as merozoite surface proteins 1 and 2 (msp, msp2) [4, 5]
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