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.Results Cpmp 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.Electronic supplementary materialThe online version of this article (10.1186/s12864-017-4260-y) contains supplementary material, which is available to authorized users.

Estimation of drug efficacy in antimalarial drug trials requires parasite genotyping to distinguish new infections from treatment failures. When using length-polymorphic molecular markers, preferential amplification of short fragments can compromise detection of coinfections, potentially leading to misclassification of treatment outcome. We quantified minority clone detectability and competition among msp1, msp2, and glurp amplicons using mixtures of Plasmodium falciparum strains and investigated the impact of template competition on genotyping outcomes in 44 paired field samples. Substantial amplification bias was detected for all three markers, with shorter fragments outperforming larger fragments. The strongest template competition was observed for the marker glurp. Detection of glurp fragments in multiclonal infections was severely compromised. Eight of 44 sample pairs were identified as new infections by all three markers. Ten pairs were defined as new infections based on one marker alone, seven of which were defined by the questionable marker glurp. The impact of size-dependent template competition on genotyping outcomes therefore calls for necessary amendments to the current WHO recommendations for PCR correction of malaria drug trial endpoints. Accuracy of genotyping outcomes could be improved by separate amplification reactions per allelic family and basing results on markers msp1 and msp2 first, with glurp only used to resolve discordant results.

29 30Amplicon deep sequencing permits sensitive detection of minority clones and improves discriminatory power 31 for genotyping multi-clone Plasmodium falciparum infections. Such high resolution is needed for molecular 32 monitoring of drug efficacy trials. Targeted sequencing of molecular marker csp and novel marker cpmp was 33 conducted in duplicate on mixtures of parasite culture strains and 37 field samples. A protocol to multiplex up 34 to 384 samples in a single sequencing run was applied. Software "HaplotypR" was developed for data 35 analysis. Cpmp was highly diverse (H e =0.96) in contrast to csp (H e =0.57). Minority clones were robustly 36 detected if their frequency was >1%. False haplotype calls owing to sequencing errors were observed below 37 that threshold. To reliably detect haplotypes at very low frequencies, experiments are best performed in 38 duplicate and should aim for coverage of >10'000 reads/amplicon. When compared to length polymorphic 39 marker msp2, highly multiplexed amplicon sequencing displayed greater sensitivity in detecting minority 40 clones. 41 42