Development of amplicon deep sequencing markers and data analysis pipeline for genotyping multi-clonal malaria infections

Background: Amplicon 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. Methods: Targeted 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 (H-e = 0.96) in contrast to csp (H-e = 0.57). Minority clones were robustly detected if their frequency was > 1%. False haplotype calls owing to sequencing errors were observed below that threshold. Conclusions: To 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.