MalDeepSeq panel: A targeted ultra-deep sequencing approach to trace drug resistance markers in Plasmodium falciparum

The emergence of drug-resistant Plasmodium falciparum highlights the need for tools to detect minor parasite subpopulations before resistant lineages expand. We developed and validated a targeted ultra-deep sequencing framework for the full-length sequences of 48 antimalarial resistance genes. Performance was evaluated using 3D7:Dd2 mock mixtures and clinical samples after selective whole-genome amplification. The panel achieved high depth and breadth, including for dried blood spot samples. Using Dd2-fixed markers, Mutect2 and HaplotypeCaller reproduced expected variant-allele frequencies, with Mutect2 exhibiting lower global bias and error. A conservative truth set derived from Dd2-pure controls yielded perfect recall and no false-positives in 3D7 controls, with high recovery of true Dd2 variants in mock mixtures for both callers. In clinical samples, the panel captured within-host diversity and minority resistance alleles. Overall, these results demonstrate that targeted deep sequencing offers a cost-efficient, high-resolution approach for routine molecular surveillance of emerging drug resistance.