Marker-specific reference primers.

Molecular epidemiologic studies of malaria parasites and other pathogens commonly employ amplicon deep sequencing (AmpSeq) of marker genes derived from dried blood spots (DBS) to answer public health questions related to topics such as transmission and drug resistance. As these methods are increasingly employed to inform direct public health action, it is important to rigorously evaluate the risk of false positive and false negative haplotypes derived from clinically-relevant sample types. We performed a control experiment evaluating haplotype recovery from AmpSeq of 5 marker genes (ama1, csp, msp7, sera2, and trap) from DBS containing mixtures of DNA from 1 to 10 known P. falciparum reference strains across 3 parasite densities in triplicate (n = 270 samples). While false positive haplotypes were present across all parasite densities and mixtures, we optimized censoring criteria to remove 83% (148/179) of false positives while removing only 8% (67/859) of true positives. Post-censoring, the median pairwise Jaccard distance between replicates was 0.83. We failed to recover 35% (477/1365) of haplotypes expected to be present in the sample. Haplotypes were more likely to be missed in low-density samples with