Targeted next-generation sequencing offers the potential for consistent, deep coverage of information-rich genomic regions to characterize polyclonal Plasmodium falciparum infections. However, methods to identify and sequence these genomic regions are currently limited.
A bioinformatic pipeline and multiplex methods were developed to identify and simultaneously sequence 100 targets and applied to dried blood spot (DBS) controls and field isolates from Mozambique. For comparison, whole-genome sequencing data were generated for the same controls.
Using publicly available genomes, 4465 high-diversity genomic regions suited for targeted sequencing were identified, representing the P. falciparum heterozygome. For this study, 93 microhaplotypes with high diversity (median expected heterozygosity = 0.7) were selected along with 7 drug resistance loci. The sequencing method achieved very high coverage (median 99%), specificity (99.8%), and sensitivity (90% for haplotypes with 5% within sample frequency in dried blood spots with 100 parasites/µL). In silico analyses revealed that microhaplotypes provided much higher resolution to discriminate related from unrelated polyclonal infections than biallelic single-nucleotide polymorphism barcodes.
A novel bioinformatics pipeline was created to select and characterize the most diverse and tractable short-range sequences (microhaplotypes) in the Plasmodium falciparum genome, followed by a robust polymerase chain reaction–based multiplexing method to simultaneously amplify and sequence these microhaplotypes.