Long-read amplicon sequencing of polymorphic malaria antigens

Many vaccine candidate proteins in the malaria parasite Plasmodium falciparum are under strong selective pressure to diversify in terms of antigenicity. We present a sequencing and data analysis platform for the genomic and epidemiological surveillance of the indel-rich antigen MSP2 from P. falciparum using long-read circular consensus sequencing (CCS) in multiclonal malaria isolates. Our platform uses 40 PCR primers to asymmetrically barcode and identify multiclonal infections in pools of up to 384 samples. We validated the method using 235 mock infections combining 10 synthetic variants at different concentrations and infection complexities, as well as 95 isolates from P. falciparum-infected residents of Nyamisati, Tanzania. We also constructed a fully automated analysis pipeline that streamlines the processing and interpretation of epidemiological and antigenic diversity data for msp2 from demultiplexed FASTQ files. Finally, this platform can be easily adapted to other polymorphic antigens of interest in Plasmodium or any other human pathogen.