Long-read amplicon sequencing of polymorphic malaria antigens. A genomic surveillance platform for indel-rich genes from Plasmodium spp. using long-read amplicon sequencing

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 antigens MSP1, MSP2, GLURP and CSP from P. falciparum using long-read circular consensus sequencing (CCS) in multiclonal malaria isolates. Our platform uses 40 PCR primers per antigen 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 282 clinical samples 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 from demultiplexed FASTQ files. Finally, this platform can be easily adapted to other polymorphic antigens of interest in Plasmodium or any other human pathogen.