304V Poster Online - Virtual Posters
Tuesday June 07, 11:00 AM - 3:00 PM

Authors:
Emily R. Ebel ^1,2; Bernard Y. Kim ²; Marina McDew-White ³; Elizabeth S. Egan ¹; Timothy J.C. Anderson ³; Dmitri A. Petrov ²

Affiliations:
1) Stanford University School of Medicine, Stanford, CA; 2) Stanford University, Stanford, CA; 3) Texas Biomedical Research Institute, San Antonio, TX

Keywords:
Molecular Evolution

Antigenic variation in the malaria parasite Plasmodium falciparum is mediated by the var gene family, named for its variability. The high diversity of var types in natural populations is thought to result from frequent structural mutations—especially recombination events—that generate novel, chimeric coding sequences. To better understand the mechanisms by which P. falciparum generates var diversity, we performed long-read sequencing of clonal, haploid parasite populations derived from a 6-month-long mutation accumulation experiment.

We used PacBio reads (7-10 kb in length) to produce high-quality genome assemblies for each clonal lineage of P. falciparum. Surprisingly, each assembly contained multiple contigs with inconsistent haplotypes at certain var loci. To clarify this result, we generated much longer Nanopore reads (30-400 kb) and developed a homology-based method to visualize gene organization on individual reads. This analysis revealed extensive genetic diversity at four var loci within most clonal samples. Individual reads displayed one of several possible haplotypes per locus, which varied from one another by large indels that appeared to create novel, chimeric var genes. Notably, the limited set of possible haplotypes at each locus was largely shared across the 17 samples; and the minor haplotype frequencies within each sample were fairly high (3-45%). These observations are inconsistent with the premise that de novo mutations generate diversity in each clonal lineage independently. Instead, we hypothesized that multiple var haplotypes per locus could be maintained in single P. falciparum cells through extrachromosomal DNA (ecDNA), as has recently been shown for a drug-resistance locus. Using quantitative ddPCR, we observed that a var locus on chr12 is present at ~1.3 times the chromosomal copy number, consistent with its presence on ecDNA. Furthermore, by combining ddPCR with an enzyme that digests only linear DNA, we found that circular DNA constructs are strongly enriched for the same var locus. This discovery that antigenic variation in P. falciparum can be maintained on ecDNA has major implications for our understanding of mutation, selection, and the evolution of immune evasion in malaria parasites.