Direct long-read visualization reveals hidden variation in GCH1 gene copy number and precise expansion steps

Abstract

Background: Increases in the copy number of large genomic regions, termed amplifications, are an important adaptive strategy for many organisms. Numerous amplifications across the AT-rich Plasmodium falciparum genome contribute directly to drug resistance or impact the fitness of this protozoan parasite. During the characterization of malaria parasites selected with a dihydroorotate dehydrogenase (DHODH) inhibitor that targets pyrimidine biosynthesis, we detected increased copies of a genomic region that encompassed 3 genes (~ 5 kb) including GTP cyclohydrolase I (GCH1 amplicon). While amplification of this gene is reported in antifolate-resistant parasites, GCH1 amplicons had not previously been implicated in DHODH inhibitor resistance. Results: Here, we explored the expansion of the GCH1 locus in this family of parasite lines using long-read sequencing and single-read visualization. We directly quantified higher numbers of tandem GCH1 amplicons in selected parasite lines (up to 9 GCH1 amplicons) compared to parental P. falciparum parasites (strictly 3 GCH1 amplicons). Because each read represents DNA from an individual genome, we were able to appreciate hidden variation within a single parasite line (3, to 5, to 7 amplicons) that was not reflected in other DNA-based analysis methods. While all GCH1 amplicons shared a consistent structure, expansions arose in precise 2-unit steps within selected lines. We found conserved AT-rich sequences at amplicon boundaries, which is consistent with the Plasmodium model of CNV formation. Parasite lines with expanded GCH1 also had DHODH amplicons on a separate chromosome. When we evaluated prior DHODH inhibitor selections, we observed that GCH1 amplification was not required for resistance; however, selection outcomes suggest that pre-existing GCH1 amplicons may support amplification at the DHODH locus. Conclusions: We identified previously undetected heterogeneity in gene copy number by viewing long pieces of DNA from individual genomes. This approach was possible due to the amplicon’s tandem orientation and relatively small size that can be spanned by a single long ONT read. The positive association between DHODH and GCH1 copy number, combined with the metabolic connection between P. falciparum pyrimidine and folate biosynthesis, justifies further investigation into the adaptive evolution of these two genomic loci.