Journal
DNA RESEARCH
Volume 23, Issue 4, Pages 339-351Publisher
OXFORD UNIV PRESS
DOI: 10.1093/dnares/dsw022
Keywords
Plasmodium falciparum; AT-biased; long-read sequencing; de novo assembly; structural variation
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Funding
- European Research Council [ERC AdG PlasmoEscape 250320]
- French Parasitology consortium ParaFrap [ANR-11-LABX0024]
- European Molecular Biology Organization Long-Term Fellowship
- Marie Sklodowska-Curie International Incoming Fellowship [FP7-MC-IIF-302451]
- Institut Pasteur Bourse Roux post-doctoral fellowship
- ERC
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The application of next-generation sequencing to estimate genetic diversity of Plasmodium falciparum, the most lethal malaria parasite, has proved challenging due to the skewed ATrichness [similar to 80.6% (A broken vertical bar T)] of its genome and the lack of technology to assemble highly polymorphic subtelomeric regions that contain clonally variant, multigene virulence families (Ex: var and rifin). To address this, we performed amplification-free, single molecule, real-time sequencing of P. falciparum genomic DNA and generated reads of average length 12 kb, with 50% of the reads between 15.5 and 50 kb in length. Next, using the Hierarchical Genome Assembly Process, we assembled the P. falciparum genome de novo and successfully compiled all 14 nuclear chromosomes telomere-to-telomere. We also accurately resolved centromeres [similar to 90-99% (A_T)] and subtelomeric regions and identified large insertions and duplications that add extra var and rifin genes to the genome, along with smaller structural variants such as homopolymer tract expansions. Overall, we show that amplification-free, long-read sequencing combined with de novo assembly overcomes major challenges inherent to studying the P. falciparum genome. Indeed, this technology may not only identify the polymorphic and repetitive subtelomeric sequences of parasite populations from endemic areas but may also evaluate structural variation linked to virulence, drug resistance and disease transmission.
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