Complete telomere-to-telomere de novo assembly of the Plasmodium falciparum genome through long-read (>11 kb), single molecule, real-time sequencing

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 AT-richness [∼80.6% (A + T)] of its genome and the lack of technology to assemble highly polymorphic subtelomeric regions t...

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Bibliographic Details
Published in:DNA research 2016-08, Vol.23 (4), p.339-351
Main Authors: Vembar, Shruthi Sridhar, Seetin, Matthew, Lambert, Christine, Nattestad, Maria, Schatz, Michael C, Baybayan, Primo, Scherf, Artur, Smith, Melissa Laird
Format: Article
Language:English
Subjects:
Contig Mapping
Genome, Protozoan
Plasmodium falciparum
Plasmodium falciparum - genetics
Polymorphism, Genetic
Sequence Analysis, DNA
Telomere - genetics
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Summary: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 AT-richness [∼80.6% (A + 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 [∼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.
ISSN:1340-2838
1756-1663
DOI:10.1093/dnares/dsw022