title: Complete telomere-to-telomere de novo assembly of the Plasmodium falciparum genome through long-read (>11 kb), single molecule, real-time sequencing.
abstract: 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.
authors.affiliation: Unité Biologie des Interactions Hôte-Parasite, Département de Parasites et Insectes Vecteurs, Institut Pasteur, Paris 75015, France CNRS, ERL 9195, Paris 75015, France INSERM, Unit U1201, Paris 75015, France shruthi-sridhar.vembar@pasteur.fr mlaird@pacificbiosciences.com.
authors.forename: Shruthi Sridhar
authors.initials: SS
authors.lastname: Vembar
authors.affiliation: Pacific Biosciences, Menlo Park, CA, USA.
authors.forename: Matthew
authors.initials: M
authors.lastname: Seetin
authors.affiliation: Pacific Biosciences, Menlo Park, CA, USA.
authors.forename: Christine
authors.initials: C
authors.lastname: Lambert
authors.affiliation: Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA.
authors.forename: Maria
authors.initials: M
authors.lastname: Nattestad
authors.affiliation: Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA Department of Computer Science, Johns Hopkins University, Baltimore, MD, USA.
authors.forename: Michael C
authors.initials: MC
authors.lastname: Schatz
authors.affiliation: Pacific Biosciences, Menlo Park, CA, USA.
authors.forename: Primo
authors.initials: P
authors.lastname: Baybayan
authors.affiliation: Unité Biologie des Interactions Hôte-Parasite, Département de Parasites et Insectes Vecteurs, Institut Pasteur, Paris 75015, France CNRS, ERL 9195, Paris 75015, France INSERM, Unit U1201, Paris 75015, France.
authors.forename: Artur
authors.initials: A
authors.lastname: Scherf
authors.affiliation: Pacific Biosciences, Menlo Park, CA, USA shruthi-sridhar.vembar@pasteur.fr mlaird@pacificbiosciences.com.
authors.forename: Melissa Laird
authors.initials: ML
authors.lastname: Smith
mesh_terms: D020451:Contig Mapping; D018503:Genome, Protozoan; D010963:Plasmodium falciparum / Q000235:genetics*; D011110:Polymorphism, Genetic; D017422:Sequence Analysis, DNA; D016615:Telomere / Q000235:genetics*
keywords: AT-biased; Plasmodium falciparum; de novo assembly; long-read sequencing; structural variation