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Tuesday, April 21, 2020

A chromosome-scale assembly of the major African malaria vector Anopheles funestus.

Anopheles funestus is one of the 3 most consequential and widespread vectors of human malaria in tropical Africa. However, the lack of a high-quality reference genome has hindered the association of phenotypic traits with their genetic basis in this important mosquito.Here we present a new high-quality A. funestus reference genome (AfunF3) assembled using 240× coverage of long-read single-molecule sequencing for contigging, combined with 100× coverage of short-read Hi-C data for chromosome scaffolding. The assembled contigs total 446 Mbp of sequence and contain substantial duplication due to alternative alleles present in the sequenced pool of mosquitos from the FUMOZ colony. Using…

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Tuesday, April 21, 2020

Progression of the canonical reference malaria parasite genome from 2002-2019.

Here we describe the ways in which the sequence and annotation of the Plasmodium falciparum reference genome has changed since its publication in 2002. As the malaria species responsible for the most deaths worldwide, the richness of annotation and accuracy of the sequence are important resources for the P. falciparum research community as well as the basis for interpreting the genomes of subsequently sequenced species. At the time of publication in 2002 over 60% of predicted genes had unknown functions. As of March 2019, this number has been significantly decreased to 33%. The reduction is due to the inclusion of…

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Wednesday, February 26, 2020

Advances in sequence consensus and clustering algorithms for effective de novo assembly and haplotyping applications.

One of the major applications of DNA sequencing technology is to bring together information that is distant in sequence space so that understanding genome structure and function becomes easier on a large scale. The Single Molecule Real Time (SMRT) Sequencing platform provides direct sequencing data that can span several thousand bases to tens of thousands of bases in a high-throughput fashion. In contrast to solving genomic puzzles by patching together smaller piece of information, long sequence reads can decrease potential computation complexity by reducing combinatorial factors significantly. We demonstrate algorithmic approaches to construct accurate consensus when the differences between reads…

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Wednesday, February 26, 2020

Complete telomere-to-telomere de novo assembly of the Plasmodium falciparum genome using long-read sequencing

Sequence-based estimation of genetic diversity of Plasmodium falciparum, the most lethal malarial parasite, has proved challenging due to a lack of a complete genomic assembly. The skewed AT-richness (~80.6% (A+T)) of its genome and the lack of technology to assemble highly polymorphic sub-telomeric regions that contain clonally variant, multigene virulence families (i.e. var and rifin) have confounded attempts using short-read NGS technologies. Using single molecule, real-time (SMRT) sequencing, we successfully compiled all 14 nuclear chromosomes of the P. falciparum genome from telomere-to-telomere in single contigs. Specifically, amplification-free sequencing generated reads of average length 12 kb, with =50% of the reads…

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Sunday, September 22, 2019

Single molecule RNA sequencing uncovers trans-splicing and improves annotations in Anopheles stephensi.

Single molecule real-time (SMRT) sequencing has recently been used to obtain full-length cDNA sequences that improve genome annotation and reveal RNA isoforms. Here, we used one such method called isoform sequencing from Pacific Biosciences (PacBio) to sequence a cDNA library from the Asian malaria mosquito Anopheles stephensi. More than 600 000 full-length cDNAs, referred to as reads of insert, were identified. Owing to the inherently high error rate of PacBio sequencing, we tested different approaches for error correction. We found that error correction using Illumina RNA sequencing (RNA-seq) generated more data than using the default SMRT pipeline. The full-length error-corrected PacBio…

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Sunday, September 22, 2019

Plasmodium knowlesi: a superb in vivo nonhuman primate model of antigenic variation in malaria.

Antigenic variation in malaria was discovered in Plasmodium knowlesi studies involving longitudinal infections of rhesus macaques (M. mulatta). The variant proteins, known as the P. knowlesi Schizont Infected Cell Agglutination (SICA) antigens and the P. falciparum Erythrocyte Membrane Protein 1 (PfEMP1) antigens, expressed by the SICAvar and var multigene families, respectively, have been studied for over 30 years. Expression of the SICA antigens in P. knowlesi requires a splenic component, and specific antibodies are necessary for variant antigen switch events in vivo. Outstanding questions revolve around the role of the spleen and the mechanisms by which the expression of these…

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Sunday, September 22, 2019

A reference genome and methylome for the Plasmodium knowlesi A1-H.1 line.

Plasmodium knowlesi, a common parasite of macaques, is recognised as a significant cause of human malaria in Malaysia. The P. knowlesi A1H1 line has been adapted to continuous culture in human erythrocytes, successfully providing an in vitro model to study the parasite. We have assembled a reference genome for the PkA1-H.1 line using PacBio long read combined with Illumina short read sequence data. Compared with the H-strain reference, the new reference has improved genome coverage and a novel description of methylation sites. The PkA1-H.1 reference will enhance the capabilities of the in vitro model to improve the understanding of P.…

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Sunday, September 22, 2019

Comparative heterochromatin profiling reveals conserved and unique epigenome signatures linked to adaptation and development of malaria parasites.

Heterochromatin-dependent gene silencing is central to the adaptation and survival of Plasmodium falciparum malaria parasites, allowing clonally variant gene expression during blood infection in humans. By assessing genome-wide heterochromatin protein 1 (HP1) occupancy, we present a comprehensive analysis of heterochromatin landscapes across different Plasmodium species, strains, and life cycle stages. Common targets of epigenetic silencing include fast-evolving multi-gene families encoding surface antigens and a small set of conserved HP1-associated genes with regulatory potential. Many P. falciparum heterochromatic genes are marked in a strain-specific manner, increasing the parasite’s adaptive capacity. Whereas heterochromatin is strictly maintained during mitotic proliferation of asexual blood stage…

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Sunday, September 22, 2019

RTS,S/AS01 malaria vaccine mismatch observed among Plasmodium falciparum isolates from southern and central Africa and globally.

The RTS,S/AS01 malaria vaccine encompasses the central repeats and C-terminal of Plasmodium falciparum circumsporozoite protein (PfCSP). Although no Phase II clinical trial studies observed evidence of strain-specific immunity, recent studies show a decrease in vaccine efficacy against non-vaccine strain parasites. In light of goals to reduce malaria morbidity, anticipating the effectiveness of RTS,S/AS01 is critical to planning widespread vaccine introduction. We deep sequenced C-terminal Pfcsp from 77 individuals living along the international border in Luapula Province, Zambia and Haut-Katanga Province, the Democratic Republic of the Congo (DRC) and compared translated amino acid haplotypes to the 3D7 vaccine strain. Only 5.2%…

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Sunday, September 22, 2019

Genomes of all known members of a Plasmodium subgenus reveal paths to virulent human malaria.

Plasmodium falciparum, the most virulent agent of human malaria, shares a recent common ancestor with the gorilla parasite Plasmodium praefalciparum. Little is known about the other gorilla- and chimpanzee-infecting species in the same (Laverania) subgenus as P. falciparum, but none of them are capable of establishing repeated infection and transmission in humans. To elucidate underlying mechanisms and the evolutionary history of this subgenus, we have generated multiple genomes from all known Laverania species. The completeness of our dataset allows us to conclude that interspecific gene transfers, as well as convergent evolution, were important in the evolution of these species. Striking…

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Sunday, September 22, 2019

Multi-population genomic analysis of malaria parasites indicates local selection and differentiation at the gdv1 locus regulating sexual development.

Parasites infect hosts in widely varying environments, encountering diverse challenges for adaptation. To identify malaria parasite genes under locally divergent selection across a large endemic region with a wide spectrum of transmission intensity, genome sequences were obtained from 284 clinical Plasmodium falciparum infections from four newly sampled locations in Senegal, The Gambia, Mali and Guinea. Combining these with previous data from seven other sites in West Africa enabled a multi-population analysis to identify discrete loci under varying local selection. A genome-wide scan showed the most exceptional geographical divergence to be at the early gametocyte gene locus gdv1 which is essential…

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Sunday, September 22, 2019

Genomic and transcriptomic comparisons of closely related malaria parasites differing in virulence and sequestration pattern.

Background: Malaria parasite species differ greatly in the harm they do to humans. While P. falciparum kills hundreds of thousands per year, P. vivax kills much less often and P. malariae is relatively benign. Strains of the rodent malaria parasite Plasmodium chabaudi show phenotypic variation in virulence during infections of laboratory mice. This make it an excellent species to study genes which may be responsible for this trait. By understanding the mechanisms which underlie differences in virulence we can learn how parasites adapt to their hosts and how we might prevent disease. Methods: Here we present a complete reference genome…

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Saturday, September 21, 2019

PacBio assembly of a Plasmodium knowlesi genome sequence with Hi-C correction and manual annotation of the SICAvar gene family.

Plasmodium knowlesi has risen in importance as a zoonotic parasite that has been causing regular episodes of malaria throughout South East Asia. The P. knowlesi genome sequence generated in 2008 highlighted and confirmed many similarities and differences in Plasmodium species, including a global view of several multigene families, such as the large SICAvar multigene family encoding the variant antigens known as the schizont-infected cell agglutination proteins. However, repetitive DNA sequences are the bane of any genome project, and this and other Plasmodium genome projects have not been immune to the gaps, rearrangements and other pitfalls created by these genomic features.…

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Friday, July 19, 2019

Differing patterns of selection and geospatial genetic diversity within two leading Plasmodium vivax candidate vaccine antigens.

Although Plasmodium vivax is a leading cause of malaria around the world, only a handful of vivax antigens are being studied for vaccine development. Here, we investigated genetic signatures of selection and geospatial genetic diversity of two leading vivax vaccine antigens–Plasmodium vivax merozoite surface protein 1 (pvmsp-1) and Plasmodium vivax circumsporozoite protein (pvcsp). Using scalable next-generation sequencing, we deep-sequenced amplicons of the 42 kDa region of pvmsp-1 (n?=?44) and the complete gene of pvcsp (n?=?47) from Cambodian isolates. These sequences were then compared with global parasite populations obtained from GenBank. Using a combination of statistical and phylogenetic methods to assess…

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Friday, July 19, 2019

Evolution of mosquito preference for humans linked to an odorant receptor.

Female mosquitoes are major vectors of human disease and the most dangerous are those that preferentially bite humans. A ‘domestic’ form of the mosquito Aedes aegypti has evolved to specialize in biting humans and is the main worldwide vector of dengue, yellow fever, and chikungunya viruses. The domestic form coexists with an ancestral, ‘forest’ form that prefers to bite non-human animals and is found along the coast of Kenya. We collected the two forms, established laboratory colonies, and document striking divergence in preference for human versus non-human animal odour. We further show that the evolution of preference for human odour…

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