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Long read assemblies of geographically dispersed Plasmodium falciparum isolates reveal highly structured subtelomeres.

ABSTRACT: Background: Although thousands of clinical isolates of Plasmodium falciparum are being sequenced and analysed by short read technology, the data do not resolve the highly variable subtelomeric regions of the genomes that contain polymorphic gene families involved in immune evasion and pathogenesis. There is also no current standard definition of the boundaries of these variable subtelomeric regions. Methods: Using long-read sequence data (Pacific Biosciences SMRT technology), we assembled and annotated the genomes of 15 P. falciparum isolates, ten of which are newly cultured clinical isolates. We performed comparative analysis of the entire genome with particular emphasis on the subtelomeric regions and the internal var genes clusters. Results: The nearly complete sequence of these 15 isolates has enabled us to define a highly conserved core genome, to delineate the boundaries of the subtelomeric regions, and to compare these across isolates. We found highly structured variable regions in the genome. Some exported gene families purportedly involved in release of merozoites show copy number variation. As an example of ongoing genome evolution, we found a novel CLAG gene in six isolates. We also found a novel gene that was relatively enriched in the South East Asian isolates compared to those from Africa. Conclusions: These 15 manually curated new reference genome sequences with their nearly complete subtelomeric regions and fully assembled genes are an important new resource for the malaria research community. We report the overall conserved structure and pattern of important gene families and the more clearly defined subtelomeric regions.

SUBMITTER: Otto TD

PROVIDER: S-EPMC5964635 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Long read assemblies of geographically dispersed Plasmodium falciparum isolates reveal highly structured subtelomeres.

Otto Thomas D TD Böhme Ulrike U Sanders Mandy M Reid Adam A Bruske Ellen I EI Duffy Craig W CW Bull Pete C PC Pearson Richard D RD Abdi Abdirahman A Dimonte Sandra S Stewart Lindsay B LB Campino Susana S Kekre Mihir M Hamilton William L WL Claessens Antoine A Volkman Sarah K SK Ndiaye Daouda D Amambua-Ngwa Alfred A Diakite Mahamadou M Fairhurst Rick M RM Conway David J DJ Franck Matthias M Newbold Chris I CI Berriman Matt M

Wellcome open research 20180503

Background: Although thousands of clinical isolates of Plasmodium falciparum are being sequenced and analysed by short read technology, the data do not resolve the highly variable subtelomeric regions of the genomes that contain polymorphic gene families involved in immune evasion and pathogenesis. There is also no current standard definition of the boundaries of these variable subtelomeric regions. Methods: Using long-read sequence data (Pacific Biosciences SMRT technology) ...[more]

PMID: 29862326

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Project description:Mechanisms regulating gene expression in malaria parasites are not well understood. Little is known about how this parasite regulates its gene expression during transition from one developmental stage to another and in response to various environmental conditions. Parasites in a diseased host face environments which differ from the static, well adapted in vitro conditions in culture. Parasites thus need to adapt quickly and effectively to these conditions by establishing transcriptional states which is best suited for better survival. With the discovery of natural antisense transcripts (NATs) in this parasite and considering the various proposed mechanisms by which NATs might regulate gene expression, it has been speculated that these might be playing a critical role in gene regulation. We report here the diversity of NATs that exist in this parasite, using isolates taken directly from patients with differing clinical symptoms caused by malaria infection. A total of 797 NATs targeted against annotated loci have been detected using a custom designed strand specific microarray. Out of these, 545 are unique to this study. The majority of NATs were positively correlated with the expression pattern of the sense transcript. Many were found to be differentially regulated in response to disease conditions. Antisense transcripts mapped to a wide variety of biochemical/ metabolic pathways, especially pathways pertaining to the central carbon metabolism and stress related pathways. Our data strongly suggests that a large group of NATs detected here are unannotated transcription units antisense to annotated gene models. The results reveal a previously unknown set of NATs that prevails in this parasite, their differential regulation in disease conditions and mapping to functionally well annotated genes. The results detailed here call for studies to deduce the possible mechanism of action, which would help in understanding the in vivo pathological adaptations of these parasites.

Project description:BackgroundSegmental duplications (SD) have been found in genomes of various organisms, often accumulated at the ends of chromosomes. It has been assumed that the sequence homology in-between the SDs allow for ectopic interactions that may contribute to the emergence of new genes or gene variants through recombinatorial events.MethodsIn silico analysis of the 3D7 Plasmodium falciparum genome, conducted to investigate the subtelomeric compartments, led to the identification of subtelomeric SDs. Sequence variation and copy number polymorphisms of the SDs were studied by DNA sequencing, real-time quantitative PCR (qPCR) and fluorescent in situ hybridization (FISH). The levels of transcription and the developmental expression of copy number variant genes were investigated by qPCR.ResultsA block of six genes of >10 kilobases in size, including var, rif, pfmc-2tm and three hypothetical genes (n-, o- and q-gene), was found duplicated in the subtelomeric regions of chromosomes 1, 2, 3, 6, 7, 10 and 11 (SD1). The number of SD1 per genome was found to vary from 4 to 8 copies in between different parasites. The intragenic regions of SD1 were found to be highly conserved across ten distinct fresh and long-term cultivated P. falciparum. Sequence variation was detected in a approximately 23 amino-acid long hypervariable region of a surface-exposed loop of PFMC-2TM. A hypothetical gene within SD1, the n-gene, encoding a PEXEL/VTS-containing two-transmembrane protein was found expressed in ring stage parasites. The n-gene transcription levels were found to correlate to the number of n-gene copies. Fragments of SD1 harbouring two or three of the SD1-genes (o-gene, pfmc-2tm, q-gene) were also found in the 3D7 genome. In addition a related second SD, SD2, of approximately 55% sequence identity to SD1 was found duplicated in a fresh clinical isolate but was only present in a single copy in 3D7 and in other P. falciparum lines or clones.ConclusionPlasmodium falciparum carries multiple sequence conserved SDs in the otherwise highly variable subtelomeres of its chromosomes. The uniqueness of the SDs amongst plasmodium species, and the conserved nature of the genes within, is intriguing and suggests an important role of the SD to P. falciparum.

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Long read assemblies of geographically dispersed Plasmodium falciparum isolates reveal highly structured subtelomeres.

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Long read assemblies of geographically dispersed <i>Plasmodium falciparum</i> isolates reveal highly structured subtelomeres.

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