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.

Strains used in whole organism Plasmodium falciparum vaccine trials differ in genome structure, sequence, and immunogenic potential

“…Several other large structural variants impact regions housing non-multi-gene family members, although none is known to be involved in pre-erythrocytic immunity. Examples include a 31-kbp-long tandem expansion of a region of chromosome 12 in the 7G8 assembly (also present in the previously published assembly for 7G8 [32]) and a 22.7-kbp-long repeat expansion of a region of chromosome 5 in NF135.C10, both of which are supported by~200 PacBio reads. The former is a segmental duplication containing a vacuolar iron transporter (PF3D7_1223700), a putative citrate/oxoglutarate carrier protein (PF3D7_1223800), a putative 50S ribosomal protein L24 (PF3D7_1223900), GTP cyclohydrolase I (PF3D7_1224000), and three conserved Plasmodium proteins of unknown function (PF3D7_1223500, PF3D7_ 1223600, PF3D7_1224100).…”

“…In particular, the 7G8 assembly was several hundred thousand base-pairs smaller than the other three assemblies. To confirm that this was not an assembly error, we compared 7G8 to a previously published 7G8 PacBio-based assembly [32]. The two assemblies were extremely close in overall genome structure, differing only by~25 kbp in cumulative length, and also shared a very similar number of SNP and small indel variants relative to 3D7 (Additional file 2: Table S4).…”

“…However, the high AT content and repetitive nature of the P. falciparum genome greatly complicates genome assembly methods [30]. Recently, longread sequencing technologies have been used to overcome some of these assembly challenges, as was shown with assemblies for 3D7, 7G8, and several other cultureadapted P. falciparum strains generated using Pacific Biosciences (PacBio) technology [31][32][33]. However, NF166.C8 and NF135.C10 still lack whole-genome assemblies; in addition, while an assembly for 7G8 is available [32], it is important to characterize the specific 7G8 clone used in heterologous CHMI, from Sanaria's working bank, as strains can undergo genetic changes over time in culture [34].…”

“…Recently, longread sequencing technologies have been used to overcome some of these assembly challenges, as was shown with assemblies for 3D7, 7G8, and several other cultureadapted P. falciparum strains generated using Pacific Biosciences (PacBio) technology [31][32][33]. However, NF166.C8 and NF135.C10 still lack whole-genome assemblies; in addition, while an assembly for 7G8 is available [32], it is important to characterize the specific 7G8 clone used in heterologous CHMI, from Sanaria's working bank, as strains can undergo genetic changes over time in culture [34]. Here, reference assemblies for NF54, 7G8, NF166.C8, and NF135.C10 (hereafter referred to as PfSPZ strains) were generated using approaches to take advantage of the resolution power of long-read sequencing data and the low error rate of short-read sequencing platforms.…”

Sensitive, highly multiplexed sequencing of microhaplotypes from thePlasmodium falciparumheterozygome

“…A. Bioinformatics workflow for the identification of the heterozygome. *13 high-quality assembled and annotated genomes of P. falciparum isolates [31]. The variable region covered was determined as the first position to the last position within the window with a SNP or INDEL >=0.5%.…”