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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 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.


Introduction
Plasmodium falciparum, the causative agent of the most lethal form of malaria, uses a complex developmental programme to propagate within the human host and mosquito vector. 1 Disease pathogenesis in humans correlates with asexual development when the parasite resides within mature erythrocytes and mitotically replicates its haploid genome. In addition to meiotic recombination during diploid stages in mosquitoes, 2 it is during mitosis that the parasite expands its genetic diversity via homologous recombination, leading to the acquisition of new variants of virulence-associated surface adhesion molecules such as erythrocyte membrane protein 1 [P. falciparum Erythrocyte Membrane Protein 1(PfEMP1); encoded by var genes]. [3][4][5] Importantly, this is the stage at which the parasite evolves drug resistance. 6,7 In fact, it has been estimated that for each cycle of intraerythrocytic replication, laboratory-adapted parasites can tolerate a single nucleotide polymorphism (SNP) mutation rate of approximately 0.5-1 Â 10 À9 per base, 3,4 and that nearly 0.2% of daughter parasites carry a new chimeric PfEMP1 molecule. 4 Furthermore, the heterogeneity within the human host can be amplified by multiple mosquito bites, which may harbour genetically diverse parasite populations. There is, therefore, great interest in evaluating the genetic complexity of the infectious pool of parasites in malaria-endemic regions, with the specific aims of improving surveillance and intervention strategies.
The $23 Mb P. falciparum genome is organized into 14 chromosomes that range in size from 0.65 to 3.4 Mb. The draft sequence of the P. falciparum genome, which was first reported in 2002 using shotgun-sequencing methods for the laboratory-adapted strain 3D7, 8 revealed that the genome has an overall (A þ T) composition of 80.6%, making it one of the most AT-rich genomes identified to date. The complexity of the genome is further underscored by the presence of extended tracts of As, Ts and TAs, in introns, intergenic and centromeric regions [up to 99% (A þ T) content]; subtelomeric, hypervariable multigene virulence families, including the $60-member var gene family 9 ; and large segments of repetitive sequences, especially in subtelomeric regions. Given these unique features, not only has accurate sequencing of P. falciparum DNA presented a technical challenge for most next-generation sequencing (NGS) technologies, [10][11][12][13][14] it has been suggested that the use of the current 3D7 genome sequence as a reference for clinical isolates results in incomplete estimates of genetic diversity. 15,16 To date, researchers have primarily used PCR-based whole genome amplification (WGA) methods to prepare short read sequencing libraries of P. falciparum laboratory-adapted and clinical strains, 3,4,6,[17][18][19][20][21] with Nair et al. applying this to single cell sequencing. 22 More recently, Oyola et al. used u29 DNA polymerase-based multiple displacement alignment (MDA), in the presence of the detergent tetramethylammonium chloride, to analyse the P. falciparum genome and showed that very low quantities of genomic DNA ($10 pg) were sufficient to generate multiplexed Illumina libraries. 11 However, the introduction of errors and bias during PCR-based WGA, 23 and the subsequent alignment-based mapping of short reads to the reference 3D7 genome 8 (http://genedb.org; 23.3 Mb assembly) may have led to an overestimation of SNPs in the sequencing data. Indeed, Oyola et al. observed that MDA introduces several per cent (2-6%) of de novo SNP calls as compared to a non-amplified library. 11 Furthermore, none of these studies analysed larger structural variants, except for Bopp et al. 3 and Claessens et al. 4 Therefore, we have a fragmented view of P. falciparum genome plasticity, in which SNPs are evaluated at high frequency but polymorphisms such as insertions and deletions, copy number variants, chromosomal rearrangements and structural variants in hypervariable and highly repetitive regions, are often underestimated or largely ignored. 16 One solution that may overcome all of these caveats is the utilization of amplification-free long-read NGS technologies to sequence the P. falciparum genome. Single molecule real-time (SMRT) sequencing, which was the first such technology described, 24 generates long-reads with little to no sequence context bias, 13,14,25 with the most recent version of the DNA polymerase (P6) combined with C4 sequencing chemistry producing reads of average length 10-15 kb. 25 Numerous studies have shown that by oversampling a genome, structural variants can be detected with confidence, 26,27 and de novo assembly can be performed with high accuracy. [28][29][30] Attempts to analyse P. falciparum genomic DNA with early SMRT sequencing chemistry (P1-C1) generated $700-1,500 base long-reads, which did not allow for complete de novo assembly or evaluation of structural variations. 13,14,31 Therefore, to develop a robust long-read sequencing and de novo assembly protocol to analyse the P. falciparum genome, we utilized the Pacific Biosciences RS II System with P6-C4 chemistry. Accordingly, we sequenced the genome of the strain 3D7 ( Supplementary Fig. S1), and generated sequencing reads that had an average read length of 11-13 kb (maximum 45-50 kb), comprising over 5.26 Gb of data. The resulting sequences were assembled de novo into a highly accurate P. falciparum genome using the Hierarchical Genome Assembly Process (HGAP), 29 with all 14 chromosomes resolved into single contigs. Even extremely AT-rich regions, including the centromeres, were resolved with uniform coverage and for the first time, subtelomeric regions of all chromosomes were successfully assembled in a single run. We present an initial analysis of the de novo-assembled P. falciparum genome and discuss the advances that can now be made with regards to estimating P. falciparum genetic diversity using long-read sequencing technologies.

Growth of P. falciparum
Blood stages of the P. falciparum laboratory strain 3D7 were grown according to Trager and Jensen 32 with a few changes. Briefly, a mixed stage culture of P. falciparum was grown in white blood cell (WBC)-free Oþ human erythrocytes (prepared from whole blood by treatment with leucocyte-specific filters) at a haematocrit of 4% in Roswell Park Memorial Institute 1640 medium containing L-glutamine (Invitrogen) supplemented with 10% v/v Albumax II (Invitrogen) and 200 lm hypoxanthine (C.C.Pro). The cultures were grown in a gas environment of 5% CO 2 , 1% O 2 and 94% N 2 to a parasitaemia of 3-8% before harvesting for downstream analysis. For synchronization, knob-positive parasites were selected by gelatin flotation using Plasmion (Fresenius Kabi) 33 and after re-invasion, treated twice with 5% sorbitol (Sigma) 34 to obtain parasites that were synchronized within a window of approximately 6 h, as evaluated by Giemsa staining.

Genomic DNA isolation
Infected human erythrocytes at different parasitaemia were harvested and 1 ml or 5 ml aliquots were frozen at À 20 C. Subsequently, genomic DNA was prepared using the DNeasy Blood and Tissue kit (Qiagen) or the Genomic Tip kit (Qiagen) according to manufac-turer's instructions. For the DNeasy kit, free parasites that were obtained from the infected human erythrocyte pellet by saponin lysis 35 were resuspended in phosphate-buffered saline (PBS) and treated as per manufacturer's instructions. For the Genomic Tip kit, nuclei were prepared directly from the infected human erythrocyte pellet as per manufacturer's instructions.

DNA purification prior to library preparation
To remove heme and its derivatives from P. falciparum genomic DNA, the DNA was purified using one of the three independent methods: (i) magnetic bead-based cleanup; (ii) electrophoretic DNA extraction using the Aurora platform (Boreal Genomics) or (iii) phenol-chloroform extraction. The starting amount of DNA used for each method is indicated in Fig. 1B. For (i), AMPure PB magnetic beads (Pacific Biosciences) were mixed with P. falciparum genomic DNA at a 1:1 (vol:vol) ratio and incubated for 20 min at room temperature (RT) with gentle end-over-end rotation. Following this, the beads were washed twice with 1.5 ml 70% ethanol and allowed to dry briefly at RT before elution with 100 ll of Pacific Biosciences Elution Buffer (EB). For (ii), P. falciparum DNA was electrophoretically purified using the Aurora System (Boreal Genomics), following manufacturer's instructions in the 'Experienced User Protocol'. For (iii), P. falciparum genomic DNA was initially mixed with 500 ll buffer containing 1 M NaCl and 2 mM ethylenediaminetetraacetic acid (EDTA). Next, an equal volume of phenol:chloroform:isoamyl alcohol (24:23:1) was added to the DNA mixture, inverted to mix and spun in a microcentrifuge at 10,000 g for 10 min at RT. The upper aqueous phase was transferred to a new microcentrifuge tube, mixed with an equal volume of chloroform:isoamyl alcohol (24:1) and spun at 10,000 g for 10 min at RT. Then, to remove excess polysaccharide, 0.3 volumes of 99.99% ethanol was added to the upper aqueous phase, the mixture was inverted and spun at 10,000 g for 15 min at RT. Finally, DNA present in the upper aqueous phase was precipitated by adding 1.7 volumes of 99.99% ethanol and spinning at 10,000 g for 15 min at RT, followed by two 70% ethanol washes. The DNA pellet was allowed to air-dry at RT for up to 5 min and resuspended in 100 ml of Pacific Biosciences EB.

SMRTbell library preparation and sequencing
Three SMRTbell libraries were constructed for genomic DNA purified with each of the methods described above (Supplementary Fig.  S1). Each library was constructed using $4 lg of purified DNA and the SMRTbell Template Prep Kit 1.0, according to the protocol described in 'Procedure & Checklist-20 kb Template Preparation Using BluePippin TM Size-Selection System' (Pacific Biosciences). Briefly, P. falciparum DNA was sheared for 5 min at 3000g or 5,500 rpm using a g-TUBE (Covaris), concentrated with AMPure PB beads and subjected to DNA damage repair and ligation of SMRTbell adapters. Following ligation, extraneous DNA was digested with exonucleases and the SMRTbell library was cleaned and concentrated with AMPure PB beads. The libraries were then subjected to a 20 kb DNA size-selection step using the BluePippin System (SageScience) to remove shorter DNA inserts with a size cutoff of 15 kb. Library quality and quantity were assessed using the Agilent 12000 DNA Kit and 2100 Bioanalyzer System (Agilent Technologies), as well as the Qubit dsDNA Broad Range Assay kit and Qubit Fluorometer (Thermo Fisher). Sequencing primer and P6 polymerase were annealed and bound, respectively, to the SMRTbell libraries as recommended by the manufacturer (Pacific Biosciences). To identify the library concentration that would achieve optimal Poisson loading on the SMRT Cell (i.e. $40% of zero mode waveguides loaded with a single DNA molecule), loading titrations were performed for each library. Based on this analysis, polymerasebound SMRTbell libraries were loaded at a concentration of 200 pM for libraries cleaned up using magnetic beads and electrophoretic extraction, and at 100 pM for the phenol-chloroform extracted library to achieve comparable sequencing efficiencies. SMRT sequencing was performed on the Pacific Biosciences RS II System using the C4 sequencing kit (Pacific Biosciences), with magnetic bead loading and 240 min movies. Three SMRT cells were run per library type, providing a total of nine SMRT cells worth of data to be used for downstream analysis. Prior to data analysis, raw reads with a predicted polymerase read quality less than 0.80 were filtered out.

Data analysis
De novo assembly of the P. falciparum genome was carried out using the RS_HGAP_Assembly.3 protocol within Pacific Biosciences' SMRT Analysis Portal 2.3.0.p2 as previously described 29 ( Supplementary Fig. S1). All parameters were set at their default values with the following exceptions: (i) the minimum subread length was set to 13,000 based on the size distribution of the reads ( Fig. 2A) and for computational expediency, so as to not exceed 100-fold coverage going into the analysis; (ii) the minimum seed read length was increased to 20,000 from a default of 6,000-this was relative to the value of the minimum subread length; (iii) the genome size was set to 24,000,000 and (iv) the target coverage parameter was increased to 30 from a default of 25 to enhance coverage in the preassembly process (described below), in turn improving the quality of the finished assembly. The first step in RS_HGAP_Assembly.3, i.e. preassembly, utilizes a directed acyclic graph-based consensus procedure to align shorter reads to the longest reads in the sequencing data, thus generating corrected, continuous preassembled reads. 29 Next, the preassembled reads are assembled into larger contigs with the AssembleUnitig algorithm (Pacific Biosciences; https://github. com/PacificBiosciences/SMRT-Analysis/wiki/SMRT-Pipe-Reference-Guide), which incorporates elements of Celera Assembler 36 for overlapping and layout. Finally, the filtered reads are mapped back to the contigs using Blasr, 37 error-corrected with Quiver 29 to generate a high-quality consensus (referred to here as the SMRT assembly), and then visualized using SMRT View 2.3.0 (Pacific Biosciences).
To assemble the $32 kb P. falciparum apicoplast genome, reads that did not align to contigs in the SMRT assembly (which comprised only the nuclear genome) were reanalysed in SMRT Analysis Portal 2.3.0.p2 resulting in an apicoplast contig. For the $6 kb P. falciparum mitochondrial genome, Blasr was used to align the raw sequencing data to the M76611 mitochondrial sequence (http://plasmodb. org/plasmo/showRecord.do?name=SequenceRecordClasses. SequenceRecordClass&project_id=PlasmoDB&primary_key= M76611) and assembly performed with reads that presented partial or full homology to M76611 mitochondrial DNA.
Dot plots comparing the SMRT assembly to the P. falciparum reference genome, Pf3D7_v3.0 (http://genedb.org), were rendered with Gepard 38 using default parameters and a word length of 30. To identify structural variations in the genome, we used our new algorithm Assemblytics. 39 Briefly, Assemblytics analyses the whole genome alignment computed by MUMmer 40 and applies a unique length filtering approach to robustly identify structural variations in six classes of variants-insertions, deletions, tandem expansions, tandem contractions, repeat expansions and repeat contractions. Based on the size of the P. falciparum genome, the 'minimum size of variant' was set to 2 bp and the 'minimum unique sequence length to anchor an alignment' was set to 10 kb, which also becomes the maximum variant size to avoid calling variants above the size of the uniquely mapped sequence. Next, the presence of poly-A, poly-T and poly-AT in the smaller insertions (less than 10 bp) was determined by the following rules: (i) insertion must only contain the repeat sequence (for instance ATA for poly-AT) and (ii) either the 10 bp on the left or the 10 bp on the right must contain at least 6 bp of the repeat (for example, ATATAT in the case of the poly-AT repeat, AAAAAA in the case of poly-A). Finally, to determine whether a variant overlapped with a genomic feature, we performed a left outer join intersect using BEDTools 41 against Pf3D7_v3.0, annotation release 13.0 from plasmodb.org.
Direct alignment of the raw sequencing reads to Pf3D7_v3.0 was carried out using Blasr 37 with default settings. The resulting coverage obtained was 166-fold.

High molecular weight genomic DNA was prepared from different P. falciparum intraerythrocytic stages
Critical to obtaining long-reads with Pacific Biosciences' SMRT sequencing technology is the extraction of high quality, high molecular weight genomic DNA, with a recommended size distribution of 50-150 kb (http://www.pacb.com/wp-content/uploads/2015/09/ User-Bulletin-Guidelines-for-Preparing-20-kb-SMRTbell-Templates. pdf). To achieve this, we prepared genomic DNA from the P. falciparum laboratory strain 3D7 cultured in human blood using two different Qiagen kits: the DNeasy Blood and Tissue kit, which is routinely used by malaria researchers, 35 and the Genomic Tip kit. As shown in Supplementary Fig. S2A, the size distribution of genomic DNA prepared using the DNeasy kit was between 33.5 and 48.5 kb, in contrast to a size distribution of >50 kb for genomic DNA prepared with the Genomic Tip kit. We also determined the output of the Genomic Tip kit for 3D7 parasites synchronized at ring (6-18 h post-invasion; non-replicating), trophozoite (28-35 h post-invasion, when DNA replication is initiated) and schizont stages (38-45 h post-invasion, when DNA replication is complete) and found that 3. 1 Â 10 8 ring stage parasites yielded $2.5 lg of genomic DNA, while >15 lg of genomic DNA could be extracted from 0.8 Â 10 8 schizonts ( Supplementary Fig. S2B). Extrapolating from these results, we conclude that >10 lg of high molecular weight genomic DNA can be extracted from a P. falciparum schizont culture growing in 500 ll of blood at a parasitaemia of $2% with Qiagen's Genomic Tip kit, which is sufficient for downstream library preparation, size-selection and sequencing.
3.2. SMRT sequencing of P. falciparum genomic DNA yielded reads of average length $12 kb Genomic DNA prepared from blood stage P. falciparum parasites is routinely contaminated with heme and its derivatives. Because these contaminants adversely affect downstream analyses such as PCR and sequencing, [42][43][44] we examined the efficiency of three different purification methods to clean up P. falciparum genomic DNA isolated from an asynchronous 3D7 culture, prior to SMRTbell library preparation ( Supplementary Fig. S1). These included: (i) magnetic bead-based cleanup, using a 1:1 ratio of AMPure PB beads, (ii) electrophoretic DNA extraction using the Aurora System from Boreal Genomics and (iii) phenol-chloroform extraction. We found that all three purification methods efficiently removed heme and other contaminants and did not affect the size distribution of the genomic DNA ( Fig. 1A; 'no shear'). Notably, the magnetic bead-based purification method demonstrated the highest per cent DNA recovery values (54% versus 33 and 36% for phenol-chloroform extraction and Aurora System clean up, respectively) (Fig. 1B). Subsequently, we prepared sequencing libraries and observed that DNA purification method did not impact shearing ( Fig. 1A; 'shear'), SMRTbell library yield (Fig. 1B) and BluePippin 20 kb size-selection (Figs 1B and C), and efficiently generated size-selected SMRTbell libraries with average insert lengths of $21 kb (Figs 1B and C). Of note, the SMRTbell libraries prepared from phenol-chloroform extracted DNA showed the highest recovery (44%) after size-selection (Fig. 1B).
Thereafter, we used the Pacific Biosciences P6 polymerase, C4 sequencing chemistry and the RS II Sequencing System to analyse the size-selected P. falciparum SMRTbell libraries (Supplementary Fig.  S1). For libraries derived from each purification method, we ran three SMRT cells for 4 h, resulting in 525,996 raw sequencing reads that totalled 5.26 Gb from nine pooled SMRT cells ( Fig. 2A). SMRTbell libraries derived from phenol-chloroform extracted DNA provided greater sequencing yields at lower loading requirements, although average read lengths were slightly shorter (not statistically significant) than libraries generated from other purification methods. On average, 58,444 reads were generated per SMRT cell, with a read length of 12.1 kb (Fig. 2A), and a tail in the read length distribution reaching close to 50 kb (Fig. 2B). Moreover, 50% of the sequenced bases originated from reads that were !15.5 kb long ( Fig.  2A; read N50), suggesting that the Pacific Biosciences P6 polymerase may be capable of sequencing through long stretches of highly ATrich genomic content, confirming prior reports of little to no sequence-context bias of SMRT sequencing. 13 Table S1). Thirteen of the 21 contigs were complete, individual P. falciparum chromosomes (chr. [2][3][4][5][6][7][8][9][10][11][12][13][14] ranging in length from 0.943 to 3.286 Mb (Fig. 3B), while the remaining eight contigs represented a 620 kb portion of chromosome 1 (contig 20) and 20-60 kb repeat regions that aligned to chromosome ends. One of these repeats corresponded to the left arm of chromosome 1 (contig 64; discussed in detail below), but the remaining six contigs could not be correctly assigned to a single chromosome end (Supplementary Table S1; Supplementary Figs. S3P-U). We consider these to be spurious contigs that may have arisen from high coverage of repetitive regions and do not have any discernible protein-coding or non-coding RNA (ncRNA) features.
Each of the 13 fully resolved chromosomes featured distinct edges at either end, where coverage abruptly stopped and beyond which reads did not extend ( Fig. 3B and Supplementary Fig. S3A-M). These ends are highly homologous to each other, both within the same chromosome and between different chromosomes, and contain several copies of the telomeric repetitive element CCCTNAA, indicating complete telomere-to-telomere assembly of chr. 2-14. A representative coverage plot for contig 12, i.e. chr. 7, is shown (Fig. 3C). In the case of chr. 1, we observed that the 620 kb contig 20 began with a gradual increase in coverage (left end) and ended with a distinct edge containing telomeric repeats (right end) ( Fig. 3B and Supplementary Fig. S3O). We, therefore, searched for a contig that could be contiguous with contig 20 and identified the 37 kb contig 64 ( Fig. 3B and Supplementary Fig. S3N), which had a distinct telomeric repeat-containing edge at its left end and a gradual drop off in coverage at its right end. The manual joining of these two contigs yielded full-length chr. 1. Finally, because the genomes of the mitochondria [$6 kb; 68.4% (A þ T) content] and apicoplast [$32 kb; 85.8% (A þ T) content] were absent from the SMRT assembly, we selected reads that did not align to the nuclear chromosomes from the raw sequencing data and reanalysed them with independent HGAP calculations (detailed in Materials and Methods). In doing so, we completely assembled the genomes of these key organelles ( Supplementary Figs S3W and S3X). Of note, the mitochondrial genome was assembled as a $38 kb contig ( Supplementary Fig. S3W), indicating that it exists as circular tandem duplications of itself: in this case, as six copies of the 6 kb genome.
We compared our SMRT assembly to other de novo assemblies of the P. falciparum genome that were generated using data from Roche 454 pyrosequencing, 45 Sanger shotgun sequencing 46 or Illumina-based sequencing by synthesis 47 of different P. falciparum strains (Table 1). We found that our assembly was the most complete: it comprised 23.6 Mb and contained the least number of contigs, with an average contig N50 of 1.71 Mb. Moreover, in comparison to previous studies that performed SMRT sequencing of P. falciparum genomic DNA, 13,14,31 we generated over 10-fold longer reads with an average genome coverage of Â 94; indeed, we believe that our HGAP-derived SMRT assembly is the first of its kind for P. falciparum.

Analysis of centromeres and subtelomeric regions
Given the telomere-to-telomere nature of the SMRT assembly, we were specifically interested in the coverage of two challenging P. falciparum genomic regions: (i) centromeres, which have elevated AT-richness [between 90 and 99% (A þ T) content] 8,48,49 and (ii) subtelomeric regions, which contain six varieties of telomereassociated repeats, multigene families such as var, rifin and stevor, 8 and where much of the chromosomal length variation occurs. 50 We observed that in the case of all centromeres, depth of coverage was Â80-140 (Supplementary Table S1 and Supplementary Fig. S3-see the green line that indicates centromere position): for example, the region of chr. 14 that is marked by the centromeric histone PfCenH3, PF3D7_14:1070851-1075311, 48 was sequenced with a coverage of $Â140, as was the core $2.5 kb centromere (Fig. 4A). This indicated that the highly AT-rich nature of centromeres, as well as the presence of atypical repeats such as AATTAA, 48 did not impede the processivity of the polymerase during sequencing. Similarly, we observed even coverage of most telomeric and subtelomeric ends (Â80-140; Fig. 4B and Supplementary Fig. S3), suggesting that the >11 kb length of the SMRT reads is sufficient to differentiate between regions of very high homology.

Novel genomic features and structural variants were resolved in the SMRT assembly
To determine if this new SMRT assembly could enhance our understanding of parasite genome organization, we compared our data to the reference 3D7 genome 8 and noted that the total size of the chromosomal contigs in the SMRT assembly, i.e. 23,294,534 bp, was comparable to the 23,292,622 bp resolved in the reference genome (Supplementary Table S1). Moreover, the sizes of most chromosomes were similar between the two assemblies with the maximum increase in size presented by the stitched SMRT chr. 1 at 3% and the maximum decrease in size presented by chr. 8 at 1.2% (Supplementary  Table S1). When we visualized the comparison using dot plots, as shown in the representative dot plot for chr. 7 (Fig. 5A) and other chromosomes ( Supplementary Fig. S4), we found that while the majority of assembled sequences aligned well with the reference genome, the increase in chr. 1 size in the SMRT assembly was due to the lengthening of its left arm ( Supplementary Fig. S4A), and the decrease in chr. 8 size was due to a shortening of its right arm ( Supplementary Fig. S4H). Upon closer analysis, we concluded that both of these size discrepancies originated from changes in the lengths of subtelomeric repeat sequences.
Furthermore, in select cases, the longer reads generated by SMRT sequencing allowed for the resolution of genomic features that were not previously described for the reference. These included large duplications and insertions (Figs 5B-D). For example, at position 1,612,060 of chromosome 10, a 14 kb duplication was apparent in the SMRT assembly relative to the reference (Fig. 5B), which results in the addition of three more rifin genes to the P. falciparum genome; PfRifins are virulence molecules that are expressed on the surface of infected erythrocytes, mediate the binding of infected erythrocytes to the vasculature 51 and undergo antigenic variation. 52 Another example is position 939,044 of chromosome 4, where a 29 kb insertion relative to the reference results in the duplication of the following features to the P. falciparum genome: three var genes, one ncRNAencoding open reading frame (ORF), one rif and one gene encoding a conserved protein of unknown function (Fig. 5C). However, this region shows a substantial pileup of extra coverage, indicative of highly repetitive sequences, and may contain additional features that were not fully resolved in the SMRT assembly. One way to address this would be to analyse 40 kb size-selected SMRTbell libraries of P. falciparum DNA. Furthermore, adjacent to this insertion, we Contig N50 is a weighted median statistic such that 50% of the entire assembly is contained in contigs or scaffolds equal to or larger than this value, and QV50 indicates an estimated accuracy of 99.999% for the assembly. (B) All 14 P. falciparum chromosomes were assembled end-to-end using HGAP, without any gaps. The only exception was chr. 1, whose left arm was assembled as separate contig of 0.35 kb. The contig name in the SMRT assembly, its size, the corresponding chromosome and its coverage are indicated. The scale of coverage is between Â0 (blue arrowhead) and 150 (red arrowhead) except for contig 64, where the scale is from 0 to 100. The centromere position in each contig is indicated with the green line. (C) Contig 12 of the SMRT assembly, which aligns to P. falciparum chr. 7, was completely assembled using HGAP and showed a depth of coverage between Â70 and Â140. observed a 1.5 kb stretch of DNA that was novel compared to the corresponding 2.5 kb stretch in the reference (Fig. 5C; 'zoomnovel') indicating that this subtelomeric region of chromosome 4 may be more complex than previously annotated. As a third example, we detected a 16 kb duplication at position 1,707,528 of chromosome 12 of the SMRT assembly (Fig. 5D), which results in the addition of 2 var genes and one ncRNA-encoding ORF to the P. falciparum annotation.
In addition to the features described above that span several kilobases (>10 kb), we utilized our assembly comparison tool called Assemblytics 39 to identify additional structural variants in our SMRT assembly relative to the reference genome. As summarized in    Figs 6A and B. Interestingly, homopolymer tract expansions, i.e. poly-A, poly-T and poly-AT expansions, of < 10 bp in size account for $61% of all insertions identified (Table 2 and  Supplementary Table S3 Table S2). These changes affect genic, intergenic and subtelomeric regions of chromosomes: for example, the 117 bp deletion at position 754,796 of chromosome 13 is within the intronless gene PF3D7_1318300, which encodes a conserved Plasmodium  protein of unknown function, while the 280 bp deletion in chr. 9 affects an intergenic region. Similarly, the 4,131 bp tandem expansion at position 1,543,910 of chr. 10 is within the intronless gene PF3D7_1038400, which encodes the gametocyte-specific protein Pf11-1, while the 378 bp tandem contraction in chr. 4 is in a subtelomeric region. Therefore, these variants will need to be rigorously curated to understand their impact on parasite biology. Given that the SMRT assembly and the reference match well in contiguity and have nearly the same total sequence length ( Fig. 6C and Supplementary Table S1), and given its high-quality score ( Fig. 3A; QV50), we can conclude that our SMRT assembly and the reference genome are of equal quality and that the structural variants identified here are significant to understanding the complexity of the parasite genome.

Discussion
In malaria-endemic areas, P. falciparum parasite genomes are constantly in flux. 16,53 However, our knowledge of P. falciparum genomic variability, which is built on PCR-based genotyping, microarray analyses and short-read NGS, is largely restricted to SNPs. 16 To characterize other genomic variants, particularly those that occur in Figure 6. The majority of variations in the SMRT assembly relative to the reference are small insertions. Variants ranging from 2 to 10 kb in size were called using Assemblytics. 39  multigene virulence families and regulatory regions characterized by low sequence complexity such as subtelomeric repeats, there is an urgent need to apply new methods to analyse the P. falciparum genome. Therefore, we adapted the amplification-free, single molecule, long-read sequencing technology developed by Pacific Biosciences to analyse P. falciparum genomic DNA and compiled a new HGAPderived 23.6 Mb genomic assembly that comprised 14 contiguous end-to-end chromosome sequences. In particular, we accurately resolved repetitive and polymorphic chromosome ends and identified large duplication and insertion events in subtelomeric regions and intra-chromosomal regions (adjacent to coding sequences) with high confidence, suggesting a greater complexity of the P. falciparum genome. In addition, we identified smaller insertions, deletions and tandem expansions in coding and non-coding regions of the genome, all of which can be used to correct and complete the P. falciparum reference genome 8 . It is noteworthy that these variants would have been missed if we had directly aligned the SMRT reads to the reference genome, thereby strongly supporting a hypothesis-free de novo assembly approach, not bound by reference bias.
In the past five yrs, the adaptation of NGS to study P. falciparum clinical isolates in Africa and South-East Asia has provided genomewide estimates of allele frequency distribution, population structure and linkage disequilibrium in malaria-endemic regions. 18,20 In addition, NGS analysis of the genetic architecture of drug-resistant subpopulations of P. falciparum in Cambodia has identified SNPs that show high levels of differentiation, and other genomic correlates of the spread of resistance. 19 Complementarily, longitudinal studies that measured the genomic plasticity of laboratory-adapted P. falciparum parasites have determined the rates at which var gene polymorphisms are generated during intraerythrocytic development. 3,4 However, all of these studies used PCR-based WGA combined with short-read NGS and relied on the alignment-based mapping of sequencing reads to the reference 3D7 genome 8 to assess genetic diversity. While this method will prove invaluable to analysing SNPs in clinical settings where researchers mostly dispose of low sample volumes, we believe that the read length limitations of these approaches will make the identification of other critical structural variants extremely difficult. Given the lack of sequence bias in SMRT sequencing, 24 length of SMRT reads (>11 kb; compared to the $8 kb size of a var ORF) and our complete resolution of chromosomal ends and copy number variants of virulence genes such as var and rifin, we argue that long-read sequencing will provide complete reference-grade genomes for P. falciparum field isolates. Such analyses, in combination with other approaches to document SNP variation, will comprehensively determine the geographical diversity of P. falciparum populations and monitor longitudinal population changes of both P. falciparum and other human malaria species.
We recognize that one limitation of our whole genome sequencing approach may be the amount of starting material ($10 lg) that is necessary to prepare high quality, size-selected SMRT sequencing libraries for de novo assembly. For example, if a malaria patient presents with a parasitaemia of >3% rings, around 4 ml of blood will be required to yield $10 lg of P. falciparum genomic DNA, after eliminating human genomic DNA contamination. 54 In contrast, for cases with lower parasitaemia, $200-500 ll of patient blood will have to be frozen and recultured with fresh WBC-free blood for one or two generations, or until the cultures reach a parasitaemia of 2% schizonts, before being processed for DNA extraction. Nonetheless, because several studies have shown that clinical isolates can be cultured in vitro from frozen stocks for short periods of time without losing their multiplication and erythrocyte invasion properties, 55,56 we believe that the latter option is feasible to compile SMRT sequencingbased whole genomes of P. falciparum clinical isolates. Furthermore, as sequencing technologies progress and develop, we anticipate that system requirements will change rapidly, consequently leading to a reduction in initial sample input, cost and time to project completion.
In conclusion, our study emphasizes the value of long-read sequencing technologies and de novo genome assembly to fully resolve pathogen genome architecture and complexity, paving the way for comprehensively assessing genetic variation at all size scales. In the specific case of Plasmodium, not only will it provide information about within-host P. falciparum diversity, but it will fill existing gaps in the genome sequences of laboratory-adapted strains as well as of field isolates. Furthermore, the resolution and understanding of P. falciparum structural variants, in particular of multigene families involved in adhesion and immune evasion (Ex: PfEMP1 and PfRifin), and genes involved in erythrocyte invasion (vaccine candidates such as PfAMA1 and PfRh proteins) and sexual stage biology (vaccine candidates such as P48/45, P25 and P28), will provide genetic correlates of parasite virulence and transmissibility. Given the recent assembly of the haploid human genome using SMRT sequencing, 28 we propose that long-read sequencing technologies will be crucial to combining Plasmodium genomic epidemiology with human population genetics to obtain a comprehensive view of parasite behaviour, genetic predisposition of humans to malaria and the co-evolution of parasite and host at a molecular level. Finally, SMRT technology can be used to identify epigenomic variations (Ex: genome-wide DNA methylation patterns) based on the kinetics of the DNA synthesis reaction performed by the polymerase, concurrent with sequencing. 57 Because this method is being utilized to examine both bacterial and eukaryotic epigenomes 58-60 and given the tractable size of the P. falciparum genome, future work could investigate if dynamic changes in DNA methylation 61 of P. falciparum clinical isolates are linked to varying degrees of severe malaria.