A new method for sequencing the hypervariable Plasmodium falciparum gene var2csa from clinical samples
© The Author(s) 2017
Received: 1 April 2017
Accepted: 4 August 2017
Published: 17 August 2017
VAR2CSA, a member of the Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family, mediates the binding of P. falciparum-infected erythrocytes to chondroitin sulfate A, a surface-associated molecule expressed in placental cells, and plays a central role in the pathogenesis of placental malaria. VAR2CSA is a target of naturally acquired immunity and, as such, is a leading vaccine candidate against placental malaria. This protein is very polymorphic and technically challenging to sequence. Published var2csa sequences, mostly limited to specific domains, have been generated through the sequencing of cloned PCR amplicons using capillary electrophoresis, a method that is both time consuming and costly, and that performs poorly when applied to clinical samples that are commonly polyclonal. A next-generation sequencing platform, Pacific Biosciences (PacBio), offers an alternative approach to overcome these issues.
PCR primers were designed that target a 5 kb segment in the 5′ end of var2csa and the resulting amplicons were sequenced using PacBio sequencing. The primers were optimized using two laboratory strains and were validated on DNA from 43 clinical samples, extracted from dried blood spots on filter paper or from cryopreserved P. falciparum-infected erythrocytes. Sequence reads were assembled using the SMRT-analysis ConsensusTools module.
Here, a PacBio sequencing-based approach for recovering a segment encoding the majority of VAR2CSA’s extracellular region is described; this segment includes the totality of the first four domains in the 5′ end of var2csa (~5 kb), from clinical malaria samples. The feasibility of the method is demonstrated, showing a high success rate from cryopreserved samples and more limited success from dried blood spots stored at room temperature, and characterized the genetic variation of the var2csa locus.
This method will facilitate a detailed analysis of var2csa genetic variation and can be adapted to sequence other hypervariable P. falciparum genes.
Placental malaria (PM) is characterized by the massive accumulation of Plasmodium falciparum-infected erythrocytes in the placental intervillous space. The sequestration of infected erythrocytes in host tissue, such as the placenta, is mediated by adhesin proteins in the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family, which are encoded by the highly diverse var gene family . PfEMP1s are a complex family of proteins ranging from 300 to 350 kDa that are composed of two to nine Duffy binding-like (DBL) domains and one to two cysteine-rich interdomain regions (CIDR) [2, 3]. Parasites sequester in many different organs, including the brain, heart, lungs, liver, and placenta (reviewed in ). Plasmodium falciparum-infected erythrocytes collected from placentas display a unique cyto-adherence phenotype , and they bind to glycosaminoglycan chondroitin-4-sulfate (CSA) expressed in the placenta, but not to endothelial receptors such as CD36 or ICAM-1 [6, 7]. The ligand mediating this interaction with CSA is a PfEMP1 protein known as VAR2CSA . VAR2CSA is a target of naturally acquired immunity [9–12], making it an attractive vaccine candidate against placental malaria.
Sequencing var2csa from clinical isolates can provide a framework for VAR2CSA-based vaccine development, but var2csa’s genetic complexity and large size have limited its successful sequencing from field samples. Capillary sequencing, also known as Sanger sequencing, has been used to sequence a limited number of full-length var2csa genes from clinical isolates [2, 25]. Most sequences have been generated using Sanger sequencing and span only single, specific var2csa domains [25–27], even though there is no consensus on which domain(s) is important for a sub-unit vaccine. This method requires a priori knowledge of the sequence of primer-binding regions, limiting the number of variants that can be obtained and analysed. It is also time consuming, as it requires cloning of PCR products prior to sequencing. In the case of polyclonal infections, which are common in clinical isolates, this method is even less efficient and may lead to the generation of chimeric sequences. Ilumina next-generation sequencing has been used to generate whole genome sequence data of P. falciparum. However, the difficulty of assembling var genes from short read next-generation whole genome sequence data, such as those generated with Illumina sequencing technologies, has resulted in limited success in generating full-length, or even large fragments of, var2csa. Therefore, alternative ways to sequence full-length var2csa, or a large critical fragment (spanning several domains) of var2csa, from clinical specimens are needed.
A next-generation sequencing platform from Pacific Biosciences (PacBio) has the potential to generate complete var2csa sequences due to its ability to generate long reads. PacBio sequencing is also appealing because it is high-throughput, as samples can be multiplexed. More importantly, the assembly does not require a reference sequence making it suitable for a highly polymorphic gene such as var2csa. Targeted PacBio sequencing has been used to sequence complex loci in humans , as well as in P. falciparum . This approach was modified here to develop a new sequencing assay for var2csa, which combines long-range PCR with PacBio sequencing and assembly. This amplicon sequencing of the var2csa N-terminal region captures approximately half of the full-length var2csa, including ID1-ID2a, the primary focus of the current VAR2CSA-based phase I vaccine trial. The results of this novel assay are reported, together with an evaluation of the genetic diversity of this segment of VAR2CSA. The methods described can be adapted to other hypervariable genes or gene families.
Sites and samples
As part of International Centers of Excellence for Malaria Research (ICEMR) and malaria-in-pregnancy (MIP) studies, samples were collected from children, men and women in Ndirande, a peri-urban township of Blantyre, in Malawi, where malaria transmission occurs throughout the year with a seasonal peak during a rainy season from December to March. Forty-three specimens, including 19 blood-spotted Whatman 3MM filter papers that had been stored at room temperature and 24 cryopreserved parasites, were randomly selected. Genomic DNA was extracted using a Qiagen Midi Kit according to manufacturer’s instructions. Four positive controls were used: 3D7, HB3, and synthetic mixtures of 70% 3D7 plus 30% HB3 and vice versa. 3D7 and HB3 each had an initial concentration of 1 ng/µl.
Primer design and sequencing template preparation
Primers that flank the region spanning the var2csa upstream promoter (position 500 bp upstream of start codon) to the DBLepam4 domain were designed using 20 aligned var2csa sequences from public databases (GenBank and VarDom) along with 12 var2csa upstream promoter (UPSE) sequences obtained previously . Each secondary PCR primer was labelled using PacBio barcode sequences (listed in Additional file 1: Table S1) to identify sequences from individual samples. High-fidelity Takara LA Taq® Polymerase (TAKARA BIO INC, Shiga, Japan) was used for the PCR with conditions listed in Additional file 1: Tables S2 and S3. An expected secondary PCR product of 5364 bp was resolved on 0.8% agarose gel and visualized using Gel Doc XR+ System (Bio-Rad, Hercules, CA, USA). Successfully amplified samples were purified using a MultiScreen filter plate (MultiScreen, Tullagreen, Germany). DNA concentration was measured using a Quant-iT PicoGreen dsDNA assay (Life Technologies, Carlsbad, CA, USA) according to manufacturer’s instructions. Subsequently, 48 equimolar barcoded amplicons were pooled to a final total of 2 µg of DNA.
Purified, pooled amplicons were sequenced using Single Molecule Real Time (SMRT) technology on a PacBio RS II sequencer (Menlo Park, Pacific Biosciences, California, USA), at the Genomics Resource Center of the Institute for Genome Sciences, University of Maryland School of Medicine, USA. Briefly, libraries were constructed by ligating SMRTbellTM adapters to the barcoded-pooled amplicon template. One SMRT™ cell with P4-C2 chemistry (P4 polymerase with C2 sequencing chemistry) was used. A 180-min movie was performed on PacBio RS II to generate the reads.
The secondary analysis was performed with the PacBio SMRT Analysis v2.3.0 package, using the long amplicon analysis (LAA) algorithm with the following options—minLength 3250—minSnr 4. This algorithm enables read clustering, phasing and consensus calling, and consensus filtering. The pooled amplicon data were de-multiplexed by barcode. The predicted accuracy of 95%, which is defined as the threshold below which a haplotype (consensus sequence) is considered as noise, was used to filter high-quality consensus sequences for downstream analysis.
To determine the sequence accuracy of the positive controls, amplicon consensus sequences were aligned to coding sequences of the respective strains from which the amplicons were generated. The corresponding test coding sequences were extracted and the phylogenetic tree was built using the Maximum Likelihood method implemented in MEGA 6.0 .
Sequences from the same sample that shared 99% identity at the nucleotide level were clustered using CD-HIT . This conservative threshold was chosen assuming that the residual sequencing error was 1% based on data from the resequenced positive controls. Sequences with depth of coverage equal to or greater than 100× were considered for downstream analysis. This 100× threshold was chosen because this was the minimum depth of coverage at which a perfect consensus sequence of the reference 3D7 was obtained. Then the amplicon sequences were fed into OrfPredictor  to predict open reading frames, and corresponding amino acid sequences were generated. Amino acid sequences were used to annotate constituent domains using the VarDom 1.0 server . Multiple alignments were performed using MAFFT with L-INS-i accuracy-oriented options. The region in the multiple alignments corresponding to the FCR3 variant of ID1-ID2a was extracted (1158–3075 corresponding to amino acid position N386 to D1025).
For the genetic diversity analysis, sequences corresponding to the coding regions were extracted from the full-length amplicon. Likely frameshift errors were corrected with AlignWise . Sequences were then aligned with MAFFT with the L-iNSi option. DnaSP v5  was used to compute population genetic parameters, including nucleotide diversity, which is the average number of nucleotide differences per site between any two given sequences; haplotype diversity, which is the probability that two randomly sampled alleles are different; and Tajima’s D neutrality test, which is used to infer the selective forces acting on a locus based on the difference between the number of segregating sites and the average number of nucleotide differences.
Robustness of the amplification protocol on clinical samples
The amplification protocol was developed using genomic DNA from two laboratory parasite reference strains, 3D7 and HB3. The primers are designed to bind to the VAR2CSA-specific upstream promoter, UPSE, and to the DBLepam4 domain, to amplify a segment close to 5 kb in length (Fig. 1). This primer combination readily amplified var2csa from both of these lines. To test the robustness of the protocol, it was applied to clinical samples including DNA extracted from blood-spotted filter papers and from cryopreserved specimens obtained from 81 clinical malaria samples from Malawi. A long PCR amplicon of the expected size was obtained from both of these sample types. The PCR was successful for all cryopreserved specimens (24/24), whereas only 33% (19/57) of the tested filter samples yielded visible PCR products by gel electrophoresis. Therefore, the starting combined pool consisted of 43 amplicons, in addition to the four positive controls from 3D7 and HB3 (described in “Methods” section).
PacBio-generated long sequence reads
Validation of var2csa reconstruction protocol
Comparison between reconstructed PacBio sequences and the respective reference var2csa alleles in 3D7 and HB3
Reference var2csa gene
Sequence identity (%)
Deletion length (bp)
Simulated 3D7 amplicon assembly at different coverage levels
Number of INDELs
Sequence identity (%)
Application of the sequencing method to clinical samples
The parameter settings used for sub-read assembly, optimized during the reconstruction of the reference 3D7 allele, were then applied to the clinical samples. After filtering, read clustering, and chimeric sequence removal, 135 consensus sequences were generated from the 43 samples. After collapsing nearly identical sequences (see “Methods”) from the same sample using a threshold for minimum nucleotide sequence identity of 99%, which is a cut-off based on the re-sequenced positive control (Table 1), the number of sequences was reduced to 99. A subsequent filter, using a minimum coverage threshold of 100-fold, yielded 51 sequences from 40 samples, with a mean coverage of 280-fold (100–500) (Additional file 2: Figure S2). All samples but one yielded one or more unique amplicon sequences (Additional file 3).
To further evaluate the specificity of the new protocol, in particularly whether in fact var2csa sequences were amplified, the translated sequences were annotated using the VarDom 1.0 server , which uses a Hidden Markov Model to identify PfEMP1 protein domains. var2csa-specific homology blocks (HB) and domains (DBLpam1, DBLpam2, CIDRpam, and DBLpam3) were detected in all samples with a single, intact open reading frame (30 sequences with length ≥1400 amino acids). The remaining 21 amplicons were also var2csa sequences, validated as such using blast searches against NCBI’s non-redundant sequence database, with frameshift mutations that resulted in in-frame stop codons. These results validate our approach to generating var2csa sequences (Additional file 2: Figure S3).
Genetic diversity of var2csa
Genetic variation across the whole segment and in individual domains
Haplotype diversity (Hd)
Nucleotide diversity (π)
Tajima’s D p value
To better understand the evolutionary forces that shape the genetic variation of var2csa, we estimated the distribution of two different statistics across the this segment, namely the ratio π N/π S, which measures the ratio of non-synonymous to synonymous mutations per site, with expected value of zero when the types of mutations are randomly distributed; and Tajima’s D statistic, which assesses the evolutionary forces operating on a locus, with expected value of zero under neutral evolution. A π N/π S ratio greater than 1 is suggestive of positive selection, whereas a ratio of less than 1 suggests purifying selection. On antigenic loci, positive values of Tajima’s D are suggestive of balancing selection, while negative values are likely due to purifying selection. None of the statistics showed a statistically significant departure from neutrality either across the whole 5 kb segment or on individual domains (Table 3). The overall ratio π N/π S was 0.89, indicating a slight excess of synonymous over non-synonymous polymorphism.
To determine whether specific short regions of the gene show evidence of positive selection, these two statistics were estimated using a sliding window analysis, with a window size of 99 nucleotides and a sliding step size of 30 nucleotides (Fig. 4). The Tajima’s D analysis identified some regions where the distribution of genetic variation departed from neutrality (p < 0.05), corresponding to positions 666–764 in the DBLpam1 domain; 3563–3694 in the CIDRpam domain; and 3866–3964 and 4391–4573 in the DBLpam3 domain (Fig. 4a). One striking π N/π S peak was present, which mapped to DBLpam3 (alignment positions 4041–4139 with πN/πS ratio of 6.47) but with relatively low overall π, suggesting that whatever polymorphism there is in this region is non-synonymous. Two intermediate peaks were also observed, which mapped to the C-terminal regions of the CIDRpam domain (alignment positions 3246–3344 with πN/πS of 2.73) and the DBLpam3 domain (4707–4856 with πN/πS ratio of 3.92), each with a significantly high value of Tajima’s D, suggesting that balancing selection is acting at these regions (Fig. 4). Taken together, these results showed that the N-terminal of var2csa is highly polymorphic. To put var2csa genetic diversity in context, its nucleotide diversity is ten times higher than that in the P. falciparum apical membrane antigen 1 (AMA1), which itself is one of the most diverse loci in P. falciparum . The high values of Tajima’s D in the DBLpam1, the CIDRpam and the DBLpam3 domains of the protein are consistent with balancing selection. These findings are in line with those from other studies that found that variable blocks in DBLpam1, DBLpam2 and DBLpam3 are evolving under positive selection [26, 36, 37]. Taken together, these analyses suggest that host immune pressure drives the diversity in these segments, likely under a selective regimen of frequency-dependent selection that favours rare alleles.
The minimal CSA-binding region, a current vaccine target, is highly diverse
Sequence diversity of the CSA binding region, located between coordinates 1185–3297 in Fig. 4, was characterized since this region is a target of the current VAR2CSA sub-unit vaccine . The overall amino acid sequence identity was 74% and the average nucleotide diversity was 14.6% (Table 3), with a proportion of segregating sites of 53%. The nucleotide diversity in this region is higher than any of the values calculated for individual VAR2CSA domains.
A better understanding of the genetic variation of VAR2CSA is critical for development of a broadly effective vaccine against placental malaria. However, characterization of its encoding gene from clinical samples has had limited success due to difficulties in amplifying and assembling this hypervariable member of a multigene family from malaria infections, with issues compounded in polyclonal infections. A novel sequencing approach was developed using long range PCR combined with long PacBio sequencing of the resulting amplicon spanning ~5 kb of the extracellular region of var2csa. This strategy obviated the need for cloning and allowed us to generate sequences of large fragments of var2csa, capturing the majority of the sequence encoding the extracellular region of the var2csa locus. This approach was validated using published sequences from laboratory parasite lines. As proof-of-concept, this approach was applied to two types of field samples, albeit with different success rate.
In this study, two well-characterized laboratory strains were used to optimize the amplification and assembly protocols. 3D7 has one allele of var2csa, whereas HB3 harbors two alleles. The PacBio re-sequenced 3D7 var2csa allele, assembled from reads with high depth of coverage, is identical to the reference strain. On the other hand, the HB3 var2csa reconstructed alleles harbored some residual sequencing errors. All of the errors observed, which were associated with low coverage, were deletions in homopolymeric regions. These findings are in line with those from a previous study that used PacBio targeted sequencing for bacterial 16S rRNA  and 454 pyrosequencing amplicon sequencing in Plasmodium .
Blood-spotted dried filter papers are widely used for molecular assays in epidemiological studies, which can be easily collected and stored during clinical studies. Promisingly, a 5 kb fragment from DNA isolated from such filter papers was often amplified and sequenced, even though the amplification success rate was lower than that of DNA obtained from cryopreserved samples. The limited success of the primers on filter papers could be due to degraded and/or limited amounts of DNA. Further efforts to optimize the DNA extraction and amplification methods from dried blood spot samples are ongoing. Meanwhile, current results suggest that, for any studies that rely on the generation of long PCR amplicons from DNA extracted from filter paper, this type of sample should be kept frozen, or at minimum refrigerated, in order to prevent DNA degradation.
In field settings, multiple infections are common and may undermine the ability of the traditional Sanger platform to sequence clinical specimens directly, without cloning. To examine whether polyclonality poses a challenge for a combined approach of PCR amplification and PacBio sequencing, we artificially created a mixture of laboratory strains. This approach successfully reconstructed alleles present at lower frequency (~23%) in the mixture. However, these minor alleles had lower read coverage and, therefore, slightly more errors. Therefore, current results suggest that in order to obtain highly accurate allelic sequences, additional sequencing may be required when initial consensus sequences are inferred from low coverage levels. However, as mentioned above, all errors observed were deletions in homopolymer stretches. This new method offers a clear advantage over traditional cloning approaches, as it allowed generation of long reads, highly accurate sequences, and was suitable for clinical samples. More importantly, as the protocol uses de novo assembly of full-length amplicons to reconstruct var2csa, it allows the accurate reconstruction of var2csa sequences from polyclonal clinical specimens.
While this method shows a potential value of PacBio sequencing of amplicons for complex genes, this approach has some limitations. The results reported here were generated from only one SMRT cell, thus limiting depth of coverage and potentially precluding obtaining high-quality sequences for minor alleles, unless funding is available for additional sequencing. Although PacBio sequencing has some residual noise at low depth of coverage, it is anticipated that increasing the number of SMRT cells or decreasing the number of samples to multiplex, and the use of the upgraded P6-C4 chemistry, will improve the outcome of this approach. The sequencing protocol presented here could serve as a potential tool to study var2csa. The approach will serve as a framework to characterize the extent of the genetic diversity in different regions of VAR2CSA, information that can then be used to inform rational vaccine design. For instance, this protocol can be used to identify the most prevalent variants to be included in a vaccine that takes into consideration sequence diversity or to identify variants or motifs that are likely to have tropism for receptors overrepresented in placental tissue.
A new high-throughput method has been developed that uses a combination of long amplicon generation and single molecule sequencing for the locus encoding VAR2CSA. Results show that this method is robust in sequencing var2csa, and that the VAR2CSA N-terminal region, including the receptor-binding region, is highly diverse in Malawian samples. The method described here can be an effective approach to study highly polymorphic and complex malaria antigens that have historically been challenging to sequence with traditional platforms.
AD designed and performed the experiments, analysed the data, and wrote the manuscript. MA contributed to the experimental design, and revising the manuscript. SSD contributed to the experimental design. EFD and SA contributed to the bio-informatic analyses. MAT, MKL, CVP, and JCS contributed to the analytical design and writing the manuscript. All authors read and approved the final manuscript.
We thank the study participants in Malawi. We would also like to thank T. Lavstsen, who generously provided the 12 UPSE sequences, and Kalyn Ali, who helped copyedit.
The authors declare that they have no competing interests.
Availability of data and materials
Sequences used in this study are available in Additional file 3.
The University of Malawi College of Medicine Research and Ethics Committee and University of Maryland Institutional Review Board approved the research protocols.
This work was supported by the Howard Hughes Medical Institute and by the Fogarty International Center (Grant Number D43TW001589) and the NIH/NIAID (Contract Number HHSN272200900009C and Grant Number U19AI10820).
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