Targeted genome enrichment for efficient purification of endosymbiont DNA from host DNA

Wolbachia endosymbionts are widespread in arthropods and are generally considered reproductive parasites, inducing various phenotypes including cytoplasmic incompatibility, parthenogenesis, feminization and male killing, which serve to promote their spread through populations. In contrast, Wolbachia infecting filarial nematodes that cause human diseases, including elephantiasis and river blindness, are obligate mutualists. DNA purification methods for efficient genomic sequencing of these unculturable bacteria have proven difficult using a variety of techniques. To efficiently capture endosymbiont DNA for studies that examine the biology of symbiosis, we devised a parallel strategy to an earlier array-based method by creating a set of SureSelect? (Agilent) 120-mer target enrichment RNA oligonucleotides (“baits”) for solution hybrid selection. These were designed from Wolbachia complete and partial genome sequences in GenBank and were tiled across each genomic sequence with 60 bp overlap. Baits were filtered for homology against host genomes containing Wolbachia using BLAT and sequences with significant host homology were removed from the bait pool. Filarial parasite Brugia malayi DNA was used as a test case, as the complete sequence of both Wolbachia and its host are known. DNA eluted from capture was size selected and sequencing samples were prepared using the NEBNext® Sample Preparation Kit. One-third of a 50 nt paired-end sequencing lane on the HiSeq? 2000 (Illumina) yielded 53 million reads and the entirety of the Wolbachia genome was captured. We then used the baits to isolate more than 97.1 % of the genome of a distantly related Wolbachia strain from the crustacean Armadillidium vulgare, demonstrating that the method can be used to enrich target DNA from unculturable microbes over large evolutionary distances.     

Efficacy of magnetic capture in comparison with conventional DNA isolation in a survey of Toxoplasma gondii in wild house mice

Toxoplasma gondii is a zoonotic parasite with a world-wide distribution. House mice (Mus musculus) play an important role as a reservoir host in the parasite life cycle. However, their detection in mouse brain is limited because the host potentially harbours only a few tissue cysts. In order to improve the diagnosis, we tested a novel protocol for T. gondii detection in mice and compared this technique to a standard PCR-based protocol using a commercial kit for DNA isolation. Efficacy of magnetic capture for isolation of T. gondii DNA from whole host brains was tested in brain samples of laboratory mice spiked with 1 up to 10⁴ tachyzoites. Real-time PCR revealed that even 1–5 tachyzoites can be detected after magnetic capture. Also this method is suitable to quantify parasite numbers in mouse brains with more than 10 tachyzoite equivalents. To assess the two techniques in wild mice, we employed a dataset consisting of 243 individuals. The prevalence of T. gondii detected by magnetic capture and qPCR and by commercial isolation and PCR was 1.2% and 0%, respectively. The magnetic capture and quantitative PCR seems to be a highly sensitive and specific diagnostic method for both laboratory research and wild population surveys.     

The identification of a new Giardia duodenalis assemblage in marine vertebrates and a preliminary analysis of G. duodenalis population biology in marine systems

Giardia duodenalis is an intestinal parasite of many vertebrates. The presence of G. duodenalis in the marine environment due to anthropogenic and wildlife activity is well documented, including the contributions from untreated sewage and storm water, agricultural run-off and droppings from terrestrial animals. Recently, studies have detected this protistan parasite in the faeces of marine vertebrates such as whales, dolphins, seals and shore birds. To explore the population biology of G. duodenalis in marine life, we determined the prevalence of G. duodenalis in two species of seal (Halichoerus grypus, Phoca vitulina vitulina and Phoca vitulina richardsi) from the east and west coasts of the USA, sequenced two loci from G. duodenalis-positive samples to assess molecular diversity and examined G. duodenalis distribution amongst these seals and other marine vertebrates along the east coast. We found a significant difference in the presence of G. duodenalis between east and west coast seal species. Only the zoonotic lineages of G. duodenalis, Assemblages A and B and a novel lineage, which we designated as Assemblage H, were identified in marine vertebrates. Assemblages A and B are broadly distributed geographically and show a lack of host specificity. Only grey seal (Halichoerus grypus) samples and one gull sample (Larus argentatus) from a northern location of Cape Cod, Massachusetts, USA, showed the presence of Assemblage H haplotypes; only one other study of harbour seals from the Puget Sound region of Washington, USA previously recorded the presence of an Assemblage H haplotype. Assemblage H sequences form a monophyletic clade that appears as divergent from the other seven Assemblages of G. duodenalis as these assemblages are from each other. The discovery of a previously uncharacterised lineage of G. duodenalis suggests that this parasite has more genetic diversity and perhaps a larger host range than previously believed.     

Prevalence patterns of avian haemosporida on Hispaniola

We used PCR to screen for the presence of haemosporidian parasites (Phylum: Apicomplexa; Order: Haemosporida) in avian blood samples, and sequenced the parasite mitochondrial cytochrome b gene from infected hosts, to study patterns in the prevalence of haemosporidians in 1,166 individuals of 50 species in four habitats along an elevation gradient in the Sierra de Bahoruco, Dominican Republic, island of Hispaniola. We found an overall prevalence of 0.44 among species with ≥10 individuals sampled per year, but this varied considerably among species. We found no difference in infection rates between years, between males and females, between second‐year (<1 y old) and older birds, or among members of different foraging guilds. Prevalence differed significantly among migratory, endemic resident, and non‐endemic resident species, with endemics having the highest rates of infection. Prevalence also varied among habitats, decreasing with increasing elevation, but the pattern was confounded by variation in the host species present at each elevation. From 215 sequenced parasites from 17 species of avian hosts, we recovered multiple examples of 12 lineages of Haemoproteus (Parahaemoproteus), two lineages of a Columbiformes‐specific clade of H. (Haemoproteus), and 10 lineages of Plasmodium, with an additional seven lineages sampled only once. A single parasite lineage was responsible for 34.4% of all infections, but five more lineages made up 41.8% of all infections. Several lineages were broadly distributed across multiple host species, but six lineages, all H. (Haemoproteus) or H. (Parahaemoproteus), were recorded from at least five individuals of a single host, suggesting host specialization. The number of host species from which each parasite lineage was recovered varied from one to nine; several host species harbored as many as 5–9 parasite lineages. Longitudinal data suggest that while hosts might harbor the same parasite lineage for more than a year, some hosts appear to clear infections from their circulating blood, while others manifested infections by a different parasite lineage.     

Long‐read sequence capture of the haemoglobin gene clusters across codfish species

Combining high‐throughput sequencing with targeted sequence capture has become an attractive tool to study specific genomic regions of interest. Most studies have so far focused on the exome using short‐read technology. These approaches are not designed to capture intergenic regions needed to reconstruct genomic organization, including regulatory regions and gene synteny. Here, we demonstrate the power of combining targeted sequence capture with long‐read sequencing technology for comparative genomic analyses of the haemoglobin (Hb) gene clusters across eight species separated by up to 70 million years. Guided by the reference genome assembly of the Atlantic cod (Gadus morhua) together with genome information from draft assemblies of selected codfishes, we designed probes covering the two Hb gene clusters. Use of custom‐made barcodes combined with PacBio RSII sequencing led to highly continuous assemblies of the LA (~100 kb) and MN (~200 kb) clusters, which include syntenic regions of coding and intergenic sequences. Our results revealed an overall conserved genomic organization of the Hb genes within this lineage, yet with several, lineage‐specific gene duplications. Moreover, for some of the species examined, we identified amino acid substitutions at two sites in the Hbb1 gene as well as length polymorphisms in its regulatory region, which has previously been linked to temperature adaptation in Atlantic cod populations. This study highlights the use of targeted long‐read capture as a versatile approach for comparative genomic studies by generation of a cross‐species genomic resource elucidating the evolutionary history of the Hb gene family across the highly divergent group of codfishes.     

Whole genome capture of vector-borne pathogens from mixed DNA samples: a case study of Borrelia burgdorferi

Background

Rapid and accurate retrieval of whole genome sequences of human pathogens from disease vectors or animal reservoirs will enable fine-resolution studies of pathogen epidemiological and evolutionary dynamics. However, next generation sequencing technologies have not yet been fully harnessed for the study of vector-borne and zoonotic pathogens, due to the difficulty of obtaining high-quality pathogen sequence data directly from field specimens with a high ratio of host to pathogen DNA.

Results

We addressed this challenge by using custom probes for multiplexed hybrid capture to enrich for and sequence 30 Borrelia burgdorferi genomes from field samples of its arthropod vector. Hybrid capture enabled sequencing of nearly the complete genome (~99.5 %) of the Borrelia burgdorferi pathogen with 132-fold coverage, and identification of up to 12,291 single nucleotide polymorphisms per genome.

Conclusions

The proprosed culture-independent method enables efficient whole genome capture and sequencing of pathogens directly from arthropod vectors, thus making population genomic study of vector-borne and zoonotic infectious diseases economically feasible and scalable. Furthermore, given the similarities of invertebrate field specimens to other mixed DNA templates characterized by a high ratio of host to pathogen DNA, we discuss the potential applicabilty of hybrid capture for genomic study across diverse study systems.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1634-x) contains supplementary material, which is available to authorized users.     

Whole genome sequencing of Theileria parva using target capture

Protozoan parasite isolation and purification are laborious and time-consuming processes required for high quality genomic DNA used in whole genome sequencing. The objective of this study was to capture whole Theileria parva genomes directly from cell cultures and blood samples using RNA baits. Cell culture material was bait captured or sequenced directly, while blood samples were all captured. Baits had variable success in capturing T. parva genomes from blood samples but were successful in cell cultures. Genome mapping uncovered extensive host contamination in blood samples compared to cell cultures. Captured cell cultures had over 81 fold coverage for the reference genome compared to 0–33 fold for blood samples. Results indicate that baits are specific to T. parva, are a good alternative to conventional methods and thus ideal for genomic studies. This study also reports the first whole genome sequencing of South African T. parva.     

Long-Range Genomic Enrichment,Sequencing, and Assembly to Determine Unknown Sequences Flanking a Known microRNA

Conserved plant microRNAs (miRNAs) modulate important biological processes but little is known about conserved cis-regulatory elements (CREs) surrounding MIRNA genes. We developed a solution-based targeted genomic enrichment methodology to capture, enrich, and sequence flanking genomic regions surrounding conserved MIRNA genes with a locked-nucleic acid (LNA)-modified, biotinylated probe complementary to the mature miRNA sequence. Genomic DNA bound by the probe is captured by streptavidin-coated magnetic beads, amplified, sequenced and assembled de novo to obtain genomic DNA sequences flanking MIRNA locus of interest. We demonstrate the sensitivity and specificity of this enrichment methodology in Arabidopsis thaliana to enrich targeted regions spanning 10–20 kb surrounding known MIR166 and MIR165 loci. Assembly of the sequencing reads successfully recovered all targeted loci. While further optimization for larger, more complex genomes is needed, this method may enable determination of flanking genomic DNA sequence surrounding a known core (like a conserved mature miRNA) from multiple species that currently don''t have a full genome assembly available.     

Deep amplicon sequencing highlights low intra-host genetic variability of Echinococcus multilocularis and high prevalence of the European-type haplotypes in coyotes and red foxes in Alberta,Canada

Echinococcus multilocularis (Em) is a zoonotic parasite considered a global emergent pathogen. Recent findings indicate that the parasite is expanding its range in North America and that European-type haplotypes are circulating in western Canada. However, genetic analyses are usually conducted only on a few parasites out of thousands of individuals within each definitive host, likely underestimating the prevalence of less common haplotypes. Moreover, mixed infections with several mtDNA haplotypes in the same host have been reported, but their relative abundance within the host was never estimated. We aimed to 1) estimate the frequency of co-infections of different Em haplotypes in coyotes (Canis latrans) and red foxes (Vulpes vulpes) from western Canada and their relative abundance within the definitive hosts, 2) detect less prevalent haplotypes by sampling a larger proportion of the parasite subpopulation per host, and 3) investigate differences in the distribution of Em haplotypes in these main definitive hosts; foxes and coyotes. We extracted DNA from ~10% of the worm subpopulation per host (20 foxes and 47 coyotes) and used deep amplicon sequencing (NGS technology) on four loci, targeting the most polymorphic regions from the mitochondrial genes cox1 (814 bp), nad1 (344 bp), and cob (387 bp). We detected the presence of mixed infections with multiple Em haplotypes and with different Echinococcus species including Em and E. granulosus s.l. genotypes G8/G10, low intraspecific diversity of Em, and a higher abundance of the European-type haplotypes in both hosts. Our results suggest a population expansion of the European over the North American strain in Alberta and a limited distribution of some European-type haplotypes. Our findings indicate that deep amplicon sequencing represents a valuable tool to characterize Em in multiple hosts, to assess the current distribution and possible origins of the European strain in North America. The potential use of next-generation sequencing technologies is particularly important to understand the patterns of geographic expansion of this parasite.     

Metabarcoding Gastrointestinal Nematodes in Sympatric Endemic and Nonendemic Species in Ranomafana National Park,Madagascar

Sympatric species are known to host the same parasites species. Nevertheless, surveys examining parasite assemblages in sympatric species are rare. To understand how parasite assemblages in sympatric host species differ in a given locality, we used a noninvasive identification method based on high-throughput sequencing. We collected fecal samples from sympatric species in Ranomafana National Park, Madagascar, from September to December in 2010, 2011, and 2012 and identified their parasites by metabarcoding, sequencing a region of the small ribosomal subunit (18S) gene. Our survey included 11 host species, including endemic primates, rodents, frogs, gastropods, and nonendemic rats and dogs. We collected 872 samples, of which 571 contained nematodes and 249 were successfully sequenced. We identified nine putative species of parasites, although their correspondence to actual parasite species is not clear as the resolution of the marker gene differs between nematode clades. For the host species that we successfully sampled with 10 or more positive occurrences of nematodes, i.e., mouse lemurs (Microcebus rufus), black rats (Rattus rattus), and frogs (Anura), the parasite assemblage compositions differed significantly among host species, sampling sites, and sampling years. Our metabarcoding method shows promise in interrogating parasite assemblages in sympatric host species and our results emphasize the importance of choosing marker regions for parasite identification accuracy.     

Positioning the Red Deer (Cervus elaphus) Hunted by the Tyrolean Iceman into a Mitochondrial DNA Phylogeny

In the last years several phylogeographic studies of both extant and extinct red deer populations have been conducted. Three distinct mitochondrial lineages (western, eastern and North-African/Sardinian) have been identified reflecting different glacial refugia and postglacial recolonisation processes. However, little is known about the genetics of the Alpine populations and no mitochondrial DNA sequences from Alpine archaeological specimens are available. Here we provide the first mitochondrial sequences of an Alpine Copper Age Cervus elaphus. DNA was extracted from hair shafts which were part of the remains of the clothes of the glacier mummy known as the Tyrolean Iceman or Ötzi (5,350–5,100 years before present). A 2,297 base pairs long fragment was sequenced using a mixed sequencing procedure based on PCR amplifications and 454 sequencing of pooled amplification products. We analyzed the phylogenetic relationships of the Alpine Copper Age red deer''s haplotype with haplotypes of modern and ancient European red deer. The phylogenetic analyses showed that the haplotype of the Alpine Copper Age red deer falls within the western European mitochondrial lineage in contrast with the current populations from the Italian Alps belonging to the eastern lineage. We also discussed the phylogenetic relationships of the Alpine Copper Age red deer with the populations from Mesola Wood (northern Italy) and Sardinia.     

Local parasite lineage sharing in temperate grassland birds provides clues about potential origins of Galapagos avian Plasmodium

Oceanic archipelagos are vulnerable to natural introduction of parasites via migratory birds. Our aim was to characterize the geographic origins of two Plasmodium parasite lineages detected in the Galapagos Islands and in North American breeding bobolinks (Dolichonyx oryzivorus) that regularly stop in Galapagos during migration to their South American overwintering sites. We used samples from a grassland breeding bird assemblage in Nebraska, United States, and parasite DNA sequences from the Galapagos Islands, Ecuador, to compare to global data in a DNA sequence registry. Homologous DNA sequences from parasites detected in bobolinks and more sedentary birds (e.g., brown‐headed cowbirds Molothrus ater, and other co‐occurring bird species resident on the North American breeding grounds) were compared to those recovered in previous studies from global sites. One parasite lineage that matched between Galapagos birds and the migratory bobolink, Plasmodium lineage B, was the most common lineage detected in the global MalAvi database, matching 49 sequences from unique host/site combinations, 41 of which were of South American origin. We did not detect lineage B in brown‐headed cowbirds. The other Galapagos‐bobolink match, Plasmodium lineage C, was identical to two other sequences from birds sampled in California. We detected a close variant of lineage C in brown‐headed cowbirds. Taken together, this pattern suggests that bobolinks became infected with lineage B on the South American end of their migratory range, and with lineage C on the North American breeding grounds. Overall, we detected more parasite lineages in bobolinks than in cowbirds. Galapagos Plasmodium had similar host breadth compared to the non‐Galapagos haemosporidian lineages detected in bobolinks, brown‐headed cowbirds, and other grassland species. This study highlights the utility of global haemosporidian data in the context of migratory bird–parasite connectivity. It is possible that migratory bobolinks bring parasites to the Galapagos and that these parasites originate from different biogeographic regions representing both their breeding and overwintering sites.     

Geographic and host‐mediated population genetic structure in a cestode parasite of the three‐spined stickleback

Comparative studies of genetic diversity and population structure can shed light on the ecological and evolutionary factors that influence host–parasite interactions. Here we examined whether geography, time and genetic variation in Alaskan three‐spined stickleback (Gasterosteus aculeatus Linneaus) hosts shape the population genetic structure of the diphyllobothridean cestode parasite Schistocephalus solidus (Müller, 1776). Host lineages and haplotypes were identified by sequencing the mitochondrial cytochrome b gene, and host population structure was assessed by Bayesian clustering analysis of allelic variation at 11 microsatellite loci. Parasite population structure was characterized according to allelic variation at eight microsatellite loci. Mantel tests and canonical redundancy analysis were conducted to evaluate the proportion of parasite genetic variation attributable to time and geography vs. host lineage, haplotype, and genotypic cluster. Host and parasite population structure were largely discordant across the study area, probably reflecting differences in gene flow, environmental influences external to the host, and genomic admixture among host lineages. We found that geography explained the greatest proportion of parasite genetic variation, but that variation also reflects time, host lineage, and host haplotype. Associations with host haplotypes suggest that one parasite genotypic cluster exhibits a narrower host range, predominantly infecting the most common host haplotypes, whereas the other parasite cluster infects all haplotypes equally, including rare haplotypes. Although experimental infection trials might prove otherwise, distributional differences in hosts preferentially infected by S. solidus could underlie the observed pattern of population structure.     

Identification of sequence variants in genetic disease-causing genes using targeted next-generation sequencing

Background

Identification of gene variants plays an important role in research on and diagnosis of genetic diseases. A combination of enrichment of targeted genes and next-generation sequencing (targeted DNA-HiSeq) results in both high efficiency and low cost for targeted sequencing of genes of interest.

Methodology/Principal Findings

To identify mutations associated with genetic diseases, we designed an array-based gene chip to capture all of the exons of 193 genes involved in 103 genetic diseases. To evaluate this technology, we selected 7 samples from seven patients with six different genetic diseases resulting from six disease-causing genes and 100 samples from normal human adults as controls. The data obtained showed that on average, 99.14% of 3,382 exons with more than 30-fold coverage were successfully detected using Targeted DNA-HiSeq technology, and we found six known variants in four disease-causing genes and two novel mutations in two other disease-causing genes (the STS gene for XLI and the FBN1 gene for MFS) as well as one exon deletion mutation in the DMD gene. These results were confirmed in their entirety using either the Sanger sequencing method or real-time PCR.

Conclusions/Significance

Targeted DNA-HiSeq combines next-generation sequencing with the capture of sequences from a relevant subset of high-interest genes. This method was tested by capturing sequences from a DNA library through hybridization to oligonucleotide probes specific for genetic disorder-related genes and was found to show high selectivity, improve the detection of mutations, enabling the discovery of novel variants, and provide additional indel data. Thus, targeted DNA-HiSeq can be used to analyze the gene variant profiles of monogenic diseases with high sensitivity, fidelity, throughput and speed.     

Occurrence of five distinct clades of mermithid nematodes (Nematoda: Mermithidae) infecting black fly larvae (Diptera: Simuliidae) in tropical streams in Malaysia

Mermithids are the most common parasites of black flies and are associated with host feminization and sterilization in infected hosts. However, information on the species / lineage of black fly mermithids in Southeast Asia, including Malaysia requires further elucidation. In this study, mermithids were obtained from black fly larvae collected from 138 freshwater stream sites across East and West Malaysia. A molecular approach based on nuclear-encoded 18S ribosomal RNA (18S rRNA) gene was used to identify the species identity / lineage of 77 nematodes successfully extracted and sequenced from the specimens collected. Maximum likelihood and neighbor-joining phylogenetic analyses demonstrated five distinct mermithid lineages. Four species delimitation analyses: automated simultaneous analysis phylogenetics (ASAP), maximum likelihood Poisson tree processes with Bayesian inferences (bPTP_ML), generalized mixed yule coalescent (GMYC) and single rate Poisson tree processes (PTP) were applied to delimit the species boundaries of mermithid lineages in this data set along with genetic distance analysis. Data analysis supports five distinct lineages or operational taxonomic units for mermithids in the present study, with two requiring further investigation as they may represent intraspecific variation or closely related taxa. One mermithid lineage was similar to that previously observed in Simulium nigrogilvum from Thailand. Co-infection with two mermithids of different lineages was observed in one larva of Simulium trangense. This study represents an important first step towards exploring other aspects of host – parasite interactions in black fly mermithids.     

Genetic diagnosis of autosomal dominant polycystic kidney disease by targeted capture and next-generation sequencing: Utility and limitations

Mutation-based molecular diagnostics of autosomal dominant polycystic kidney disease (ADPKD) is complicated by genetic and allelic heterogeneity, large multi-exon genes, and duplication sequences of PKD1. Recently, targeted resequencing by pooling long-range polymerase chain reaction (LR-PCR) amplicons has been used in the identification of mutations in ADPKD. Despite its high sensitivity, specificity and accuracy, LR-PCR is still complicated. We performed whole-exome sequencing on two unrelated typical Chinese ADPKD probands and evaluated the effectiveness of this approach compared with Sanger sequencing. Meanwhile, we performed targeted gene and next-generation sequencing (targeted DNA-HiSeq) on 8 individuals (1 patient from one family, 5 patients and 2 normal individuals from another family). Both whole-exome sequencing and targeted DNA-HiSeq confirmed c.11364delC (p.H3788QfsX37) within the unduplicated region of PKD1 in one proband; in the other family, targeted DNA-HiSeq identified a small insertion, c.401_402insG (p.V134VfsX79), in PKD2. These methods do not overcome the screening complexity of homology. However, the true positives of variants confirmed by targeted gene and next-generation sequencing were 69.4%, 50% and 100% without a false positive in the whole coding region and the duplicated and unduplicated regions, which indicated that the screening accuracy of PKD1 and PKD2 can be largely improved by using a greater sequencing depth and elaborate design of the capture probe.     

Deep Sequencing of the Trypanosoma cruzi GP63 Surface Proteases Reveals Diversity and Diversifying Selection among Chronic and Congenital Chagas Disease Patients

Background

Chagas disease results from infection with the diploid protozoan parasite Trypanosoma cruzi. T. cruzi is highly genetically diverse, and multiclonal infections in individual hosts are common, but little studied. In this study, we explore T. cruzi infection multiclonality in the context of age, sex and clinical profile among a cohort of chronic patients, as well as paired congenital cases from Cochabamba, Bolivia and Goias, Brazil using amplicon deep sequencing technology.

Methodology/ Principal Findings

A 450bp fragment of the trypomastigote TcGP63I surface protease gene was amplified and sequenced across 70 chronic and 22 congenital cases on the Illumina MiSeq platform. In addition, a second, mitochondrial target—ND5—was sequenced across the same cohort of cases. Several million reads were generated, and sequencing read depths were normalized within patient cohorts (Goias chronic, n = 43, Goias congenital n = 2, Bolivia chronic, n = 27; Bolivia congenital, n = 20), Among chronic cases, analyses of variance indicated no clear correlation between intra-host sequence diversity and age, sex or symptoms, while principal coordinate analyses showed no clustering by symptoms between patients. Between congenital pairs, we found evidence for the transmission of multiple sequence types from mother to infant, as well as widespread instances of novel genotypes in infants. Finally, non-synonymous to synonymous (dn:ds) nucleotide substitution ratios among sequences of TcGP63Ia and TcGP63Ib subfamilies within each cohort provided powerful evidence of strong diversifying selection at this locus.

Conclusions/Significance

Our results shed light on the diversity of parasite DTUs within each patient, as well as the extent to which parasite strains pass between mother and foetus in congenital cases. Although we were unable to find any evidence that parasite diversity accumulates with age in our study cohorts, putative diversifying selection within members of the TcGP63I gene family suggests a link between genetic diversity within this gene family and survival in the mammalian host.     

Targeted resequencing of candidate genes using selector probes

Targeted genome enrichment is a powerful tool for making use of the massive throughput of novel DNA-sequencing instruments. We herein present a simple and scalable protocol for multiplex amplification of target regions based on the Selector technique. The updated version exhibits improved coverage and compatibility with next-generation-sequencing (NGS) library-construction procedures for shotgun sequencing with NGS platforms. To demonstrate the performance of the technique, all 501 exons from 28 genes frequently involved in cancer were enriched for and sequenced in specimens derived from cell lines and tumor biopsies. DNA from both fresh frozen and formalin-fixed paraffin-embedded biopsies were analyzed and 94% specificity and 98% coverage of the targeted region was achieved. Reproducibility between replicates was high (R² = 0, 98) and readily enabled detection of copy-number variations. The procedure can be carried out in <24 h and does not require any dedicated instrumentation.     

The origin of malarial parasites in orangutans

Background

Recent findings of Plasmodium in African apes have changed our perspectives on the evolution of malarial parasites in hominids. However, phylogenetic analyses of primate malarias are still missing information from Southeast Asian apes. In this study, we report molecular data for a malaria parasite lineage found in orangutans.

Methodology/Principal Findings

We screened twenty-four blood samples from Pongo pygmaeus (Kalimantan, Indonesia) for Plasmodium parasites by PCR. For all the malaria positive orangutan samples, parasite mitochondrial genomes (mtDNA) and two antigens: merozoite surface protein 1 42 kDa (MSP-1₄₂) and circumsporozoite protein gene (CSP) were amplified, cloned, and sequenced. Fifteen orangutans tested positive and yielded 5 distinct mitochondrial haplotypes not previously found. The haplotypes detected exhibited low genetic divergence among them, indicating that they belong to one species. We report phylogenetic analyses using mitochondrial genomes, MSP-1₄₂ and CSP. We found that the orangutan malaria parasite lineage was part of a monophyletic group that includes all the known non-human primate malaria parasites found in Southeast Asia; specifically, it shares a recent common ancestor with P. inui (a macaque parasite) and P. hylobati (a gibbon parasite) suggesting that this lineage originated as a result of a host switch. The genetic diversity of MSP-1₄₂ in orangutans seems to be under negative selection. This result is similar to previous findings in non-human primate malarias closely related to P. vivax. As has been previously observed in the other Plasmodium species found in non-human primates, the CSP shows high polymorphism in the number of repeats. However, it has clearly distinctive motifs from those previously found in other malarial parasites.

Conclusion

The evidence available from Asian apes indicates that these parasites originated independently from those found in Africa, likely as the result of host switches from other non-human primates.