Long range PCR-based deep sequencing for haplotype determination in mixed HCMV infections

Background Short read sequencing has been used extensively to decipher the genome diversity of human cytomegalovirus (HCMV) strains, but falls short to reveal individual genomes in mixed HCMV strain populations. Novel third-generation sequencing platforms offer an extended read length and promise to resolve how distant polymorphic sites along individual genomes are linked. In the present study, we established a long amplicon PacBio sequencing workflow to identify the absolute and relative quantities of unique HCMV haplotypes spanning over multiple hypervariable sites in mixtures. Initial validation of this approach was performed with defined HCMV DNA templates derived from cell-culture enriched viruses and was further tested for its suitability on patient samples carrying mixed HCMV infections. Results Total substitution and indel error rate of mapped reads ranged from 0.17 to 0.43% depending on the stringency of quality trimming. Artificial HCMV DNA mixtures were correctly determined down to 1% abundance of the minor DNA source when the total HCMV DNA input was 4 × 104 copies/ml. PCR products of up to 7.7 kb and a GC content < 55% were efficiently generated when DNA was directly isolated from patient samples. In a single sample, up to three distinct haplotypes were identified showing varying relative frequencies. Alignments of distinct haplotype sequences within patient samples showed uneven distribution of sequence diversity, interspersed by long identical stretches. Moreover, diversity estimation at single polymorphic regions as assessed by short amplicon sequencing may markedly underestimate the overall diversity of mixed haplotype populations. Conclusions Quantitative haplotype determination by long amplicon sequencing provides a novel approach for HCMV strain characterisation in mixed infected samples which can be scaled up to cover the majority of the genome by multi-amplicon panels. This will substantially improve our understanding of intra-host HCMV strain diversity and its dynamic behaviour.

Comparative Phylogeography of Southern African Bird Species Suggests an Ephemeral Speciation Model

Southern Africa is remarkably rich in avian species diversity; however, the evolutionary and biogeographic mechanisms responsible for that diversity are, in general, poorly understood, and this is particularly true with respect to the many species that are endemic or near-endemic to the region. Here, we used mtDNA to assess genetic structure in three southern African bird species to determine whether each was genetically panmictic, or whether there was standing genetic variation upon which abiotic factors (e.g., climate, biome boundaries, geographic features) could have acted to drive lineage diversification. Haplotype diversity was partitioned into two (two species) or three (one species) distinct haplotype clusters that did not reflect biogeographic or biome partitioning; instead, haplotype clusters overlapped in central South Africa. Population and demographic analyses, along with ecological niche modeling and Bayesian Skyline Plots, indicated that each of the three species were likely isolated in refugia during Pleistocene climatic perturbations, with subsequent expansions from refugia resulting in present-day overlapping distributions. Collectively, our analyses suggest that an ephemeral speciation model is operating in southern Africa, driven by the dynamic climatic oscillations that characterize the region. At least some of the regional endemic bird species (e.g., White-eyes, Zosterops spp.) may be the result of sufficiently long periods in refugia as opposed to the distinct but ephemeral clusters recovered within our three focal species.

Evidence of Low Chloroplast Genetic Diversity in Two Carpinus Species in the Northern Balkans

Genetic diversity and differentiation in two Carpinus species (C. betulus and C. orientalis) occurring in Romania was investigated by using three chloroplast Simple Sequence Repeat markers (cpSSRs). A total of 96 and 32 individuals were sampled in eighteen C. betulus and six C. orientalis populations, respectively. A total of four chloroplast haplotypes were observed. Two haplotypes were specific for C. betulus and two for C. orientalis. Most of C. betulus populations were fixed for the predominant haplotype (H1), which was observed in 82% of the individuals. All C. orientalis populations were fixed for one haplotype or the other. Populations with haplotype (H3) are spread in southern Romania and the haplotype (H4) was observed at the northern limit of C. orientalis natural distribution range. Genetic differentiation among populations was moderate in C. betulus (GST = 0.422), compared to the high value observed in C. orientalis (GST = 1.000), which can be explained by the occurrence of a distinct haplotype in the peripheral population. RST values for both species suggest low levels of recurrent gene flow through seeds among populations. Our data on geographic distribution of chloroplast DNA haplotypes may be useful for the identification and conservation of distinct genetic resources of the two Carpinus species.

Selfish evolution of cytonuclear hybrid incompatibility in Mimulus

Intraspecific coevolution between selfish elements and suppressors may promote interspecific hybrid incompatibility, but evidence of this process is rare. Here, we use genomic data to test alternative models for the evolution of cytonuclear hybrid male sterility in Mimulus . In hybrids between Iron Mountain (IM) Mimulus guttatus × Mimulus nasutus , two tightly linked M. guttatus alleles ( Rf 1 /Rf 2) each restore male fertility by suppressing a local mitochondrial male-sterility gene (IM-CMS). Unlike neutral models for the evolution of hybrid incompatibility loci, selfish evolution predicts that the Rf alleles experienced strong selection in the presence of IM-CMS. Using whole-genome sequences, we compared patterns of population-genetic variation in Rf at IM to a neighbouring population that lacks IM-CMS. Consistent with local selection in the presence of IM-CMS, the Rf region shows elevated F ST , high local linkage disequilibrium and a distinct haplotype structure at IM, but not at Cone Peak (CP), suggesting a recent sweep in the presence of IM-CMS. In both populations, Rf 2 exhibited lower polymorphism than other regions, but the low-diversity outliers were different between CP and IM. Our results confirm theoretical predictions of ubiquitous cytonuclear conflict in plants and provide a population-genetic mechanism for the evolution of a common form of hybrid incompatibility.

WHOLE GENOME IDENTITY-BY-DESCENT DETERMINATION

High-throughput single nucleotide polymorphism genotyping assays conveniently produce genotype data for genome-wide genetic linkage and association studies. For pedigree datasets, the unphased genotype data is used to infer the haplotypes for individuals, according to Mendelian inheritance rules. Linkage studies can then locate putative chromosomal regions based on the haplotype allele sharing among the pedigree members and their disease status. Most existing haplotyping programs require rather strict pedigree structures and return a single inferred solution for downstream analysis. In this research, we relax the pedigree structure to contain ungenotyped founders and present a cubic time whole genome haplotyping algorithm to minimize the number of zero-recombination haplotype blocks. With or without explicitly enumerating all the haplotyping solutions, the algorithm determines all distinct haplotype allele identity-by-descent (IBD) sharings among the pedigree members, in linear time in the total number of haplotyping solutions. Our algorithm is implemented as a computer program iBDD. Extensive simulation experiments using 2 sets of 16 pedigree structures from previous studies showed that, in general, there are trillions of haplotyping solutions, but only up to a few thousand distinct haplotype allele IBD sharings. iBDD is able to return all these sharings for downstream genome-wide linkage and association studies.

Genetic determinants of plasma von Willebrand factor antigen levels: a target gene SNP and haplotype analysis of ARIC cohort

Abstractvon Willebrand factor (VWF) is an essential component of hemostasis and has been implicated in thrombosis. Multimer size and the amount of circulating VWF are known to impact hemostatic function. We associated 78 VWF single nucleotide polymorphisms (SNPs) and haplotypes constructed from those SNPs with VWF antigen level in 7856 subjects of European descent. Among the nongenomic factors, age and body mass index contributed 4.8% and 1.6% of VWF variation, respectively. The SNP rs514659 (tags O blood type) contributed 15.4% of the variance. Among the VWF SNPs, we identified 18 SNPs that are associated with levels of VWF. The correlative SNPs are either intronic (89%) or silent exonic (11%). Although SNPs examined are distributed throughout the entire VWF gene without apparent cluster, all the positive SNPs are located in a 50-kb region. Exons in this region encode for VWF D2, D′, and D3 domains that are known to regulate VWF multimerization and storage. Mutations in the D3 domain are also associated with von Willebrand disease. Fifteen of these 18 correlative SNPs are in 2 distinct haplotype blocks. In summary, we identified a cluster of intronic VWF SNPs that associate with plasma levels of VWF, individually or additively, in a large cohort of healthy subjects.