Haplotyping using a combination of polymerase chain reaction–single-strand conformational polymorphism analysis and haplotype-specific PCR amplification

A single nucleotide polymorphism (SNP) may have an impact on phenotype, but it may also be influenced by multiple SNPs within a gene; hence, the haplotype or phase of multiple SNPs needs to be known. Various methods for haplotyping SNPs have been proposed, but a simple and cost-effective method is currently unavailable. Here we describe a haplotyping approach using two simple techniques: polymerase chain reaction–single-strand conformational polymorphism (PCR–SSCP) and haplotype-specific PCR. In this approach, individual regions of a gene are analyzed by PCR–SSCP to identify variation that defines sub-haplotypes, and then extended haplotypes are assembled from the sub-haplotypes either directly or with the additional use of haplotype-specific PCR amplification. We demonstrate the utility of this approach by haplotyping ovine FABP4 across two variable regions that contain seven SNPs and one indel. The simplicity of this approach makes it suitable for large-scale studies and/or diagnostic screening.     

Direct molecular haplotyping by melting curve analysis of hybridization probes: beta 2-adrenergic receptor haplotypes as an example

Direct determination of the association of multiple genetic polymorphisms, or haplotyping, in individual samples is challenging because of chromosome diploidy. Here, we describe the ability of hybridization probes, commonly used as genotyping tools, to establish single nucleotide polymorphism (SNP) haplotypes in a single step. Three haplotypes found in the beta 2-adrenergic receptor (β2AR) gene and characterized by three different SNPs combinations are presented as examples. Each combination of SNPs has a unique stability, recorded by its melting temperature, even when intervening sequences from the template must loop out during probe hybridization. In the course of this study, two haplotypes in β2AR not described previously were discovered. This approach provides a tool for molecular haplotyping that should prove useful in clinical molecular genetics diagnostics and pharmacogenetic research where methods for direct haplotyping are needed.     

Algorithm for haplotype inference via galled-tree networks with simple galls

The problem of determining haplotypes from genotypes has gained considerable prominence in the research community. Here the focus is on determining sets of SNP values on individual chromosomes since such information captures the genetic causes of diseases. The most efficient algorithmic tool for haplotyping is based on perfect phylogenetic trees. A drawback of this method is that it cannot be applied in situations when the data contains homoplasies (multiple mutations of the same character) or recombinations. Recently, Song et al. ( 2005 ) studied the two cases: haplotyping via imperfect phylogenies with a single homoplasy and via galled-tree networks with one gall. In Gupta et al. ( 2010 ), we have shown that the haplotyping via galled-tree networks is NP-hard, even if we restrict to the case when every gall contains at most 3 mutations. We present a polynomial algorithm for haplotyping via galled-tree networks with simple galls (each having two mutations) for genotype matrices which satisfy a natural condition which is implied by presence of at least one 1 in each column that contains a 2. In the end, we give the experimental results comparing our algorithm with PHASE on simulated data.     

A Recombination Hotspot in a Schizophrenia-Associated Region of GABRB2

Background

Schizophrenia is a major disorder with complex genetic mechanisms. Earlier, population genetic studies revealed the occurrence of strong positive selection in the GABRB2 gene encoding the β₂ subunit of GABA_A receptors, within a segment of 3,551 bp harboring twenty-nine single nucleotide polymorphisms (SNPs) and containing schizophrenia-associated SNPs and haplotypes.

Methodology/Principal Findings

In the present study, the possible occurrence of recombination in this ‘S1–S29’ segment was assessed. The occurrence of hotspot recombination was indicated by high resolution recombination rate estimation, haplotype diversity, abundance of rare haplotypes, recurrent mutations and torsos in haplotype networks, and experimental haplotyping of somatic and sperm DNA. The sub-segment distribution of relative recombination strength, measured by the ratio of haplotype diversity (H_d) over mutation rate (θ), was indicative of a human specific Alu-Yi6 insertion serving as a central recombining sequence facilitating homologous recombination. Local anomalous DNA conformation attributable to the Alu-Yi6 element, as suggested by enhanced DNase I sensitivity and obstruction to DNA sequencing, could be a contributing factor of the increased sequence diversity. Linkage disequilibrium (LD) analysis yielded prominent low LD points that supported ongoing recombination. LD contrast revealed significant dissimilarity between control and schizophrenic cohorts. Among the large array of inferred haplotypes, H26 and H73 were identified to be protective, and H19 and H81 risk-conferring, toward the development of schizophrenia.

Conclusions/Significance

The co-occurrence of hotspot recombination and positive selection in the S1–S29 segment of GABRB2 has provided a plausible contribution to the molecular genetics mechanisms for schizophrenia. The present findings therefore suggest that genome regions characterized by the co-occurrence of positive selection and hotspot recombination, two interacting factors both affecting genetic diversity, merit close scrutiny with respect to the etiology of common complex disorders.     

A simple, bead-based approach for multi-SNP molecular haplotyping

Single nucleotide polymorphisms (SNPs) within a gene region have often been studied to determine their effect on phenotype. Although a single base pair change can produce a phenotypic change, phenotype is often influenced by the presence of multiple polymorphisms and their relative positions within a given region. For example, if multiple changes occur in a promoter region, how they influence gene expression will depend on their cis/trans configuration. As such, it is essential to consider the haplotype, or the alignment of multiple SNP alleles on each chromosome when attempting to associate genomic changes with phenotype. Unfortunately, no method of high-throughput molecular haplotyping of multiple SNPs currently exists. In response to this unmet need, we have developed an inexpensive, reliable bead-based capture-based haplotyping (CBH) assay to determine the phase, or haplotype, of multiple SNP alleles in a high-throughput manner. The CBH assay requires minimal setup and handling, requires no centrifugation steps and can be performed in <1 h. Data collection is performed via flow cytometry and the assay yields plus/minus results allowing for automated calling by a simple computer application. We will present data demonstrating the molecular haplotyping of 11 SNPs within exon 2 of the N-acetyltransferase-2 (NAT2) gene, which expresses an important drug-metabolizing enzyme. This assay has applications in diagnostic testing, promoter analysis, association studies and pharmacogenetic analysis.     

Protein variation in Adh and Adh-related in Drosophila pseudoobscura. Linkage disequilibrium between single nucleotide polymorphisms and protein alleles

A 3.5-kb segment of the alcohol dehydrogenase (Adh) region that includes the Adh and Adh-related genes was sequenced in 139 Drosophila pseudoobscura strains collected from 13 populations. The Adh gene encodes four protein alleles and rejects a neutral model of protein evolution with the McDonald-Kreitman test, although the number of segregating synonymous sites is too high to conclude that adaptive selection has operated. The Adh-related gene encodes 18 protein haplotypes and fails to reject an equilibrium neutral model. The populations fail to show significant geographic differentiation of the Adh-related haplotypes. Eight of 404 single nucleotide polymorphisms (SNPs) in the Adh region were in significant linkage disequilibrium with three ADHR protein alleles. Coalescent simulations with and without recombination were used to derive the expected levels of significant linkage disequilibrium between SNPs and 18 protein haplotypes. Maximum levels of linkage disequilibrium are expected for protein alleles at moderate frequencies. In coalescent models without recombination, linkage disequilibrium decays between SNPs and high frequency haplotypes because common alleles mutate to haplotypes that are rare or that reach moderate frequency. The implication of this study is that linkage disequilibrium mapping has the highest probability of success with disease-causing alleles at frequencies of 10%.     

Construction of mutant and chimeric genes using the polymerase chain reaction.

In the polymerase chain reaction (PCR) the specific amplification of a small segment of DNA within a complex DNA sample is effected by repeated cycles of DNA denaturation and enzymatic synthesis primed by two oligonucleotides complementary to regions within opposite strands of the DNA. In this report a simple and efficient method is described in which PCR methodology is used to introduce specific mutations into a double stranded DNA molecule. In this procedure a supercoiled plasmid DNA serves as template for a PCR in which a primer bearing the mutated sequence is incorporated into the amplified product. The presence of convenient restriction sites in the mutagenic primer and in the amplified DNA permit direct replacement of a wild type DNA segment with the mutated segment by treating the PCR mixture with the appropriate restriction endonucleases followed by DNA ligase. Using this procedure, a single amino acid replacement, a 16 amino acid deletion and a replacement of four amino acids with a twelve amino acid segment from another membrane protein were introduced into the amino terminal signal segment of rat hepatic cytochrome P450b (P450IIB1).     

Molecular haplotyping at high throughput

Reconstruction of haplotypes, or the allelic phase, of single nucleotide polymorphisms (SNPs) is a key component of studies aimed at the identification and dissection of genetic factors involved in complex genetic traits. In humans, this often involves investigation of SNPs in case/control or other cohorts in which the haplotypes can only be partially inferred from genotypes by statistical approaches with resulting loss of power. Moreover, alternative statistical methodologies can lead to different evaluations of the most probable haplotypes present, and different haplotype frequency estimates when data are ambiguous. Given the cost and complexity of SNP studies, a robust and easy-to-use molecular technique that allows haplotypes to be determined directly from individual DNA samples would have wide applicability. Here, we present a reliable, automated and high-throughput method for molecular haplotyping in 2 kb, and potentially longer, sequence segments that is based on the physical determination of the phase of SNP alleles on either of the individual paternal haploids. We demonstrate that molecular haplotyping with this technique is not more complicated than SNP genotyping when implemented by matrix-assisted laser desorption/ionisation mass spectrometry, and we also show that the method can be applied using other DNA variation detection platforms. Molecular haplotyping is illustrated on the well-described β₂-adrenergic receptor gene.     

An efficient method for multi-locus molecular haplotyping

Many methods exist for genotyping—revealing which alleles an individual carries at different genetic loci. A harder problem is haplotyping—determining which alleles lie on each of the two homologous chromosomes in a diploid individual. Conventional approaches to haplotyping require the use of several generations to reconstruct haplotypes within a pedigree, or use statistical methods to estimate the prevalence of different haplotypes in a population. Several molecular haplotyping methods have been proposed, but have been limited to small numbers of loci, usually over short distances. Here we demonstrate a method which allows rapid molecular haplotyping of many loci over long distances. The method requires no more genotypings than pedigree methods, but requires no family material. It relies on a procedure to identify and genotype single DNA molecules, and reconstruction of long haplotypes by a ‘tiling’ approach. We demonstrate this by resolving haplotypes in two regions of the human genome, harbouring 20 and 105 single-nucleotide polymorphisms, respectively. The method can be extended to reconstruct haplotypes of arbitrary complexity and length, and can make use of a variety of genotyping platforms. We also argue that this method is applicable in situations which are intractable to conventional approaches.     

Long-range, high-throughput haplotype determination via haplotype-fusion PCR and ligation haplotyping

Ligation Haplotyping is a robust, novel method for experimental determination of haplotypes over long distances, which can be applied to assaying both sequence and structural variation. The simplicity and efficacy of the method for genotyping large chromosomal rearrangements and haplotyping SNPs over long distances make it a valuable and powerful addition to the methodological repertoire, which will be beneficial to studies of population genetics and evolution, disease association and inheritance, and genomic variation. We illustrate the versatility of the method both by genotyping a Yp paracentric inversion, found in approximately 60% of Northwest European males, that strongly influences the germline rate of infertility-causing XY translocations and by haplotyping two autosomal SNPs that lie 16.4 kb apart on chromosome 7, and which influence an individual's susceptibility to systemic lupus erythematosus.     

Single-reaction for SNP Genotyping on Agarose Gel by Allele-specific PCR in Black Poplar (Populus nigra L.)

The wide development of single nucleotide polymorphism (SNP) markers also in non-model species increases the need for inexpensive methods that do not require sophisticated equipment and time for optimization. This work presents a new method for polymerase chain reaction (PCR) amplification of multiple specific alleles (PAMSA), which allows efficient discrimination of SNP polymorphisms in one reaction tube with standard PCR conditions. This improved PAMSA requires only three unlabeled primers: a common reverse primer and two allele-specific primers having a tail of different length to differentiate the two SNP alleles by the size of amplification products on agarose gel. A destabilizing mismatch within the five bases of the 3′ end is also added to improve the allele specificity. To validate the accuracy of this method, 94 full-sib individuals were genotyped with three SNPs and compared to the genotypes obtained by cleaved amplified polymorphic sequence (CAPS) or derived CAPS. This method is flexible, inexpensive, and well suited for high throughput and automated genotyping.     

Direct haplotyping of bi-allelic SNPs using ARMS and RFLP analysis techniques

Haplotype analysis of single nucleotide polymorphisms (SNPs) is an important and rapidly growing approach for association studies. In recent years, statistical procedures to haplotype determination from genotypic information have employed in population studies. These procedures, even though some advantages for estimation of haplotype frequencies in large population samples, have limitations in the accuracy of the analysis. In this study, we have designed a reliable method for direct haplotyping of polymorphic sites using the amplification refractory mutation system (ARMS) and restriction fragment length polymorphism (RFLP) analysis techniques. We applied the method to determination of haplotypes composed of three SNPs within the paraoxonase1 gene promoter and found the approach can be used in many studies in population and in a variety of clinical settings.     

Sequence specific generation of a DNA panhandle permits PCR amplification of unknown flanking DNA.

We present a novel method for the PCR amplification of unknown DNA that flanks a known segment directly from human genomic DNA. PCR requires that primer annealing sites be present on each end of the DNA segment that is to be amplified. In this method, known DNA is placed on the uncharacterized side of the sequence of interest via DNA polymerase mediated generation of a PCR template that is shaped like a pan with a handle. Generation of this template permits specific amplification of the unknown sequence. Taq (DNA) polymerase was used to form the original template and to generate the PCR product. 2.2 kb of the beta-globin gene, and 657 bp of the 5' flanking region of the cystic fibrosis transmembrane conductance regulator gene, were amplified directly from human genomic DNA using primers that initially flank only one side of the region amplified. This method will provide a powerful tool for acquiring DNA sequence information.     

TAR cloning: insights into gene function, long-range haplotypes and genome structure and evolution

The structural and functional analysis of mammalian genomes would benefit from the ability to isolate from multiple DNA samples any targeted chromosomal segment that is the size of an average human gene. A cloning technique that is based on transformation-associated recombination (TAR) in the yeast Saccharomyces cerevisiae satisfies this need. It is a unique tool to selectively recover chromosome segments that are up to 250 kb in length from complex genomes. In addition, TAR cloning can be used to characterize gene function and genome variation, including polymorphic structural rearrangements, mutations and the evolution of gene families, and for long-range haplotyping.     

ADLAPH: A molecular haplotyping method based on allele-discriminating long-range PCR

We present a method, called Allele-Discriminating Long and Accurate PCR Haplotyping (ADLAPH), for directly determining haplotypes from an extended genomic region. This method uses allele-discriminating primers in long-range PCR to amplify only one of the two chromosome homologues for the region of interest. Haplotypes are then determined from these phase-separated PCR fragments by conventional single nucleotide polymorphism (SNP) genotyping methods. This simple robust procedure makes it practical for high-throughput haplotyping of unrelated individuals, and potentially allows direct observation of haplotype information for up to 40 kb or more. We demonstrate the feasibility of this molecular haplotyping procedure by generating apolipoprotein E (APOE) haplotypes from 100 unrelated subjects.     

Multiplex genotyping by melting analysis of loci-spanning probes: beta-globin as an example

Multiplexing genotyping technologies usually require as many probes as genetic variants. Oligonucleotides that span multiple loci--loci spanning probes (LSProbes)--hybridize to two or more noncontiguous DNA sequences present in a template and can be used to analyze multiple variants simultaneously. The intervening template sequence, omitted in the LSProbe, creates a bulge-loop during binding. Melting temperatures of the probe, monitored by fluorescence reading are specific to the presence or absence of the mutations. We previously described LSProbes as a molecular haplotyping tool and apply here the principle to genotype simultaneously three mutations of the beta-globin gene responsible for the corresponding hemoglobinopathies. Analysis with both labeled and unlabeled LSProbes demonstrate that the four possible alleles studied (WT, HbS, HbC, and HbE) are identifiable by the specific melting temperatures of the LSProbes. This demonstrates that, in addition to their haplotyping capabilities, LSProbes are able to genotype in a single step, loci 58 nucleotides apart.     

Genetic analysis of completely sequenced disease-associated MHC haplotypes identifies shuffling of segments in recent human history

The major histocompatibility complex (MHC) is recognised as one of the most important genetic regions in relation to common human disease. Advancement in identification of MHC genes that confer susceptibility to disease requires greater knowledge of sequence variation across the complex. Highly duplicated and polymorphic regions of the human genome such as the MHC are, however, somewhat refractory to some whole-genome analysis methods. To address this issue, we are employing a bacterial artificial chromosome (BAC) cloning strategy to sequence entire MHC haplotypes from consanguineous cell lines as part of the MHC Haplotype Project. Here we present 4.25 Mb of the human haplotype QBL (HLA-A26-B18-Cw5-DR3-DQ2) and compare it with the MHC reference haplotype and with a second haplotype, COX (HLA-A1-B8-Cw7-DR3-DQ2), that shares the same HLA-DRB1, -DQA1, and -DQB1 alleles. We have defined the complete gene, splice variant, and sequence variation contents of all three haplotypes, comprising over 259 annotated loci and over 20,000 single nucleotide polymorphisms (SNPs). Certain coding sequences vary significantly between different haplotypes, making them candidates for functional and disease-association studies. Analysis of the two DR3 haplotypes allowed delineation of the shared sequence between two HLA class II-related haplotypes differing in disease associations and the identification of at least one of the sites that mediated the original recombination event. The levels of variation across the MHC were similar to those seen for other HLA-disparate haplotypes, except for a 158-kb segment that contained the HLA-DRB1, -DQA1, and -DQB1 genes and showed very limited polymorphism compatible with identity-by-descent and relatively recent common ancestry (<3,400 generations). These results indicate that the differential disease associations of these two DR3 haplotypes are due to sequence variation outside this central 158-kb segment, and that shuffling of ancestral blocks via recombination is a potential mechanism whereby certain DR-DQ allelic combinations, which presumably have favoured immunological functions, can spread across haplotypes and populations.     

Multiplex restriction amplicon sequencing: a novel next‐generation sequencing‐based marker platform for high‐throughput genotyping

To enable rapid selection of traits in marker‐assisted breeding, markers must be technically simple, low‐cost, high‐throughput and randomly distributed in a genome. We developed such a technology, designated as Multiplex Restriction Amplicon Sequencing (MRASeq), which reduces genome complexity by polymerase chain reaction (PCR) amplification of amplicons flanked by restriction sites. The first PCR primers contain restriction site sequences at 3’‐ends, preceded by 6‐10 bases of specific or degenerate nucleotide sequences and then by a unique M13‐tail sequence which serves as a binding site for a second PCR that adds sequencing primers and barcodes to allow sample multiplexing for sequencing. The sequences of restriction sites and adjacent nucleotides can be altered to suit different species. Physical mapping of MRASeq SNPs from a biparental population of allohexaploid wheat (Triticum aestivum L.) showed a random distribution of SNPs across the genome. MRASeq generated thousands of SNPs from a wheat biparental population and natural populations of wheat and barley (Hordeum vulgare L.). This novel, next‐generation sequencing‐based genotyping platform can be used for linkage mapping to screen quantitative trait loci (QTL), background selection in breeding and many other genetics and breeding applications of various species.     

Mutation Scanning in Wheat by Exon Capture and Next-Generation Sequencing

Targeted Induced Local Lesions in Genomes (TILLING) is a reverse genetics approach to identify novel sequence variation in genomes, with the aims of investigating gene function and/or developing useful alleles for breeding. Despite recent advances in wheat genomics, most current TILLING methods are low to medium in throughput, being based on PCR amplification of the target genes. We performed a pilot-scale evaluation of TILLING in wheat by next-generation sequencing through exon capture. An oligonucleotide-based enrichment array covering ~2 Mbp of wheat coding sequence was used to carry out exon capture and sequencing on three mutagenised lines of wheat containing previously-identified mutations in the TaGA20ox1 homoeologous genes. After testing different mapping algorithms and settings, candidate SNPs were identified by mapping to the IWGSC wheat Chromosome Survey Sequences. Where sequence data for all three homoeologues were found in the reference, mutant calls were unambiguous; however, where the reference lacked one or two of the homoeologues, captured reads from these genes were mis-mapped to other homoeologues, resulting either in dilution of the variant allele frequency or assignment of mutations to the wrong homoeologue. Competitive PCR assays were used to validate the putative SNPs and estimate cut-off levels for SNP filtering. At least 464 high-confidence SNPs were detected across the three mutagenized lines, including the three known alleles in TaGA20ox1, indicating a mutation rate of ~35 SNPs per Mb, similar to that estimated by PCR-based TILLING. This demonstrates the feasibility of using exon capture for genome re-sequencing as a method of mutation detection in polyploid wheat, but accurate mutation calling will require an improved genomic reference with more comprehensive coverage of homoeologues.