Single nucleotide polymorphism genotyping in polyploid wheat with the Illumina GoldenGate assay

Single nucleotide polymorphisms (SNPs) are indispensable in such applications as association mapping and construction of high-density genetic maps. These applications usually require genotyping of thousands of SNPs in a large number of individuals. Although a number of SNP genotyping assays are available, most of them are designed for SNP genotyping in diploid individuals. Here, we demonstrate that the Illumina GoldenGate assay could be used for SNP genotyping of homozygous tetraploid and hexaploid wheat lines. Genotyping reactions could be carried out directly on genomic DNA without the necessity of preliminary PCR amplification. A total of 53 tetraploid and 38 hexaploid homozygous wheat lines were genotyped at 96 SNP loci. The genotyping error rate estimated after removal of low-quality data was 0 and 1% for tetraploid and hexaploid wheat, respectively. Developed SNP genotyping assays were shown to be useful for genotyping wheat cultivars. This study demonstrated that the GoldenGate assay is a very efficient tool for high-throughput genotyping of polyploid wheat, opening new possibilities for the analysis of genetic variation in wheat and dissection of genetic basis of complex traits using association mapping approach. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

High-throughput genotyping of single nucleotide polymorphisms using new biplex invader technology

The feasibility of large-scale genome-wide association studies of complex human disorders depends on the availability of accurate and efficient genotyping methods for single nucleotide polymorphisms (SNPs). We describe a new platform of the invader assay, a biplex assay, where both alleles are interrogated in a single reaction tube. The assay was evaluated on over 50 different SNPs, with over 20 SNPs genotyped in study cohorts of over 1500 individuals. We assessed the usefulness of the new platform in high-throughput genotyping and compared its accuracy to genotyping results obtained by the traditional monoplex invader assay, TaqMan genotyping and sequencing data. We present representative data for two SNPs in different genes (CD36 and protein tyrosine phosphatase 1β) from a study cohort comprising over 1500 individuals with high or low-normal blood pressure. In this high-throughput application, the biplex invader assay is very accurate, with an error rate of <0.3% and a failure rate of 1.64%. The set-up of the assay is highly automated, facilitating the processing of large numbers of samples simultaneously. We present new analysis tools for the assignment of genotypes that further improve genotyping success. The biplex invader assay with its automated set-up and analysis offers a new efficient high-throughput genotyping platform that is suitable for association studies in large study cohorts.     

High-density genotyping and linkage disequilibrium in the human genome using chromosome 22 as a model

Technology and genetics have advanced to the point where genotyping thousands of individuals at thousands of marker locations around the whole human genome is possible. The whole-genome scan for detection of complex disease genes is a widely discussed topic. We review some of the recent high-density genotyping experiments and discuss related details, particularly the extent and variability of linkage disequilibrium. We also discuss the quality of single nucleotide polymorphisms (SNPs) in public databases and its consequences to the number of SNPs required for large-scale genotyping projects.     

Rapid SNP allele frequency determination in genomic DNA pools by pyrosequencing

Individual genotyping of single nucleotide polymorphisms (SNPs) remains expensive, especially for linkage disequilibrium mapping strategies involving high-throughput SNP genotyping. On one hand, current methods may suit scientific and laboratory needs in regard to accuracy, reproducibility/robustness, and large-scale application. On the other hand, a cheaper and less time-consuming alternative to individual genotyping is the use of SNP allelefrequencies determined in DNA pools. We have developed an accurate and reproducible protocol for allele frequency determination using Pyrosequencing technology in large genomic DNA pools (374 individuals). The measured correlation (R2) in large DNA pools was 0.980. In the context of disease-associated SNPs studies, we compared the allele frequencies between the disease (e.g., type 2 diabetes and obesity) and control groups detected by either individual genotyping or Pyrosequencing of DNA pools. In large pools, the variation between the two methods was 1.5 +/- 0.9%. It may be concluded that the allele frequency determination protocol could reliably detect over 4% differences between populations. The method is economical in regard to amounts of DNA, PCR, and primer extension reagents required. Furthermore, it allows the rapid determination of allelefrequency differences in case/control groups for association studies and susceptibility gene discovery in complex diseases.     

Large-scale genotyping of complex DNA

Genetic studies aimed at understanding the molecular basis of complex human phenotypes require the genotyping of many thousands of single-nucleotide polymorphisms (SNPs) across large numbers of individuals. Public efforts have so far identified over two million common human SNPs; however, the scoring of these SNPs is labor-intensive and requires a substantial amount of automation. Here we describe a simple but effective approach, termed whole-genome sampling analysis (WGSA), for genotyping thousands of SNPs simultaneously in a complex DNA sample without locus-specific primers or automation. Our method amplifies highly reproducible fractions of the genome across multiple DNA samples and calls genotypes at >99% accuracy. We rapidly genotyped 14,548 SNPs in three different human populations and identified a subset of them with significant allele frequency differences between groups. We also determined the ancestral allele for 8,386 SNPs by genotyping chimpanzee and gorilla DNA. WGSA is highly scaleable and enables the creation of ultrahigh density SNP maps for use in genetic studies.     

Estimation of relative allele frequencies of single-nucleotide polymorphisms in different populations by microarray hybridization of pooled DNA

Single-nucleotide polymorphisms (SNPs) are considered useful polymorphic markers for genetic studies of polygenic traits. A new practical approach to high-throughput genotyping of SNPs in a large number of individuals is needed in association study and other studies on relationships between genes and diseases. We have developed an accurate and high-throughput method for determining the allele frequencies by pooling the DNA samples and applying a DNA microarray hybridization analysis. In this method, the combination of the microarray, DNA pooling, probe pair hybridization, and fluorescent ratio analysis solves the dual problems of parallel multiple sample analysis, and parallel multiplex SNP genotyping for association study. Multiple DNA samples are immobilized on a slide and a single hybridization is performed with a pool of allele-specific oligonucleotide probes. The results of this study show that hybridization of microarray from pooled DNA samples can accurately obtain estimates of absolute allele frequencies in a sample pool. This method can also be used to identify differences in allele frequencies in distinct populations. It is amenable to automation and is suitable for immediate utilization for high-throughput genotyping of SNP.     

PCR amplification on magnetic nanoparticles: application for high-throughput single nucleotide polymorphism genotyping

A novel approach for the genotyping of single nucleotide polymorphisms (SNPs) based on solidphase PCR on magnetic nanoparticles (MNPs) is described. PCR products were amplified directly on MNPs. The genotypes of a given SNP were differentiated by hybridization with a pair of allele-specific probes labeled with dual-color fluorescence (Cy3, Cy5). The results were analyzed by scanning the microarray printed with the denatured fluorescent probes on an unmodified glass slide. Electrophoresis analysis indicated that PCR could proceed successfully when MNPs-bound primers were used. Furthermore, nine different samples were genotyped and their fluorescent signals were quantified. Genotyping results showed that three genotypes for the locus were very easily discriminated. The fluorescent ratios (match probe:mismatch probe signal) of homozygous samples were over 9.3, whereas heterozygous samples had ratios near 1.0. Without any purification and concentration of PCR products, this new MNP-PCR based genotyping assay potentially provides a rapid, labor-saving method for genotyping of a large number of individuals.     

应用SNPstream技术检测MASP2基因的多态性

目的：应用一种高通量单核苷酸多态性（SNP）检测方法——SNPstream技术检测甘露聚糖结合凝集素相关丝氨酸蛋白酶-2（MASP2）基因的多态性。方法：收集北京汉族人群SARS病例96例和正常对照96例,用SNPstream技术检测样本的MASP2基因多态性,并用PCR产物直接测序技术对其中一个位点rs2273346进行分型,以验证SNPstream技术的准确性。结果：192例样本的MASP2基因rs2273346位点SNPstream技术分型结果与测序结果完全相符,2种方法的基因型分型结果具有很好的一致性。结论：SNPstream技术是高通量SNP检测的良好工具,准确性高,所需样本量低,在大规模人群SNP筛检中具有良好的发展前景。     

Discovery and development of exome‐based,co‐dominant single nucleotide polymorphism markers in hexaploid wheat (Triticum aestivum L.)

Globally, wheat is the most widely grown crop and one of the three most important crops for human and livestock feed. However, the complex nature of the wheat genome has, until recently, resulted in a lack of single nucleotide polymorphism (SNP)‐based molecular markers of practical use to wheat breeders. Recently, large numbers of SNP‐based wheat markers have been made available via the use of next‐generation sequencing combined with a variety of genotyping platforms. However, many of these markers and platforms have difficulty distinguishing between heterozygote and homozygote individuals and are therefore of limited use to wheat breeders carrying out commercial‐scale breeding programmes. To identify exome‐based co‐dominant SNP‐based assays, which are capable of distinguishing between heterozygotes and homozygotes, we have used targeted re‐sequencing of the wheat exome to generate large amounts of genomic sequences from eight varieties. Using a bioinformatics approach, these sequences have been used to identify 95 266 putative single nucleotide polymorphisms, of which 10 251 were classified as being putatively co‐dominant. Validation of a subset of these putative co‐dominant markers confirmed that 96% were true polymorphisms and 65% were co‐dominant SNP assays. The new co‐dominant markers described here are capable of genotypic classification of a segregating locus in polyploid wheat and can be used on a variety of genotyping platforms; as such, they represent a powerful tool for wheat breeders. These markers and related information have been made publically available on an interactive web‐based database to facilitate their use on genotyping programmes worldwide.     

Global genetic analysis

The introduction of molecular markers in genetic analysis has revolutionized medicine. These molecular markers are genetic variations associated with a predisposition to common diseases and individual variations in drug responses. Identification and genotyping a vast number of genetic polymorphisms in large populations are increasingly important for disease gene identification, pharmacogenetics and population-based studies. Among variations being analyzed, single nucleotide polymorphisms seem to be most useful in large-scale genetic analysis. This review discusses approaches for genetic analysis, use of different markers, and emerging technologies for large-scale genetic analysis where millions of genotyping need to be performed.     

Towards the Genotyping of Fungi: Methods,Benefits and Challenges

Several fungi have recently been described as capable to recombine and drive large genetic diversity in clinical samples and in the environment. Among the genotyping methods, microsatellite analysis is frequently reported as preferred for studying local epidemiology, but single nucleotide polymorphisms represent the best markers for evaluation of recombination, linkage and aneuploidy. The future of typing analyses may reside in strategies capable of cataloging the whole genome and complete microbial diversity. The present review focuses the current strategies employed for fungal genotyping and evaluation of genetic diversity, and the challenges of next generation sequencing with regard to this topic. Typing methods establish the genetic identity of fungal isolates and allow clarification of outbreaks and transmission of strains between individuals, comparison of chronic colonization versus patients carrying unrelated strains, detection of co-evolution of pathogenic and/or drug-resistant strains. The next advances in molecular mycology may revolutionize clinics and redesign concepts of microbial evolution.     

High-throughput detection of multiple genetic polymorphisms influencing drug metabolism with mismatch primers in allele-specific polymerase chain reaction

Because of genetic polymorphisms of drug-metabolizing enzyme genes, the activities of the enzymes in humans vary widely and alter the metabolism of commonly used clinical agents. Severe adverse effects or resistance to therapy may result. We have developed a rapid and high-throughput genotyping method for detecting polymorphisms of the drug-metabolizing enzyme genes CYP2C9*3, CYP2C19*2, *3, CYP2D6*2, *4, *10, *14, *21, NAT2*5, *6, *7, and TPMT*3 using allele-specific polymerase chain reaction (PCR) with mismatch primers (ASPCR-MP) and CYP2D6*5, *36, and CYP2D6xN using stepdown PCR with detection by SYBR Green I. We analyzed genomic DNA from 139 Japanese volunteers. Identical genotyping results were obtained by using ASPCR-MP, stepdown PCR, and conventional PCR. We found that the methods clearly differentiate three specific profiles with no overlap in the signals. Moreover, both ASPCR-MP and stepdown PCR for genotyping took less than 3-4h. To our knowledge, this is the first report of successful simultaneous detection of multiple genetic polymorphisms with point mutations using ASPCR-MP or multiple genetic polymorphisms with large structural alterations using stepdown PCR. In conclusion, ASPCR-MP and stepdown PCR appear to be suitable for large clinical and epidemiological studies as methods that enable highly sensitive genotyping and yield a high-throughput.     

Simultaneous analysis of multiple polymorphic loci using amplified sequence polymorphisms (ASPs)

In this paper we present a systematic approach to gene mapping and genotyping based on the simultaneous analysis of multiple amplified sequence polymorphisms (ASPs). These genetic markers measure variation in DNA sequences which have been amplified by a polymerase and/or a ligase. The amplified sequence lengths are determined by appropriate choice of oligonucleotides used in the amplification reaction. We describe three classes of ASPs: restriction site polymorphisms, sequence length polymorphisms, and DNA base pair changes not associated with restriction sites. Simultaneous analysis of multiple ASPs using a modified automated DNA sequencing apparatus should be possible because amplification with oligonucleotides provides control over the fragment lengths generated. Development of an automated ASP technology is therefore the next logical step for efficient gene mapping and genotyping of individuals. With this technology, one gel would be sufficient to indicate the most probable locations of a gene and a second gel would permit the selection of the correct location while simultaneously providing a fine structure map.     

A simple and cost-effective method for rapid genotyping of insertion/deletion polymorphisms

We developed a simple method, based on the TaqMan technology, for fast genotyping of insertion/deletion polymorphisms of known location. The genotypes of 22 CEPH individuals, previously ascertained by conventional methods, were confirmed in the new assay without manual, time-consuming, post-PCR analysis. We propose to expand the application of TaqMan probes for population screening of insertion/deletion polymorphisms in which the exact endpoints of the insertion/deletion are known. The method can be applied to polymorphisms of any size and can be used for different applications such as diagnostics, genome variation, and species identification.     

淋球菌AFLP基因分型的初步研究

采用扩增片段长度多态性技术(AFLP)对奈瑟氏淋球菌菌株进行基因分型研究。以EcoRI和MesI酶切26株淋球菌临床分离株基因组,并进行AFLP分析。同一地区的淋球菌分离株之间存在相当大的DNA多态性。AFLP是鉴别淋球菌临床分离株有用而敏感的基因分型技术,有助于了解流行淋球菌菌株的来源、流行菌株之间的克隆相关性,以及抗生素耐药性菌株的传播情况。     

Population-scale analysis of human microsatellites reveals novel sources of exonic variation

Using our microsatellite specific genotyping method, we analyzed tandem repeats, which are known to be highly variable with some recognized as biomarkers causative of disease, in over 500 individuals who were exon sequenced in a 1000 Genomes Project pilot study. We were able to genotype over 97% of the microsatellite loci in the targeted regions. A total of 25,115 variations were observed, including repeat length and single nucleotide polymorphisms, corresponding to an average of 45.6 variations per individual and a density of 1.1 variations per kilobase. Standard variant detection did not report 94.2% of the exonic repeat length variations in part because the alignment techniques are not ideal for repetitive regions. Additionally some standard variation detection tools rely on a database of known variations, making them less likely to call repeat length variations as only a small percent of these loci (~ 6000) have been accurately characterized. A subset of the hundreds of non-synonymous variations we identified was experimentally validated, indicating an accuracy of 96.5% for our microsatellite-based genotyping method, with some novel variants identified in genes associated with cancer. We propose that microsatellite-based genotyping be used as a part of large scale sequencing studies to identify novel variants.     

Characterization of cytochrome P450 2D6 alleles using the Invader system

The cytochrome p450 (CYP) superfamily comprises enzymes that play an essential role in the transformation of medically relevant compounds. Accurate genotyping of polymorphisms in members of this family is drawing increasing interest because certain allelic variants may result in either loss of efficacy or toxic accumulation of therapeutic agents. Debrisoquine 4-hydroxylase, or CYP2D6, is among the most widely studied of the CYPs. The complexity of the CYP2D6 genomic region, including pseudogenes, gene deletions, and gene duplications, has offered numerous challenges to developing a genotyping strategy. We describe a comprehensive CYP2D6 genotyping strategy that employs both a PCR/Invader genotyping assay system and an Invader genomic copy number assay The Invader system is a homogeneous, isothermal, highly specific, and robust signal amplification system. Resultsfrom II CYP2D6 assays in an alle frequency study compare well to published allele frequency values for Caucasians. Further, Invader assays provided unambiguous genotyping determinations for 100% of the 171 samples that yielded a visible PCR product on an agarose gel. A copy number assay yielded only one equivocal result in 205 samples. We identified 17 single-copy individuals and 17 three-copy (or more) individuals.     

Association mapping in forest trees and fruit crops

Association mapping (AM), also known as linkage disequilibrium (LD) mapping, is a viable approach to overcome limitations of pedigree-based quantitative trait loci (QTL) mapping. In AM, genotypic and phenotypic correlations are investigated in unrelated individuals. Unlike QTL mapping, AM takes advantage of both LD and historical recombination present within the gene pool of an organism, thus utilizing a broader reference population. In plants, AM has been used in model species with available genomic resources. Pursuing AM in tree species requires both genotyping and phenotyping of large populations with unique architectures. Recently, genome sequences and genomic resources for forest and fruit crops have become available. Due to abundance of single nucleotide polymorphisms (SNPs) within a genome, along with availability of high-throughput resequencing methods, SNPs can be effectively used for genotyping trees. In addition to DNA polymorphisms, copy number variations (CNVs) in the form of deletions, duplications, and insertions also play major roles in control of expression of phenotypic traits. Thus, CNVs could provide yet another valuable resource, beyond those of microsatellite and SNP variations, for pursuing genomic studies. As genome-wide SNP data are generated from high-throughput sequencing efforts, these could be readily reanalysed to identify CNVs, and subsequently used for AM studies. However, forest and fruit crops possess unique architectural and biological features that ought to be taken into consideration when collecting genotyping and phenotyping data, as these will also dictate which AM strategies should be pursued. These unique features as well as their impact on undertaking AM studies are outlined and discussed.