High-throughput SNP genotyping

Whole genome approaches using single nucleotide polymorphism (SNP) markers have the potential to transform complex disease genetics and expedite pharmacogenetics research. This has led to a requirement for high-throughput SNP genotyping platforms. Development of a successful high-throughput genotyping platform depends on coupling reliable assay chemistry with an appropriate detection system to maximise efficiency with respect to accuracy, speed and cost. Current technology platforms are able to deliver throughputs in excess of 100 000 genotypes per day, with an accuracy of >99%, at a cost of 20-30 cents per genotype. In order to meet the demands of the coming years, however, genotyping platforms need to deliver throughputs in the order of one million genotypes per day at a cost of only a few cents per genotype. In addition, DNA template requirements must be minimised such that hundreds of thousands of SNPs can be interrogated using a relatively small amount of genomic DNA. As such, it is predicted that the next generation of high-throughput genotyping platforms will exploit large-scale multiplex reactions and solid phase assay detection systems.     

Detection of epidermal growth factor receptor (EGFR) mutations in non-small cell lung cancer (NSCLC) using a fully automated system with a nano-scale engineered biomagnetite

A fully automated system using nano-scale engineered biomagnetite was developed to detect mutations in the epidermal growth factor receptor (EGFR) gene in non-small cell lung cancer (NSCLC). Bacterial magnetic particles (BacMPs) were isolated from the magnetic bacterium Magnetospirillum magneticum AMB-1 and conjugated to streptavidin. Biotin-labeled target PCR products were then captured with the BacMPs, hybridized with the detection probe and detected by fluorescence signaling. The process was performed using a newly designed automated processor equipped with an XYZ mobile arm containing a 96-way automated pipetter, reagent dispenser and fluorescence detector. Two types of somatic mutations (in-frame deletions and point substitutions) in the EGFR gene were successfully identified within 3.5h using this system, suggesting that this system could be used in clinical tests of EGFR gene mutations in lung cancer, and potentially other cancer, patients. Additionally, a very low mutation rate could be detected in these samples.     

High-throughput SNP detection by using DNA pooling and denaturing high performance liquid chromatography (DHPLC)

One of the critical steps in the positional cloning of a complex disease gene involves association analysis between a phenotype and a set of densely spaced diallelic markers, typically single nucleotide repeats (SNPs), covering the region of interest. However, the effort and cost of detecting sufficient numbers of SNPs across relatively large physical distances represents a significant rate-limiting step. We have explored DNA pooling, in conjunction with denaturing high performance liquid chromatography (DHPLC), as a possible strategy for augmenting the efficiency, economy, and throughput of SNP detection. DHPLC is traditionally used to detect variants in polymerase chain reaction products containing both allelic forms of a polymorphism (e.g., heterozygotes or a 1:1 mix of both alleles) via heteroduplex separation and thereby requires separate analyses of multiple individual test samples. We have adapted this technology to identify variants in pooled DNA. To evaluate the utility and sensitivity of this approach, we constructed DNA pools comprised of 20 previously genotyped individuals with a frequency representation of 0%-50% for the variant allele. Mutation detection was performed by using temperature-modulated heteroduplex formation/DHPLC and dye-terminator sequencing. Using DHPLC, we could consistently detect SNPs at lower than 5% frequency, corresponding to the detection of one variant allele in a pool of 20 alleles. In contrast, fluorescent sequencing detected variants in the same pools only if the frequency of the less common allele was at least 10%. We conclude that DNA pooling of samples for DHPLC analysis is an effective way to increase throughput efficiency of SNP detection.     

High-throughput genotyping in citrus accessions using an SNP genotyping array

We developed a 384 multiplexed SNP array, named CitSGA-1, for the genotyping of Citrus cultivars, and evaluated the performance and reliability of the genotyping. SNPs were surveyed by direct sequence comparison of the sequence tagged site (STS) fragment amplified from genomic DNA of cultivars representing the genetic diversity of citrus breeding in Japan. Among 1497 SNPs candidates, 384 SNPs for a high-throughput genotyping array were selected based on physical parameters of Illumina’s bead array criteria. The assay using CitSGA-1 was applied to a hybrid population of 88 progeny and 103 citrus accessions for breeding in Japan, which resulted in 73,726 SNP calls. A total of 351 SNPs (91 %) could call different genotypes among the DNA samples, resulting in a success rate for the assay comparable to previously reported rates for other plant species. To confirm the reliability of SNP genotype calls, parentage analysis was applied, and it indicated that the number of reliable SNPs and corresponding STSs were 276 and 213, respectively. The multiplexed SNP genotyping array reported here will be useful for the efficient construction of linkage map, for the detection of markers for marker-assisted breeding, and for the identification of cultivars.     

High-throughput SNP genotyping on universal bead arrays

We have developed a flexible, accurate and highly multiplexed SNP genotyping assay for high-throughput genetic analysis of large populations on a bead array platform. The novel genotyping system combines high assay conversion rate and data quality with >1500 multiplexing, and Array of Arrays formats. Genotyping assay oligos corresponding to specific SNP sequences are each linked to a unique sequence (address) that can hybridize to its complementary strand on universal arrays. The arrays are made of beads located in microwells of optical fiber bundles (Sentrix Array Matrix) or silicon slides (Sentrix BeadChip). The optical fiber bundles are further organized into a matrix that matches a 96-well microtiter plate. The arrays on the silicon slides are multi-channel pipette compatible for loading multiple samples onto a single silicon slide. These formats allow many samples to be processed in parallel. This genotyping system enables investigators to generate approximately 300,000 genotypes per day with minimal equipment requirements and greater than 1.6 million genotypes per day in a robotics-assisted process. With a streamlined and comprehensive assay, this system brings a new level of flexibility, throughput, and affordability to genetic research.     

Determination of microsatellite repeats in the human thyroid peroxidase (TPOX) gene using an automated gene analysis system with nanoscale engineered biomagnetite

The number of repeat in the microsatellite region (AATG)(5-14) of the human thyroid peroxidase gene (TOPX) was determined using an automated DNA analysis system with nano-scale engineered biomagnetite. Thermal melting curve analysis of DNA duplexes on biomagnetite indicated that shorter repeat sequences (less than 9 repeats) were easily discriminated. However, it was difficult to determine the number of repeats at more than nine. In order to improve the selectivity of this method for the longer repeats, a "double probe hybridization assay" was performed in which an intermediate probe was used to replace a target repeat sequence having more than 9 repeats with a shorter sequence possessing less than 9 repeats. Thermal probe melting curve analyses and Tm determination confirmed that the target with 10 repeats was converted to 5 repeats, 11 repeats converted to 4 and 12 to 3, respectively. Furthermore, rapid determination of repeat numbers was possible by measuring fluorescence intensities obtained by probe dissociation at 56 and 66 degrees C, and 40, 60 and 80 degrees C for signal normalization.     

High-throughput SNP genotyping by single-tube PCR with Tm-shift primers

Despite many recent advances in high-throughput single nucleotide polymorphism (SNP) genotyping technologies, there is still a great need for inexpensive and flexible methods with a reasonable throughput. Here we report substantial modifications and improvements to an existing homogenous allele-specific PCR-based SNP genotyping method, making it an attractive new option for researchers engaging in candidate gene studies or following up on genome-wide scans. In this advanced version of the melting temperature (Tm)-shift SNP genotyping method, we attach two GC-rich tails of different lengths to allele-specific PCR primers, such that SNP alleles in genomic DNA samples can be discriminated by the Tms of the PCR products. We have validated 306 SNP assays using this method and achieved a success rate in assay development of greater than 83% under uniform PCR conditions. We have developed a standalone software application to automatically assign genotypes directly from melting curve data. To demonstrate the accuracy of this method, we typed 592 individuals for 6 SNPs and showed a high call rate (>98%) and high accuracy (>99.9%). With this method, 6-10,000 samples can be genotyped per day using a single 384-well real-time thermal cycler with 2-4 standard 384-well PCR instruments.     

Facile SNP detection using bifunctional, cross-linking oligonucleotide probes

A facile, sensitive method for detecting specific sequences of oligonucleotides was developed. Detection of DNA sequences with single nucleotide discrimination is achieved by combining the selectivity of hybridization with an efficient cross-linking reaction. Readily synthesized bifunctional oligonucleotide probes containing a modified pyrimidine that is capable of forming interstrand cross-links under mild oxidative conditions internally, and biotin at their 5′-termini were used to discriminate between 16-nt long sites in plasmid DNA that differ by a single nucleotide. The target sequence was detected via fluorescence spectroscopy by utilizing conjugates of avidin and horseradish peroxidase in a microtiter plate assay. The method is able to detect as little as 250 fmol of target without using PCR and exhibits single nucleotide discrimination that approaches 200:1. In principle, this method is capable of probing any target sequence containing a 2′-deoxyadenosine.     

High-throughput SNP genotyping with the GoldenGate assay in maize

Single nucleotide polymorphisms (SNPs) are abundant and evenly distributed throughout the genomes of most plant species. They have become an ideal marker system for genetic research in many crops. Several high throughput platforms have been developed that allow rapid and simultaneous genotyping of up to a million SNP markers. In this study, a custom GoldenGate assay containing 1,536 SNPs was developed based on public SNP information for maize and used to genotype two recombinant inbred line (RIL) populations (Zong3 x 87-1, and B73 x By804) and a panel of 154 diverse inbred lines. Over 90% of the SNPs were successfully scored in the diversity panel and the two RIL populations, with a genotyping error rate of less than 2%. A total of 975 SNP markers detected polymorphism in at least one of the two mapping populations, with a polymorphic rate of 38.5% in Zong3 x 87-1 and 52.6% in B73 x By804. The polymorphic SNPs in B73 x By804 have been integrated with previously mapped simple sequence repeat markers to construct a high-density linkage map containing 662 markers with a total length of 1,673.7 cM and an average of 2.53 cM between two markers. The minor allelic frequency (MAF) was distributed evenly across 10 continued classes from 0.05 to 0.5, and about 16% of the SNP markers had a MAF below 10% in the diversity panel. Polymorphism rates for individual SNP markers in pair-wise comparisons of genotypes tested ranged from 0.3 to 63.8% with an average of 36.3%. Most SNPs used in this GoldenGate assay appear to be equally useful for diversity analysis, marker-trait association studies, and marker-aided breeding.     

SNP genotyping using microsphere-linked PNA and flow cytometric detection.

BACKGROUND: Single nucleotide polymorphisms (SNPs) represent the most frequent form of genetic variations. Some of the most sensitive methods for SNP genotyping employ synthetic oligonucleotides, such as the peptide nucleic acid (PNA). We introduce a new method combining allele-specific hybridization, PNA technology, and flow cytometric detection. We tested the design by genotyping a Danish basal cell carcinoma cohort of 80 individuals for an A/C SNP in exon 6 of the XPD gene. METHODS: Genomic DNA was amplified by a two-step polymerase chain reaction (PCR) in the presence of fluorescein-dyed primers and fluorescein-12-dUTP. The allele-specific PNA molecules were covalently coupled to carboxylated microspheres with and without rhodamine. Allele-specific hybridization between PCR products and immobilized PNA was carried out at 60 degrees C followed by flow cytometric detection. RESULTS: We present a fully functional two-bead genotyping system based on PNA capture and flow cytometric detection used for the correct and fast regenotyping of a Danish basal cell carcinoma cohort. CONCLUSIONS: This new assay presents a simple, rapid, and robust method for SNP genotyping for laboratories equipped with a standard flow cytometer. Moreover, this system offers potential for multiplexing and will be operational for middle-scale genotyping.     

High-throughput detection of mutations responsible for childhood hearing loss using resequencing microarrays

Background  

Despite current knowledge of mutations in 45 genes that can cause nonsyndromic sensorineural hearing loss (SNHL), no unified clinical test has been developed that can comprehensively detect mutations in multiple genes. We therefore designed Affymetrix resequencing microarrays capable of resequencing 13 genes mutated in SNHL (GJB2, GJB6, CDH23, KCNE1, KCNQ1, MYO7A, OTOF, PDS, MYO6, SLC26A5, TMIE, TMPRSS3, USH1C). We present results from hearing loss arrays developed in two different research facilities and highlight some of the approaches we adopted to enhance the applicability of resequencing arrays in a clinical setting.     

SNP detection in transforming growth factor-beta1 gene using bacterial magnetic particles

A single nucleotide polymorphism (SNP) within the transforming growth factor-beta1 (TGF-beta1) gene was detected by hybridization-based method using bacterial magnetic particles (BMPs). TGF-beta1 is commonly associated with a single base change resulting in a Leu(10)-->Pro (T(869)-->C) polymorphism and is a genetic marker for susceptibility to osteoporosis. Short (9 bases) and specific probes were designed to detect SNP in TGF-beta1. Detection probes were immobilized on BMPs using cross-linking reagents. TGF-beta1 PCR products (139 bp) were labeled with the fluorescent dye coumarin and hybridized with detection probes on BMPs. Complementary hybridized targets gave over four times higher fluorescent intensities, compared with one base mismatched hybridizations. The SNP genotype was successfully discriminated using this technique.     

基于SNP数据检测染色体拷贝数结果可信度分析

利用SNP数据检测肿瘤细胞染色体拷贝数变异是癌症相关研究的一个热点,目前已有多种方法可以通过分析SNP array数据检测染色体拷贝数。然而在某些情况下,这些检测方法检测结果与真实拷贝数具有一定错误率。目前并没有方法研究预测结果发生错误的规律。本文分别分析了GPHMM,ASCAT两种检测方法结果信息熵与检测正确率的关系,发现检测正确率与信息熵存在很强的相关性。通过对比不同肿瘤细胞比例下信息熵与正确率关系,本文发现随着肿瘤细胞比例的增大,检测结果信息熵平均值增大,方差减小;同时平均检测正确率也越来越大,方差显著减小。这些结果显示信息熵的大小可以反映出检测结果正确率的高低。最后,本文以高肿瘤细胞比例下拷贝数检测结果为例,研究了在变异类型单一,信息熵小的情况下,染色体倍性检测的正确率。结果表明信息熵可以作为衡量检测结果可信度的指标：即信息熵越高,检测结果越可信。     

High-throughput detection of knockdown resistance in Myzus persicae using allelic discriminating quantitative PCR

The peach-potato aphid Myzus persicae (Sulzer) has developed resistance to pyrethroid insecticides as a result of a mechanism conferring reduced nervous system sensitivity, termed knockdown resistance (kdr). This reduced sensitivity is caused by two mutations, L1014F (kdr) and M918T (super-kdr), in the para-type voltage-gated sodium channel. We have developed a diagnostic dose bioassay to detect kdr and provide preliminary information on the genotype present. We also developed two allelic discrimination PCR assays to determine precisely the genotypes of the two mutations (L1014F and M918T) in individual M. persicae using fluorescent Taqman MGB probes. In combination with assays for elevated carboxylesterase levels and modified acetylcholinesterase (MACE), this suite of assays allows for rapid high-throughput diagnosis, in individual aphids, of the three main resistance mechanisms of practical importance in the UK.     

Disease-driven detection of differential inherited SNP modules from SNP network

Detection of the synergetic effects between variants, such as single-nucleotide polymorphisms (SNPs), is crucial for understanding the genetic characters of complex diseases. Here, we proposed a two-step approach to detect differentially inherited SNP modules (synergetic SNP units) from a SNP network. First, SNP-SNP interactions are identified based on prior biological knowledge, such as their adjacency on the chromosome or degree of relatedness between the functional relationships of their genes. These interactions form SNP networks. Second, disease-risk SNP modules (or sub-networks) are prioritised by their differentially inherited properties in IBD (Identity by Descent) profiles of affected and unaffected sibpairs. The search process is driven by the disease information and follows the structure of a SNP network. Simulation studies have indicated that this approach achieves high accuracy and a low false-positive rate in the identification of known disease-susceptible SNPs. Applying this method to an alcoholism dataset, we found that flexible patterns of susceptible SNP combinations do play a role in complex diseases, and some known genes were detected through these risk SNP modules. One example is GRM7, a known alcoholism gene successfully detected by a SNP module comprised of two SNPs, but neither of the two SNPs was significantly associated with the disease in single-locus analysis. These identified genes are also enriched in some pathways associated with alcoholism, including the calcium signalling pathway, axon guidance and neuroactive ligand-receptor interaction. The integration of network biology and genetic analysis provides putative functional bridges between genetic variants and candidate genes or pathways, thereby providing new insight into the aetiology of complex diseases.