Multiplexed SNP genotyping using the Qbead™ system: a quantum dot-encoded microsphere-based assay

We have developed a new method using the Qbead™ system for high-throughput genotyping of single nucleotide polymorphisms (SNPs). The Qbead system employs fluorescent Qdot™ semiconductor nanocrystals, also known as quantum dots, to encode microspheres that subsequently can be used as a platform for multiplexed assays. By combining mixtures of quantum dots with distinct emission wavelengths and intensities, unique spectral ‘barcodes’ are created that enable the high levels of multiplexing required for complex genetic analyses. Here, we applied the Qbead system to SNP genotyping by encoding microspheres conjugated to allele-specific oligonucleotides. After hybridization of oligonucleotides to amplicons produced by multiplexed PCR of genomic DNA, individual microspheres are analyzed by flow cytometry and each SNP is distinguished by its unique spectral barcode. Using 10 model SNPs, we validated the Qbead system as an accurate and reliable technique for multiplexed SNP genotyping. By modifying the types of probes conjugated to microspheres, the Qbead system can easily be adapted to other assay chemistries for SNP genotyping as well as to other applications such as analysis of gene expression and protein–protein interactions. With its capability for high-throughput automation, the Qbead system has the potential to be a robust and cost-effective platform for a number of applications.     

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.     

Universal SNP genotyping assay with fluorescence polarization detection

The degree of fluorescence polarization (FP) of a fluorescent molecule is a reflection of its molecular weight (Mr). FP is therefore a useful detection methodfor homogeneous assays in which the starting reagents and products differ significantly in Mr. We have previously shown that FP is a good detection method for the single-base extension and the 5'-nuclease assays. In this report, we describe a universal, optimized single-base extension assay for genotyping single nucleotide polymorphisms (SNPs). This assay, which we named the template-directed dye-terminator incorporation assay with fluorescence polarization detection (FP-TDI), uses four spectrally distinct dye terminators to achieve universal assay conditions. Even without optimization, approximately 70% of all SNP markers tested yielded robust assays. The addition of an E. coli ssDNA-binding protein just before the FP reading significantly increased FP values of the products and brought the success rate of FP-TDI assays up to 90%. Increasing the amount of dye terminators and reducing the number of thermal cycles in the single-base extension step of the assay increased the separation of the FP values benveen the products corresponding to different genotypes and improved the success rate of the assay to 100%. In this study the genomic DNA samples of 90 individuals were typed for a total of 38 FP-TDI assays (using both the sense and antisense TDI primers for 19 SNP markers). With the previously described modifications, the FP-TDI assay gave unambiguous genotyping data for all the samples tested in the 38 FP-TDI assays. When the genotypes determined by the FP-TDI and 5'-nuclease assays were compared, they were in 100% concordance for all experiments (a total of 3420 genotypes). The four-dye-terminator master mixture described here can be used for assaying any SNP marker and greatly simplifies the SNP genotyping assay design.     

The SNPlex genotyping system: a flexible and scalable platform for SNP genotyping.

We developed the SNPlex Genotyping System to address the need for accurate genotyping data, high sample throughput, study design flexibility, and cost efficiency. The system uses oligonucleotide ligation/polymerase chain reaction and capillary electrophoresis to analyze bi-allelic single nucleotide polymorphism genotypes. It is well suited for single nucleotide polymorphism genotyping efforts in which throughput and cost efficiency are essential. The SNPlex Genotyping System offers a high degree of flexibility and scalability, allowing the selection of custom-defined sets of SNPs for medium- to high-throughput genotyping projects. It is therefore suitable for a broad range of study designs. In this article we describe the principle and applications of the SNPlex Genotyping System, as well as a set of single nucleotide polymorphism selection tools and validated assay resources that accelerate the assay design process. We developed the control pool, an oligonucleotide ligation probe set for training and quality-control purposes, which interrogates 48 SNPs simultaneously. We present performance data from this control pool obtained by testing genomic DNA samples from 44 individuals. in addition, we present data from a study that analyzed 521 SNPs in 92 individuals. Combined, both studies show the SNPlex Genotyping system to have a 99.32% overall call rate, 99.95% precision, and 99.84% concordance with genotypes analyzed by TaqMan probe-based assays. The SNPlex Genotyping System is an efficient and reliable tool for a broad range of genotyping applications, supported by applications for study design, data analysis, and data management.     

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.     

Optimization of a SNP assay for genotyping Theobroma cacao under field conditions

The tropical tree crop Theobroma cacao L. is grown commercially for its beans, which are used in the production of cocoa butter and chocolate. Although the upper Amazon region is the center of origin for cacao, 70% of the world??s supply of cacao beans currently comes from small farms in West Africa. While cacao breeding programs in producer nations are the source of improved planting material, modern marker-based breeding is difficult to perform due to the lack of genotyping facilities in these countries. While DNA extraction can be routinely performed, the equipment needed to analyze simple sequence repeats (SSRs) is seldom available, forcing the outsourcing of genotyping to foreign laboratories and delaying the breeding process. We describe a 5?? nuclease (TaqMan)-based single nucleotide polymorphism (SNP) assay for genotyping cacao plants under conditions similar to those found in most cacao-producing areas. The assay was tested under field conditions by planting open pollinated seeds of seven pods from four different maternal plants. The resulting 171 seedlings were successfully genotyped with 18 SNP markers representing 12 loci. The ability to use temperature-stable reagents and rapid DNA extraction methods is also explored. Additionally, by examining the seedling genotypes for the SNP markers and 14 additional SSR markers, we investigated whether seeds in a pod are the result of single or multiple pollination events. This simple, effective method of genotyping cacao seedlings in the field should allow for more efficient resource management of seed gardens and is currently being implemented in Ghana.     

Application of multiple response optimization design to quantum dot-encoded microsphere bioconjugates hybridization assay

The optimization of DNA hybridization for genotyping assays is a complex experimental problem that depends on multiple factors such as assay formats, fluorescent probes, target sequence, experimental conditions, and data analysis. Quantum dot-doped particle bioconjugates have been previously described as fluorescent probes to identify single nucleotide polymorphisms even though this advanced fluorescent material has shown structural instability in aqueous environments. To achieve the optimization of DNA hybridization to quantum dot-doped particle bioconjugates in suspension while maximizing the stability of the probe materials, a nonsequential optimization approach was evaluated. The design of experiment with response surface methodology and multiple optimization response was used to maximize the recovery of fluorescent probe at the end of the assay simultaneously with the optimization of target–probe binding. Hybridization efficiency was evaluated by the attachment of fluorescent oligonucleotides to the fluorescent probe through continuous flow cytometry detection. Optimal conditions were predicted with the model and tested for the identification of single nucleotide polymorphisms. The design of experiment has been shown to significantly improve biochemistry and biotechnology optimization processes. Here we demonstrate the potential of this statistical approach to facilitate the optimization of experimental protocol that involves material science and molecular biology.     

SNP genotyping using single-tube fluorescent bidirectional PCR

SNP genotyping is a well-populatedfield with a large number of assay formats offering accurate allelic discrimination. However, there remains a discord between the ultimate goal of rapid, inexpensive assays that do not require complex design considerations and involved optimization strategies. We describe the first integration of bidirectional allele-specific amplification, SYBR Green I, and rapid-cycle PCR to provide a homogeneous SNP-typing assay. Wild-type, mutant, and heterozygous alleles were easily discriminated in a single tube using melt curve profiling of PCR products alone. We demonstrate the effectiveness and reliability of this assay with a blinded trial using clinical samples from individuals with sickle cell anemia, sickle cell trait, or unaffected individuals. The tests were completed in less than 30 min without expensive fluorogenic probes, prohibiting design rules, or lengthy downstream processing for product analysis.     

Genosnip: SNP genotyping by MALDI-TOF MS using photocleavable oligonucleotides

A photocleavable o-nitrobenzyl CE phosphoramidite building-block was synthesised and incorporated within oligonucleotides. After allele-specific primer extension, desalting was performed using genostrep purification plates. Release of the SNP information containing part through photocleavage created shortened molecules that are easily accessible for MALDI-TOF analysis. Additionally, incorporation of mass modified nucleosides enables flexible design of multiplex genotyping.     

SNPWave: a flexible multiplexed SNP genotyping technology

Scalable multiplexed amplification technologies are needed for cost-effective large-scale genotyping of genetic markers such as single nucleotide polymorphisms (SNPs). We present SNPWave, a novel SNP genotyping technology to detect various subsets of sequences in a flexible fashion in a fixed detection format. SNPWave is based on highly multiplexed ligation, followed by amplification of up to 20 ligated probes in a single PCR. Depending on the multiplexing level of the ligation reaction, the latter employs selective amplification using the amplified fragment length polymorphism (AFLP) technology. Detection of SNPWave reaction products is based on size separation on a sequencing instrument with multiple fluorescence labels and short run times. The SNPWave technique is illustrated by a 100-plex genotyping assay for Arabidopsis, a 40-plex assay for tomato and a 10-plex assay for Caenorhabditis elegans, detected on the MegaBACE 1000 capillary sequencer.     

SNPWaveTM: a flexible multiplexed SNP genotyping technology

Scalable multiplexed amplification technologies are needed for cost-effective large-scale genotyping of genetic markers such as single nucleotide polymorphisms (SNPs). We present SNPWave^TM, a novel SNP genotyping technology to detect various subsets of sequences in a flexible fashion in a fixed detection format. SNPWave is based on highly multiplexed ligation, followed by amplification of up to 20 ligated probes in a single PCR. Depending on the multiplexing level of the ligation reaction, the latter employs selective amplification using the amplified fragment length polymorphism (AFLP®) technology. Detection of SNPWave reaction products is based on size separation on a sequencing instrument with multiple fluorescence labels and short run times. The SNPWave technique is illustrated by a 100-plex genotyping assay for Arabidopsis, a 40-plex assay for tomato and a 10-plex assay for Caenorhabditis elegans, detected on the MegaBACE 1000 capillary sequencer.     

SYBR green dye-based probe-free SNP genotyping: Introduction of T-Plex real-time PCR assay

Single-nucleotide polymorphism (SNP) genotyping is widely used in genetic association studies to characterize genetic factors underlying inherited traits. Despite many recent advances in high-throughput SNP genotyping, inexpensive and flexible methods with reasonable throughput levels are still needed. Real-time PCR methods for discovering and genotyping SNPs are becoming increasingly important in various fields of biology. In this study, we introduce a new, single-tube strategy that combines the tetra-primer ARMS PCR assay, SYBR Green I-based real-time PCR, and melting-point analysis with primer design strategies to detect the SNP of interest. This assay, T-Plex real-time PCR, is based on the T_m discrimination of the amplified allele-specific amplicons in a single tube. The specificity, sensitivity, and robustness of the assay were evaluated for common mutations in the FV, PII, MTHFR, and FGFR3 genes. We believe that T-Plex real-time PCR would be a useful alternative for either individual genotyping requests or large epidemiological studies.     

PanSNPdb: the Pan-Asian SNP genotyping database

The HUGO Pan-Asian SNP consortium conducted the largest survey to date of human genetic diversity among Asians by sampling 1,719 unrelated individuals among 71 populations from China, India, Indonesia, Japan, Malaysia, the Philippines, Singapore, South Korea, Taiwan, and Thailand. We have constructed a database (PanSNPdb), which contains these data and various new analyses of them. PanSNPdb is a research resource in the analysis of the population structure of Asian peoples, including linkage disequilibrium patterns, haplotype distributions, and copy number variations. Furthermore, PanSNPdb provides an interactive comparison with other SNP and CNV databases, including HapMap3, JSNP, dbSNP and DGV and thus provides a comprehensive resource of human genetic diversity. The information is accessible via a widely accepted graphical interface used in many genetic variation databases. Unrestricted access to PanSNPdb and any associated files is available at: http://www4a.biotec.or.th/PASNP.     

Development of a single-nucleotide polymorphism (SNP) assay for genotyping of Pandora neoaphidis

Pandora neoaphidis (Entomophthoromycotina, Entomophthorales) is one of the most important fungal pathogens of aphids with great potential as a biological control agent. Development of tools that allow high-resolution monitoring of P. neoaphidis in the environment is a prerequisite for the successful implementation of biological control strategies. In this study, a single-nucleotide polymorphism (SNP) assay was developed. The assay targets 13 SNPs identified in 6 genomic regions including the largest subunit of nuclear RNA polymerase II (RPB1) gene, the second-largest subunit of nuclear RNA polymerase II (RPB2) gene, the β-tubulin (BTUB) gene, the elongation factor 1α-like (EFL) gene, the large subunit (LSU) rRNA gene, and the small subunit (SSU) rRNA gene together with the internal transcribed spacer (ITS). The assay allowed the discrimination of 15 different SNP profiles among 19 P. neoaphidis isolates and 4 P. neoaphidis-infected cadavers. Results showed that the assay is applicable to DNA extracted from infected aphids allowing genotyping of the fungus without cultivation. The SNP assay provides an efficient tool for investigation of population structures and dynamics of P. neoaphidis, as well as its persistence and epidemiology in agro-ecosystems. Furthermore, it constitutes a powerful approach for monitoring potential biological control strains of P. neoaphidis in the environment.     

Simple SNP typing assay using a base-discriminating fluorescent probe

We have developed a new concept involving a single-step homogeneous method for single-nucleotide polymorphism (SNP) typing. In this method, a probe containing base-discriminating fluorescent (BDF) bases is added to a sample solution. BDF base-containing DNA usually shows only a weak fluorescence, but emits a strong blue fluorescence when it recognizes a target base at a specific site in a hybridized strand. By utilizing this feature, a simple mix-and-read SNP typing assay was achieved without any tedious probe-designing or washing processes for exclusion of hybridization error or any addition of DNA-modifying enzymes. This is very different from conventional methods. We simultaneously analyzed a number of samples with ease, with a high accuracy, using our BDF assay.     

Mutagenic primer design for mismatch PCR-RFLP SNP genotyping using a genetic algorithm

Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) is useful in small-scale basic research studies of complex genetic diseases that are associated with single nucleotide polymorphism (SNP). Designing a feasible primer pair is an important work before performing PCR-RFLP for SNP genotyping. However, in many cases, restriction enzymes to discriminate the target SNP resulting in the primer design is not applicable. A mutagenic primer is introduced to solve this problem. GA-based Mismatch PCR-RFLP Primers Design (GAMPD) provides a method that uses a genetic algorithm to search for optimal mutagenic primers and available restriction enzymes from REBASE. In order to improve the efficiency of the proposed method, a mutagenic matrix is employed to judge whether a hypothetical mutagenic primer can discriminate the target SNP by digestion with available restriction enzymes. The available restriction enzymes for the target SNP are mined by the updated core of SNP-RFLPing. GAMPD has been used to simulate the SNPs in the human SLC6A4 gene under different parameter settings and compared with SNP Cutter for mismatch PCR-RFLP primer design. The in silico simulation of the proposed GAMPD program showed that it designs mismatch PCR-RFLP primers. The GAMPD program is implemented in JAVA and is freely available at http://bio.kuas.edu.tw/gampd/.     

Multiplex SNP genotyping in pooled DNA samples by a four-colour microarray system

We selected 125 candidate single nucleotide polymorphisms (SNPs) in genes belonging to the human type 1 interferon (IFN) gene family and the genes coding for proteins in the main type 1 IFN signalling pathway by screening databases and by in silico comparison of DNA sequences. Using quantitative analysis of pooled DNA samples by solid-phase mini-sequencing, we found that only 20% of the candidate SNPs were polymorphic in the Finnish and Swedish populations. To allow more effective validation of candidate SNPs, we developed a four-colour microarray-based mini-sequencing assay for multiplex, quantitative allele frequency determination in pooled DNA samples. We used cyclic mini-sequencing reactions with primers carrying 5′-tag sequences, followed by capture of the products on microarrays by hybridisation to complementary tag oligonucleotides. Standard curves prepared from mixtures of known amounts of SNP alleles demonstrate the applicability of the system to quantitative analysis, and showed that for about half of the tested SNPs the limit of detection for the minority allele was below 5%. The microarray-based genotyping system established here is universally applicable for genotyping and quantification of any SNP, and the validated system for SNPs in type 1 IFN-related genes should find many applications in genetic studies of this important immunoregulatory pathway.     

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.