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| Research Services: Illumina® GoldenGate SNP Genotyping |
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Research Services: Illumina® GoldenGate SNP Genotyping Illumina® GoldenGate® genotyping is a high throughput SNP genotyping platform for custom and fixed content needs. It is designed to genotype 384 to 1536 loci in 480 to thousands of samples with minimal DNA expenditure. More SNP loci may be assayed using multiple iterations. If you are interested in considering the Illumina technology for your research, please contact the Genomics and Proteomics Core Laboratories to discuss your research goals, how this platform might address them, project initiation and sample submission guidelines. Project Initiation and Sample Preparation GoldenGate® projects are costly and complex. All projects must be discussed with GPCL before sample submission. Sample preparation guidelines will be discussed at that time. The GoldenGate Assay The Illumina® GoldenGate® Assay queries genomic DNA with three probes per locus and creates DNA fragments that can be amplified by standard PCR methods using three universal primers. For each locus interrogated, the oligo mix contains 2 allele specific and one locus specific probe, each incorporating a different universal primer site. Each allele probe terminates at the point of interrogation with an allele specific base. The locus specific probe hybridizes 5 to 20 bases beyond this point. This probe is also contains the specific Illumacode that will be used to identify the locus.After the probes annealto the genomic DNA, Taq polymerase is added to close the gap between them and the two fragments are ligated together. Probe fragments are amplified by PCR. Primers for allele specific probes are labeled with Cy3 and Cy5 so that dye ratio represents allele frequency. Labeled amplification products are hybridized to the SAM and scanned for dye ratio at each locus. The Sentrix Array Matrix The Sentrix Array Matrix (SAM) is an array of fiber optic bundles in 96 well plate format. A bundle contains 50K separate fiber optic fibers, each containing a bead coated with oligos complementary to Illumacodes. The bead deposition process results in a randomly ordered array of captured Illumacodes. The manufacturer performs a quality control to map the Illumacode that is represented on each bead and to verify that each Illumacode is represented a minimum of 5 times. On average there 30X probe redundancy. The Oligo Pool (OPA®) Any GoldenGate assay requires an oligo pool of allele and locus specific probes. Any single oligo pool OPA can interrogate up to 1536 different SNP loci. Multiple oligo pools can be run on sample sets to increase the number of loci queried. Oligo pools are shipped with their own information file designating which Illumacode is used to interrogate each locus as well as which allele is labeled with Cy3 and which with Cy5. Custom content oligo pools can be ordered at a minimum density of 384 SNPs and increasing in increments of 96 loci. The most efficient scales are 384, 768 and 1536 SNP loci per assay. The minimum batch size for oligo pools is enough to interrogate 480 samples. Batch size also increases in increments of 96 samples. Analysis The BeadArray reader scans the hybridized SAM and determines the signal intensities for each dye at each bead location. Software uses the information files from the SAM and the OPA to map the known location of each Illumacode on the SAM back to the locus being interrogated. These dye intensities are examined to determine the genotype of each sample. A locus returning predominantly signal from Cy3 is AA, Cy5 is BB and an even ratio represents a heterozygous individual. Data is returned in a text file containing the allele call for each locus as well as a Gencall score, representing the reliability of that genotyping call. Custom OPA Design The selection of SNPs for a project is dependent on the project goals and a series of factors based partially on the technology used to obtain the genotypes. If the goal of the genotyping is to associate a specific functional SNP with a condition, then the suspected mutation is targeted by the SNP assay. In general the factors that should be considered are based on how much information is available about a SNP locus and flanking sequence. Many potential SNPs identified through the Human Genome Project turn out to have little heterogeneity or not to be true SNPs. The use of such SNP loci is risky without further information. Many web-based tools are available which output SNP loci by genomic location or gene name. The National Center for Biotechnology Information (NCBI) and UCSC Genome Bioinformatics (UCSC) web sites include information, which allows a more effective selection. In choosing SNPs for the Illumina snp platform a list of RS numbers may be sent to Illumina. Illumina will score the SNPs based on a series of factors and return a score, which predicts the success of each locus within an assay set. Illumina® Defined OPAs Illumina® has a number of defined content panels available for performing GoldenGate analysis. These include panels for genetic linkage, Major Histochemical Compatibility, cancer SNPs and African American admixture. For up to date information on products, please see descriptions available from Illumina for focused content or linkage products. Pricing Pricing is determined based on the number of loci and the number of samples. Please contact the GPCL to discuss pricing based on your project parameters. |