The MCIC provides services for
- Capillary sequencing using the ABI 3100xl
- Genotyping: fragment analysis on Beckman CEQ8800, SNP analysis on Luminex200 or Illumina BeadXpress
- Deep genome sequencing using the Illumina Genome Analyzer II.
Genotyping
Instrumentation and assay
The Illumina BeadXpress system enables high-throughput, duel color detection of a broad range of multiplex assays. This system allows assaying 1-384 SNP in any given number of individuals. Main advantage of BeadXpress compared to the Luminex is that the costs/SNP is lower than on Luminex. However, it is not economical to run this machine for a few SNPs. BeadXpress utilizes the Golden Gate assay describe below.
Golden Gate Assay Basics
Golden Gate assay can be used to generate SNP specific PCR products that can be used for solid matrix (Bead array platforms) or in solution (BeadXpress). Three primers will be used for this: two allele specific primers (ASPs) (these will recognize you specific alleles) and a locus specific primer (LSP) which binds downstream of the SNPs. ASPs and LSP contains universal sequences (Fig. 1: fluorescently labeled P1, P2 and primer containing address sequence, illumicode, P3). Beads carry a sequence complimentary to P3. During assay a pool of primers (OPA) is hybridized simultaneously to DNA and after allele specific primer extension and ligation and P1 (Cy3 labeled) and P2 (Cy5 labeled) primers will be added to perform a PCR. These fluorescently labeled products are combined with VeraCode bar coded beads and PCR amplicons will hybridize to the respective sequence on the bead. Based on the identity of the illumicode and the florescence label, alleles will be called for each genotype.
http://www.illumina.com/support/support_protocols.ilmn
Starting a Bead Xpress project
Since these projects are time consuming and resource intensive, it is advised that you contact us before starting the project to discuss the cost and the time. However, here are some of the main steps that you need to do to start the project:
Detailed information regarding custom primer design and procedure can be found in the document 'Illumina_Custom_OPA_Design' (download PDF). or at the Illumina site www.illumina.com, then navigate to Support Tab, Protocols & Documents.
Briefly, the first step is to create a file that contains your desired SNPs. During this process, Illumina Bioinformaticians and the researcher work together to find a set of primers, which are likely to give the best data. Therefore, it is important to have more SNPs than you want to finally use in your experiment. The cost of the primer designing is included in the kit and the minimum order provide enough reagents to test 480 individuals (5 X 96 well plates). Depending on the format, user can use Assay Design Tool available from Illumina (You can get more information on this process from this document). Once you finalized the primers, the file is provided to us and this would be submitted to Illumina. Illumina provides some of the reagents, including OPA. You have two options:
- Perform the labeling of PCR products and hybridize the PCR products to the beads in your lab or in the MCIC. In this case, you give us the bead plate ready to run in the machine. Please be aware, you will need additional reagents and material to perform these steps and we are not responsible for providing them. However, you may use the equipments available in the center. You will only charge for running the machine.
- Give us high quality DNA (please see below regarding the quality and quantity of DNA) and we will perform all the steps and will give you the results. There is an additional charge for the labor and the reagents.
DNA quality and quantity:
Quantity needs to be measured with a reasonable accuracy and the sample concentration should be around 50ng/ul. Higher or lower concentration may work, but it has been shown that the lower concentrations lead to poor reproducibility and inaccurate allele calling. In addition, variability of concentrations among samples might affect the tightness of the clusters and also the actual allele call. Illumina recommend using PICO green to quantify the DNA (We don’t carry the necessary reagents for this array at this time.).
Sample submission:
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Rates for the BeadXpress
For detail pricing please visit our "Rates" page.
If you have any questions, please contact the MCIC staff.
Genotyping Order forms