The Illumina GoldenGate genotyping assay is designed for large scale SNP genotyping. The platform has a capacity of over 1,000,000 genotypes a day and enables parallel typing from 384 to 1536 SNPs. At the Innovation Centre, this platform was used to generate about 10% of the International HapMap Project (International HapMap Consortium, 2005, Nature 437: 1299-1320; see also http://www.hapmap.org).
The GoldenGate genotyping reaction consists of an initial allele-specific extension reaction followed by PCR amplification. Amplified products are hybridized to an array matrix of bead-based probe sequences where each bead is coated with universal probes (up to 1536 different bead types) and represented multiple times for increased accuracy (average of 25 times). The entire procedure is controlled by a LIMS.
Assay conversion rate for custom panels varies between 85 and 95% depending on the level of SNP validation. For pre-defined panels (linkage panels, chromosome-specific panels, etc.), the conversion rate is above 95%. The overall call rate is about 99.7% and the error rate is below 0.1% which was evaluated by comparing data between 10 different centers as part of the HapMap project. (See Oliphant et al. 2002, Biotechniques Suppl: 56-61 or http://www.illumina.com for more details).
Custom or commercial panels may be ordered. For custom panels, the number of SNPs may vary from 384 to 1536 SNPs, but should be multiples of 96. Only bi-allelic SNPs may be genotyped. The genotyping personnel are available to facilitate in the selection of SNPs based on your region and density requirements. Primer designs are conducted free of charge at the Centre.
These are the available commercial panels:
LinkageIVb Panel
- Contains more than 6000 highly informative SNP markers distributed evenly across the human genome and delivering high information optimized for linkage mapping studies.
Mouse LD Linkage (low density) Panel
- Consists of 377 loci, optimized for application to N2 and F2 mouse genetics crosses. These crosses can be used for mapping quantitative trait loci.
Mouse MD Linkage (high density) Panel
- Consists of 1,449 loci, optimized for various mapping applications including characterization of transgenic, congenic and knockout animals, and genetic mapping in advanced intercross mouse lines.
The Major Histocompatibility Complex (MHC) Panel Set :
- Consists of two pools, the MHC Mapping Panel and the MHC Exon-Centric Panel that can be used independently or combined for more comprehensive coverage.
- MHC Exon-Centric Panel: >1200 SNPs within 10 kb of coding sequences of genes in the MHC.
- MHC Mapping Panel: >1250 SNPs with average spacing of 3.8 kb).
Cancer Panel
- Consists of 1,421 thoroughly screened and validated SNP loci that have been chosen from the National Cancer Institute’s (NCI) Cancer Genome Anatomy Project SNP500 Cancer Database.
DNA Test Panel
- Consists of 360 thoroughly screened and validated SNP loci that have been chosen from Illumina's LinkageIVb Panel. This is a cost-effective SNP-based tool for pre-screening DNA samples for assay performance before conducting studies with larger numbers of loci.
Submission Requirements
DNA Samples And Plates
DNA samples must be sent in 96 well plates in the appropriate plate type (see below). The DNA plates should be properly sealed and the samples shipped frozen. Please, do not completely fill the wells with samples because this might increase the risk of samples contamination. This will ensure the preservation of the DNA integrity during the shipment and the storage. To seal the TCY plates, use a good quality adhesive film, and for the Deep-Well plates, use a compatible sealing mat.
PREFERABLY, use either of the two following plates (this facilitates the samples handling because these are compatible with the robot):
- TCY plates: Microseal PCR plates, 96-well clear (Bio-Rad; cat# MSP9601) & MicroAmp Clear Adhesive Films (Applied Biosystems; cat# 4306311).
- MIDI plates: 96-well 0.8 mL deep-well storage plate (ABgene, cat# AN-0859) and 96-well cap sealing mat (ABgene; cat# AB-0566).
- Other acceptable plates:
- Deep-well plates: 96-well, 1.1 mL deep-well plates (ProGene; cat# 24-PDW-11-C-S) and 96-well cap sealing mat (ProGene; cat# 24-AM2ML-RD).
- Any other full-skirt 96-well PCR plate or 96-well deep-well plate.
- Plates that SHOULD NOT BE USED
- 96-well Cell culture plates plate
- 96-well half-skirt PCR plate
- 96-well no-skirt PCR plate
For GoldenGate projects, please reserve three empty wells per plate (one for negative control and two for positive controls). Please pay attention to the DNA plating requirements.
For the positions reserved for negative and positive controls, the wells should be left empty (no water, no buffer).
If you cannot complete a plate, please organize the samples in successive columns or in successive rows.
Please try to regroup the samples in successive columns or in successive rows or in different plates when you have, for example:
- Different ethnicities.
- Genomic DNA and whole genome amplified samples.
- Different tissue sources.
- etc.
Note that multiples of 16 samples are required for high-throughput genotyping. It must also be noted that for custom projects, oligo synthesis is available only for multiple of 96 samples (with a minimum of 480 samples) and will be reflected in the final price.
Clearly label all your DNA plates on the left side (A01-H01) and on the front (H01-H12) of the plate. A suggested naming scheme is to incorporate your project name into the DNA plates. For instance, if your samples relate to heart disease, one way to number your plates would be HD1 (Heart Disease 1), HD2, etc.
All samples should be normalized to a concentration of 100 ng/µL by the customer. We will verify the DNA concentration by PicoGreen DNA Measurement before proceeding to the genotyping. It's recommended to dilute DNA in either TE (10 mM Tris pH 8.0 / 1 mM EDTA) or in nuclease-free water.
A minimum of 20 µL (2µg if the concentration is 100 ng/µL) should be sent for each sample. If you would like to genotype your samples with more than one panel, a minimum volume of 50 µL of DNA is required (250 ng is needed for each panel).
If the DNA samples were whole genome amplified (WGA), here are the recommendations:
- Recommended technologies:
- REPLI-g (Qiagen) (Multiple Displacement Amplification)
- OmniPlex (Rubicon Genomics)
- Phi-29 Polymerase Multiple Displacement Amplification
- DNA samples specifications;
- High quality DNA, intact DNA, within the recommended ranges of the manufacturer’s specifications should be used.
- Concerning the DNA amount in the input WGA reaction, we recommend using at least 50 ng of intact DNA and a minimum of 100-200 ng for DNA suspected or known to have undergone degradation.
- The DNA should be accurately quantified with a DNA specific method or reagent as PicoGreen not only prior to the WGA reaction but also before starting the genotyping procedure.
- It’s recommended to test the WGA samples prior to the genotyping procedure (using PCR or TaqMan for example).
If your DNA is derived from human subjects, please attach the ethics approval with Request Form 1 – General Genotyping Services. DNA from species other than human and mouse may be supported. Please contact us to see if they are.
If indicated in Request Form 1 – General Genotyping Services, excess DNA will be returned to you once the experiment is done.
Please send an electronic version of the DNA plate layout. The "SamplePlateTemplateManyCustomers.xls" file should be filled out with your DNA plate layout (up to 4 DNA plate layouts per file). Click here to fill out your DNA plate layout using the example as a guide. Name this file in the following manner: "YOUR PROJECT NAME-Plate-layout-X.xls" (example: HD-1.xls).
Mandatory fields to fill out are marked with an asterisk (*) and a plus sign (+). The columns (H & I) for the DNA volume and concentration should be filled too.
In naming your samples, please keep the following guidelines in mind: each individual must have a unique individual ID. If a sample exists more than once either on the same DNA plate or over multiple plates, use the same individual ID for all replicates but assign different sample names by adding a suffix.
In naming your pedigrees, please keep the following guidelines in mind: each family pedigree must have a unique ID. Unique pedigrees must be created for each separate family or unlinked individual. Also, don’t forget to indicate in columns F & G the individual names of the parents.
Please indicate the sex of the sample in column D (if known).
Please contact us for further inquires.
Markers
Either commercial or custom panels may be ordered. All panels must be ordered through us.
- For custom panels, the number of SNPs should be multiples of 96. Three different types of arrays are available that can handle up to 384, 768, 1536 SNPs. Only bi-allelic SNPs may be genotyped (no indels, MNPs or SSRs). Since about 10% of the SNPs normally fail at the scoring level, the customer should then provide a longer list in order to have enough SNPs with high scoring for the final selection.
- The genotyping personnel can design primers for your list of SNPs. The following is the required sequence format (Click here to create your list of SNPs, name your file as follows and send electronically (SNPlist-YOUR PROJECT NAME.xls):
- For rs SNPs (found in dbSNP) just indicate the SNP ID. There is no need for more information
- For other SNPs, it would be preferable for the name of the SNP to not exceed 15 characters.
- Provide at least 150 base pairs of flanking sequence on each side of the SNP.
- Indicate the SNP of interest in square brackets.
- The flanking sequence should be repeat masked, except for the 25 base pairs surrounding the SNP on each side.
- The sequence should include neighbouring SNPs using the IUPAC symbols listed in the table below.
| R |
A/G |
| Y |
C/T |
| M |
A/C |
| K |
G/T |
| S |
C/G |
| W |
A/T |
| B |
C/G/T |
| D |
A/G/T |
| H |
A/C/T |
| V |
A/C/G |
| N |
A/C/G/T |
- Please indicate the priority of the SNP (high or low), if applicable.
- Please indicate the expected minor allele frequency of each SNP, if it is known.
You may have regions or genes of interest in mind. In this case, relay this information by clicking here to fill out the request. Our personnel will select SNPs and design primers to suit your needs.
A design score will be returned for every SNP. Please note that a minimum score is used to determine which SNPs can or can't be included in a panel.
Note that depending on the sequence provided (the presence of repeats or low complexity sequences), it may be difficult to design appropriate primers for some SNPs. Customers will be notified of the design scores for each SNP and may be asked to replace some SNPs before proceeding with the order.
Please complete Request Form 2 – SNP and Microsatellite Markers.
Please contact us for further inquires.
