The Infinium genotyping assay is designed for whole-genome SNP analyses. The reaction consists in a whole genome amplification step followed by a fragmentation. The product is then hybridized to a bead array where each bead is coated with specific probes and represented multiple times for increased accuracy. Then the hybridized product is extended with labelled bases and detected with a scanner. Call rates are greater than 98%. (See Gunderson et al., 2005, Nat Genet 37: 549-554 or Steemers et al., 2006, Nat Methods 3: 31-33 for more details).
Various products are available:
- Human-1 Genotyping BeadChip (100K)
- Covers the entire genome with 109K SNPs with a focus on exonic regions.
- HumanHap300 Duo Genotyping BeadChip (300K)
- Contains 319K tag SNPs derived from the Phase I of the HapMap project.
- HumanHap240S Genotyping BeadChip (240K)
- Contains 240K tag SNPs derived from the Phase II of the HapMap project.
- HumanHap550 Genotyping BeadChip (550K)
- Contains the SNPs from both the 240K and 300K chips.
- HumanHap650 Genotyping BeadChip (650K)
- Contains the SNPs from the 550K chip and an additional 100K Yoruba-specific tagSNPs
- 12K coding non-synonymous polymorphism chip
Also, custom arrays from 7K to 120K can be designed. 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.
Submission Requirements
DNA Samples And Plates
If there are less than 24 samples, they can be sent in tubes. Otherwise they should be sent in 96-well plates of 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):
Please include one empty well every 24 samples (we suggest wells B12, D12, F12 and H12). Please pay attention to the DNA plating requirements.
- For the positions reserved for negative or 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.
Note that multiples of 8 samples are required for whole-genome 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 (150 ng/uL for GeneChip Universal Arrays) 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 (40 uL for GeneChip Universal Arrays) should be sent for each sample. If you would like to genotype your samples with more than one panel, a minimum volume of 30 µL of DNA is required (750 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.
Mandatory fields to fill out are marked with an asterisk (*). The columns (J & K) 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 H & I 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.
Custom panels of 15 000 or 30 000 SNPs can be ordered for the Infinium platform. 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 provide a longer list in order to have enough SNPs with good score 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.
If you have regions or genes of interest in mind, click here and use the examples to describe your needs. Our personnel will help in the SNP selection 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.
Genotyping Procedures
Validation Step
This is the first step in our genotyping protocol. The aim is to assess how a panel performs and if the quality of the DNA is high enough for the genotyping process. If the validation step yields good quality data, the rest of your DNA will be processed as part of the production step. Otherwise, we will evaluate whether the poor quality data was due to poor synthesis of primers or poor DNA quality. The following provides an overview of the validation steps for custom panels:
- Once a set of SNPs is selected, confidence scores indicating how likely each SNP will perform well will be given.
- Based on this information, the panel of SNPs will be rearranged to remove SNPs with poor confidence scores. Then, the panel will be ordered.
- All DNAs will be quantified using PicoGreen.
- One of your DNA plates will be used to test each panel of SNPs.
- If we obtain good quality data, the rest of your plates will be processed as part of the production step.
- If we obtain poor quality data, we will investigate (using the positive DNA controls) whether it is due to poor DNA quality or poor synthesis of primers.
For commercial panels, we will assess the quality of the samples using up to 24 samples in the validation step. If the data is of high enough quality, we will continue to the production step.
Production Step
This is the second step in our genotyping protocol. When good quality data is obtained from the validation step, the rest of the DNA plates will be processed as part of the production step.
