The SNPstream technology is ideal for medium sized projects where scoring between 10 and 300 SNPs on hundreds to a few thousands of DNA samples is needed. All SNPs in a panel must be of the same type, regardless of the orientation. (A panel consists of multiple of 12 or 48 SNPs). For example, one may combine GA and CT polymorphisms to create one panel, but not GA and GT polymorphisms. The genotyping personnel are available to facilitate in the selection of SNPs based on your region and density requirements.
The genotyping reaction consists of a 12-plex or a 48-plex PCR followed by a single-base extension reaction using dye-labeled dideoxynucleotides (ddNTPs). The products are then separated by hybridization on an array of tags printed on a glass surface. Although, it is designed for SNP genotyping, it can also accommodate certain insertions/deletions, but not microsatellite markers.
The GenomeLab SNPstream technology has an average assay conversion rate of about 80-85%. The call rate is about 90-95% and the error rate is less than 0.5%. See Bell et al., 2002, BioTechniques 32: S70-S77. More details can also be found at http://www.beckmancoulter.com.
Submission Requirements
DNA Samples And Plates
DNA samples must be sent in 96 well plates (see acceptable formats below). The DNA plates should be properly sealed and the samples shipped frozen. For each DNA plate we receive, we reserve specific positions for our in-house controls will be used as a control for DNA quality and reaction success.
- PREFERABLY, use either of the two following plates (this facilitates the samples handling because these are compatible with the robot):
- Human and mouse DNA – 96 well format: Actual number of customer DNA per plate is a maximum of 90. Please leave positions A1, C12, D12, G12, H1 and H12 empty for our in-house controls. If you have more than one plate you can use 92 wells and leave 4 blanks (C12, D12, G12 and H12).
- Note that multiples of 96 samples are required for SNPstream/Sequenom iPlex genotyping. 96 samples per plate will be charged regardless of how many customer DNAs per plate exist.
Clearly label all your DNA plates. 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 Heart-Disease (or HD) 1 through to the number of plates you have.
All samples will be quantified using PicoGreen before proceeding with the genotyping. Regardless, the customer is to submit the expected DNA concentrations.
The quantity of DNA required corresponds to at least three times the amount required for the whole genotyping process. 40 ng of DNA will be used for each panel.
Note that the minimum acceptable amount of DNA is 30 µL at a concentration of 20ng/ul.
If your DNA is derived from human subjects, please attach the ethics approval with Request Form 1 – General Genotyping Services.
If indicated in Request Form 1 – General Genotyping Services, excess DNA will be returned to you once the experiment is completed.
Send the DNA plate layouts electronically. Click here to download the DNA plate layout form. Place all DNA plate layouts in the same worksheet as in the example. Name this file in the following manner: YOUR PROJECT NAME-Plate-layout-X.xls.
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.
Under the Sex column, indicate if the sample is male or female (if known).
Please contact us for further inquires.
Markers
Due to the multiplex nature of the SNPstream technology, one must always select SNPs of the same type (regardless of orientation) to group into one pool. The following list provides the different SNP type combinations that one must respect:
- GA and CT polymorphisms
- GT and CA polymorphisms
- GC polymorphisms (cannot be combined with other SNP types)
- AT polymorphisms (cannot be combined with other SNP types)
For the Sequenom iPlex Gold technology there is no restriction on the SNP type
Primer designs are done in-house. For each SNP, three primers will be designed: two PCR primers and one probe. Probes are designed using softwares from Beckman Coulter.
While the actual genotyping reactions will be performed on panels of up to 12 or 48 SNPs for SNPstream, it is strongly suggested to provide an excess of SNPs as it may be difficult to design primers due to the presence of repeats, low complexity sequences or incompatibility betwen SNPs of a panel. If the list of SNPs has been exhausted, customers will be notified and be asked to replace some SNPs.
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.
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 unsuccessful assay designs and may be asked to replace some SNPs.
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.
Please complete Request Form 2 – SNP and Microsatellite Markers.
If indicated in Request Form 1 – General Genotyping Services and if applicable, primers will be returned to you once the experiment is completed.
Please contact us for further inquires.
