The FP-SBE technology is designed for small scale SNP genotyping (for scoring less than 5 SNPs). The single-plex reaction provides for genotyping flexibility as one is able to genotype SNPs of different types. The FP-SBE system can be coupled with other technologies for scoring individual SNPs in larger projects.
The genotyping reaction consists of a single-plex PCR followed by a single base pair extension reaction using a template specific probe and dye-labeled dideoxynucleotides (ddNTPs). The instrument used to analyze the results for this platform is the Analyst HT from Molecular Devices.
The FP-SBE system has an average assay conversion rate of about 85%. The genotyping call rate is about 90-95% and the error rate is about 0.5-1%.
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 FP-SBE genotyping. 96 samples per plate will be charged regardless of how many customer samples there are per plate.
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
Four primers are needed for each SNP: two PCR primers and two probes, one on the sense strand (sense orientation), the other on the antisense strand (antisense orientation).
Customers may design their own PCR primers; however it is preferable for us to take on this task. If primers already exist, we need to approve of their design first. Please contact us for more information.
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
Genotyping Procedures
Validation Step
This is the first step in our FP-SBE genotyping protocol. The aim is to find the appropriate genotyping conditions for each SNP. Only once a set of conditions is found to work well for each SNP will the genotyping commence for the rest of your DNA plates (production step). When all experimental conditions have been exhausted, the SNP will be put aside and the customer will be notified and asked to replace the SNP, if possible. Relative to the number of genotypes obtained, the validation step is more time-consuming than the production step. It is for this reason that the price per reaction done in the validation step is slightly more expensive than the production step. The following provides an overview of the FP-SBE validation steps:
- Once a set of SNPs is selected, confidence scores indicating how likely each SNP will perform well will be given.
- In-house control DNAs will be used for PCR to test the newly designed and obtained PCR primers.
- Once a set of successful PCR conditions is obtained, PCR will be done on one of your 96 well DNA plates. Note that if you only have one DNA plate, validation prices will apply.
- The extension step will be done on the 96 reactions using two orientations. Obtaining data from both orientations increases our confidence in the data as the concordance between the genotypes of both orientations is looked at. If one orientation fails, the rest of the DNA plates will be processed using the working orientation. Note that we may need to optimize the conditions of the extension step if both orientations fail. All repeated validation experiments will be charged.
- Once the validation step yields good quality data, we will move to the production step where the rest of the DNA plates will be processed.
Production Step
This is the second step in our FP-SBE 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. The price per genotype for this step is less expensive than the validation step
