McGill University and Génome Québec Innovation Centre

Genotyping

Functional Genomics

Proteomics

Sequencing

Genotyping Platform

Frequently asked questions

General questions about genotyping services

What is the procedure I should follow to request genotyping service?
Fax completed Request Form 1 – General Genotyping Services and Request Form 2 – SNP and Microsatellite Markers. Be sure to sign both forms. Once your Request Forms are reviewed, you will be contacted regarding the DNA and markers.
What should the sent DNA concentration be?
There are different requirements for the different technologies.

For microsatellite, FP-SBE and SNPstream genotyping, DNA should be sent at a minimum concentration of 20ng/ul. Note that the minimum acceptable amount of DNA is 20ul at a concentration of 20ng/ul.

For Illumina genotyping, DNA should be sent at a minimum concentration of 100ng/ul. Note that the minimum acceptable amount of DNA is 25ul at a concentration of 100ng/ul. DNA should be diluted in TE buffer (10mM Tris pH 8.0 / 1mM EDTA). If you wish, you may send lyophilized samples
I do not have enough extracted DNA, can I send whole-genome amplified DNA instead?
We have used whole-genome amplified DNA for only Illumina so far. While the assay conversion rates are not significantly different, we have observed that some SNPs cluster and behave differently between amplified and non-amplified DNA samples. Therefore, mixing amplified and non-amplified DNA samples in one experiment may not be ideal. Note however that if it is the only way, problematic SNPs will simply be removed from the analysis. In addition, about 5% amplified DNA samples failed to genotype properly on the Illumina platform.
If I wish to provide the primers, what is the required primer concentration I should send?
Primers should be at a concentration of 100uM. Note that this only applies to microsatellite, FP-SBE and SNPstream genotyping as all Illumina primers are ordered through us.
How should I send my DNA samples and primers?
Samples should be sent in 96 well plates, securely sealed with an appropriate sealer. Also include schematic plate layouts with your plates.

If applicable, primers should be sent in tubes at a concentration of 100uM.
Is there a specific layout I should use to create my DNA plate layout?
Please refer to the 'DNA' section under each technology for more information.
Do I need to create a pedigree for samples that have no pedigree?
If your whole DNA cohort is composed of sporadic individuals, then a pedigree is obviously not needed. However, if most of your DNAs have pedigree structures while only a subset are sporadic, please have an entry for each sporadic individual in the pedigree file in this case. These sporadic individuals will be unlinked to any other sample.
Is there a certain sample naming scheme to follow?
What is important is to have a unique ID for each sample on each plate. Please refer to the ‘DNA’ section under each technology for more information.
How will the genotypes be delivered?
The data will be sent in our standard output format files. The performance of each marker and the number of solid Mendelian errors will be summarized. Results will be sent as indicated on Request Form 1 – General Genotyping Services, either electronically or on CD by FedEx, as soon as they are analyzed and verified.
What are validation and production steps?
The validation step is the first step in the genotyping procedure. The aim of this step is to find the optimal working conditions for your markers of interest using a subset of samples. These conditions are then used for the second step in the genotyping procedure, the production step. If no conditions could be used to rescue failed markers in the validation step, the customer will be notified and asked to replace the markers or repeat the validation step. Only when good quality data is obtained from the validation step do we proceed to the production step.
What are validation and production genotypes?
Validation genotypes are genotypes obtained from the validation step. Production genotypes are obtained from the production step.
How long do genotyping projects take for completion?
Projects are processed on a first come, first served basis. In addition, the time needed to complete a project depends on the technology to be used and the experiment design and details. Please contact us for more information regarding your particular project.

Questions about microsatellite genotyping services

How is the paneling of microsatellite markers done?
Markers are grouped in panels of about 8 such that markers of the same dye do not overlap one another in fragment size. Markers of different dyes may overlap one another in size. Our personnel panel microsatellite markers for all customers.

Questions about FP-SBE and SNPstream genotyping services

Can I genotype a SNP on less than 48 samples?
Analysis of the SNP genotypes using the instruments at the Innovation Centre is based on clustering. The more DNA genotyped per SNP, the more easily distinguishable the different clusters (or genotypes) become, leading us to be more confident in the genotyping calls. In addition, genotyping few samples may lead us to miss low frequency alleles.

Therefore, the minimum number of samples one can process for FP-SBE is 48. Technically it is possible to genotype 48 samples on SNPstream, however, the minimum number of samples we use is 96 due to plate format restrictions.
How many SNPs must I select for genotyping using SNPstream?
Each SNPstream pool consists of 12 SNPs all of the same polymorphism, regardless of orientation. 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)
That is, one can only combine C/T with G/A SNPs and G/T with C/A SNPs. G/C and A/T SNPs must be run separately. When providing us with the list of SNPs of interest, please provide us with more than just multiples of 12 SNPs. Depending on the surrounding sequence, we may not be able to design primers for all SNPs. Providing an excess of SNPs allows us to select the best sets of 12 and design primers for them.
Do the selected SNPs have to be obtained from the Innovation Centre’s in-house database, Nanuq?
No, however, it is always best to select validated SNPs from public databases. Avoid SNPs existing in repeat regions.
How should I name the selected SNPs?
The best way to name your SNPs would be to include the rs number. If one does not exist, provide a short, informative name. Avoid names longer than 15 characters.
There are two different prices for Production FP genotypes, what does this indicate?
We design extension probes for FP in both sense and antisense orientations because of the increased confidence obtained from genotyping two orientations. The success of each orientation is evaluated at the validation step. It is not always the case that both orientations work well. For this reason, we have two different prices reflecting the number of orientations used to generate the ‘Production genotypes’.
What are the programs you use to design SNP primers?
PCR primers for FP-SBE are designed using Primer 3, and extension probes are designed using Primer Premier 5

All primers for SNPstream are designed using Autoprimer, a web based primer design software from Beckman Coulter.

Questions about Illumina genotyping services

How are panels of up to 1536 SNPs assembled?
There are three different scenarios for building a panel:
- The customer creates the panel. This means that the customer submits the SNPs of interest to us. We will obtain a design score for each SNP. Based on these scores, we will ask the customer to choose the final set of SNPs for the panel. Primers will be ordered from Illumina.
- We create the panel. If you have regions or genes of interest, we can select SNPs and design primers to suit your needs. The customer will next need to approve of the SNP selection before we order the primers.
- We build the panel together. This applies when the customer has a region of interest in addition to certain SNPs in mind. The two points above apply.
What should the minimum distance between two SNP within a panel be?
Each SNP is amplified by its own pair of PCR primers; that is, each amplicon consists of only one SNP. Therefore, two SNPs cannot be closer than 60 bp.
How many SNPs do I have to choose to make a panel of 1536 SNPs?
The Illumina algorithm assigns a confidence score to each selected SNP. A SNP with a low confidence score will have a reduced chance of producing good data, therefore, these SNPs are removed. The more SNPs you select, the better. We suggest you initially select double the number of needed SNPs so that you can be sure enough will be chosen to make up your panel
Are there any SNPs I should avoid selecting?
We strongly suggest selecting SNPs that have been validated. The following are good indicators of real SNPs: Double-hit SNPs, SNPs that have been verified by Perlegen or Illumina, SNPs that have allele frequencies associated to them, and SNPs from the HapMap database (http://www.hapmap.org/). Generally, non-validated are less reliable.
What is the ideal project size for Illumina?
The ideal project for Illumina consists of having multiples of 1536 SNPs to be run on at least 500 DNAs. Attempting to genotype less than 1000 SNPs is not cost-effective as the same amount of reagents, labour and primers would be used.
Can I order and run half the number of SNPs initially, and then continue with the other half?
No, in order to take full advantage of the cost of genotyping, we need to order primers for a complete panel of 1536 SNPs in one shot. However, we can work on creating payments schedules so that the payments could be made over a period of time. Contact us for more information.
Will Illumina eventually scale down to genotyping less than 1536 SNPs?
There are plans to create new arrays in 384 and 768 SNP formats. Refer to http://www.illumina.com/ or contact us for more information.
What is the quality of the results?
Illumina’s technique uses an array of optic fibres, where each sample is assigned to a bundle of over 50, 000 fibres. Therefore, each of the 1536 SNPs is replicated 20 to 30 times. This means that each genotype is the result of the mean intensity of all replicates. This allows us to get a reproducibility frequency of 99.99%, and a familial inheritance frequency error of less than 0.1%.