Genotyping Services Submission Requirements

To begin, we will require purified DNA samples, the SNP panel reagents, and a manifest of your sample IDs, and a pedigree file if you want Mendelian error-checking.

DNA SAMPLES

• DNA samples must be submitted in standard 96-well plates, which must be clearly labeled. Note that multiples of 16 or 96 samples are required for Illumina genotyping (except for 100k+ whole genome genotyping) and less than 16 or 96 samples per plate will be charged regardless of how many DNA are on the plate.

• The submitted DNA must be of excellent purity. We have successfully used Qiagen Flexigene, Gentra Puregene, and DNAtek Oragene kits to purify DNA for Illumina genotyping.

• The quantity of genomic DNA required for genotyping is determined by the number of SNPs to be analyzed for each sample. For custom SNP panels of up to 1,536 SNPs, we would like to receive 1 ug of DNA in a 20 ul volume (100 ng/ul concentration), and an absolute minimum of 500 ng of genomic DNA in 10 ul (50 ng/ul concentration). DNA should be diluted in TE buffer (10mM Tris pH 8.0/1mM EDTA).

• For whole genome analyses of up to 500,000 SNPs, we require at least 750 ng of genomic DNA in an 10 ul volume.

SAMPLE MANIFEST FILE

Click here to download the sample manifest file (xls, 30kb).

Please fill in your "Institute Name", "Date", "Comments", "Sample names", "Sample plates" (if >1 plate), "Species", and "sex". Note that each sample must have a unique ID, and for replicates on the same DNA plate or over multiple plates, use the same ID for all replicates. Please email a copy of the sample manifest to Colin Ross at cjross@cmmt.ubc.ca.

SAMPLE PEDIGREE FILE

If your samples consist of families, a pedigree will be required for Mendelian error checking.

Click here to download the sample pedigree file (xls, 36kb).

Use this file as an example to create a pedigree file, and remember to keep each sample ID identical to the ID in the sample manifest file, and each pedigree must be grouped together (i.e. the pedigree file must be sorted by the pedigree column), and that each sample must be linked to a father and mother ID (if the parent IDs are unknown, simply indicate these IDs with a zero, for instance, if you were to send trios consisting of parents and a child, you may indicate the grandparents of the child with zeroes). Note that every ID in this file must have its own entry regardless of whether or not the DNA is on your plates. Also, treat samples that are in duplicate as though they were independent of one another. That is, keep the unique IDs (nomenclature discussed in the DNA section) and simply group them under the same pedigree. Under the Sex column, indicate males as 1 and females as 2. These designations are essential for X-linked markers.

SNP PANEL

The reagents for fixed content SNP panels will be obtained by the CMMT through Illumina. For custom SNP panels, we will work with you to develop and synthesize your custom panel. Assume at least 2 weeks to finalize the panel and 4-8 weeks for the panel to be synthesized and shipped.

Additional notes on custom SNP panel development:

• Two SNPs on one panel cannot be closer than 60 bp. Each SNP is amplified by its own pair of PCR primers.

• How many SNPs to choose for a panel: We will assign a confidence score to each selected SNP that predicts the success rate of that SNP on the Illumina platform. A SNP with a low confidence score will have a reduced chance of producing good data. We suggest you initially select two-times the number of needed SNPs so that you can be sure enough will be chosen to make up your panel.

• 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

  Generally, non-validated are less reliable.

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