GENOTYPING SERVICES. McGill University and Génome Québec Innovation Centre JANUARY 30, Version 2.1

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1 JANUARY 30, 2012 GENOTYPING SERVICES McGill University and Génome Québec Innovation Centre Microsatellite Technology User Guide Version 2.1 Copyright 2010 McGill University and Génome Québec Innovation Centre All rights reserved.

2 Table of Contents TABLE OF CONTENTS... 2 INTRODUCTION... 3 SERVICE REQUEST... 4 QUOTE REQUEST... 4 SERVICE REQUEST... 4 METHOD OF PAYMENT... 4 SAMPLE PREPARATION AND SUBMISSION GENERAL GUIDELINES... 5 SAMPLE SUBMISSION REQUIREMENTS... 6 DNA SAMPLES AND PLATES... 6 For projects using markers already validated:... 6 Recommended 96-Well Plate Types... 6 Forbidden Plate Types... 6 For other projects... 7 General Information... 7 GENOTYPING PROCEDURE... 8 VALIDATION STEP... 8 PRODUCTION STEP... 8 For projects using markers already validated... 8 For the other projects... 8 TRANSMISSION OF RESULTS... 9 FOR MORE INFORMATION Client Management Office

3 Introduction This report covers users of the genotyping microsatellite service of the McGill University and Génome Québec Innovation Center. It contains information on the procedure and requirements for submitting a project, samples, and appropriate documents. WARNING! When special instruction is given by the personnel of the genotyping service, please follow it. 3

4 Service Request Quote request Contact the client management office for more information regarding prices or quote requests: Phone: Fax: Note: A contract is required for all services that total or exceed $25, (prior to taxes). Service request To complete a service request: 1. Download the Service Request Form. 2. Fill the form, following carefully the instructions. 3. If applicable, send alongside the service request form: Important: Note: a. A copy of the ethics review committee s approval forms if any of the submitted samples are obtained from human subjects. b. A Purchase Order, if it is the chosen method of payment c. Any documents required by the service ordered. These documents are available through hyperlinks in the appropriate service section of the Service Request Form. A purchase order number is required for services that total or exceed $2, (prior to taxes). For security purposes do not enter the credit card information in the sequencing services request form. A separate form will be sent at time of billing. The Service Request Form is an Excel file and requires macros to be enabled to allow dynamic features to work. Method of payment Payments can be made by cheque (purchase order mandatory) or credit card. The cheque must be addressed and sent to: Génome Québec 630, boulevard René Lévesque, suite 2660, Montréal (Québec) Canada H3B 1S6 4

5 Sample preparation and submission General guidelines To avoid any delay in the processing of your request, it is recommended to follow carefully the instructions provided in this User Guide. Note that samples are processed on a «first come, first served» basis, and that the lead time may vary with the project size. Therefore it is suggested to contact the Customer Management Office about processing time when submitting samples. 5

6 Sample Submission Requirements DNA Samples and Plates For projects using markers already validated: DNA samples must be sent in 96-well plates. For each DNA plate we receive, we reserve specific positions for in-house controls which will be used to assess DNA quality and reaction success. The following are the acceptable formats for the plates: Recommended 96-Well Plate Types Half-Skirt PCR Plates Any half-skirt 96-well PCR plate Full-Skirt PCR Plates are also acceptable Microseal PCR plates 96-well clear (Bio-Rad, cat# MSP9601) Any other full-skirt 96-well PCR plate Adhesive Seals for Full-Skirt and Half-Skirt PCR Plates MicroAmp Clear Adhesive Films (Applied Biosystems, cat# ) MicroSeal F Foil (Bio-Rad, cat# MSF-1001) Forbidden Plate Types 96-well cell culture plates No-skirt 96-well PCR plates 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 in-house controls. If you have more than one plate you can use 92 wells and leave 4 blanks (C12, D12, G12 and H12). Other (non-human and non-mouse) DNA 96-well format: Actual number of customer DNA, including positive controls, per plate is a maximum of 94. Please leave positions G12 and H12 empty for our negative controls. Note that it is useful to provide positive controls on each plate as a reference. All 96 samples will be charged regardless of how many customer DNAs per plate. All samples will be quantified using PicoGreen (Molecular Probes) before proceeding with the genotyping. Regardless, the customer is to submit the expected DNA concentrations. The quantity of DNA required corresponds to at least twice the amount needed for the whole genotyping process. 10 ng of DNA will be used for each PCR reaction. For example, if you request a genome-wide scan, which comprises 400 markers, we will need 400 x 10ng x 2 = 8000 ng or 800 µl at 10 ng/µl for each sample. Note that the minimum acceptable amount of DNA is 30µL at a concentration of 20ng/ul. 6

7 For other projects The PCR products must be sent in a 96-wells plate, in a container (protected from light) and properly sealed. We ask that you freeze the PCR if shipping is expected to take several days. See previous section for accepted plate formats. It is recommended to include positive and negative controls during your PCR reactions. We suggest the use of DNA NA07057 and NA06990 for human samples, 129P3/J and C57BL/6J for mice and at least one control sample for other species. Multiples of 96 samples will be charged, regardless of the number of samples per plate. Note that the minimum acceptable volume of sample is 10μL. You must also provide a picture of your agarose gel for a few samples on each plate. This will help us determine if the PCR products must be diluted before reading. General Information 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. Add indication about the markers used in the PCR reactions, if applicable. If indicated in the Service Request Form, excess DNA will be returned to you once the experiment is completed. 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 (and pedigree) 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 individuals. 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. These designations are essential for X- linked markers. Please contact us for further inquires. 7

8 Genotyping Procedure Validation Step Since January 1st 2009, the validation of markers is no longer supported by the genotyping service. Production Step For projects using markers already validated A first DNA plate will be genotyped with selected markers to verify the quality of amplification. Once good quality data are obtained, the remaining DNA plates will be processed later in the production. For the other projects The fluorescent dyes accepted are FAM, VIC, NED and PET. Four options are available; Option 1: reading only (no analysis). The plates submitted already contain the PCR products in a solution of formamide + standard molecular weight ladder. Option 2: reading + formamide (no analysis) Includes the transfer of PCR products in a plate containing the formamide-molecular weight ladder mix. Option 3: reading + formamide + analysis Same as option 2 with analysis of chromatograms. Option 4: reading + analysis Same as option 1 with analysis of chromatograms. If you want to submit PCR products already in the formamide mixture, please use the following reagents: Reagents Company PN Number n = 1 HI_DI formamide Applied Biosystems μL GeneScan 500 LIZ Size Standard Applied Biosystems μL Volume needed PCR product 2μL Contact our laboratory staff for more details in the preparation of your plates. 8

9 Transmission of Results Raw and binned data will be sent in order of chromosomal position, if provided. The performance of each marker and the number of solid Mendelian errors will be summarized. Results will be sent as indicated on Service Request Form, either electronically or on CD, as soon as they are analyzed and verified. 9

10 For More Information Client Management Office Fanny Chagnon, Ph.D. Julie Vallée, B.Sc. Frédérick Robidoux, B.Sc. McGill University and Génome Québec Innovation Centre 740, Dr. Penfield Avenue, Room 7104 Montréal (Québec) Canada H3A 0G1 Phone: Fax: