Replacing TaqMan SNP Genotyping Assays that Fail Applied Biosystems Manufacturing Quality Control. Begin

Transcription

1 User Bulletin TaqMan SNP Genotyping Assays May 2008 SUBJECT: Replacing TaqMan SNP Genotyping Assays that Fail Applied Biosystems Manufacturing Quality Control In This Bulletin Overview This user bulletin describes how to: Search the NCBI dbsnp Database for Alternate SNPs Search for Alternate TaqMan SNP Genotyping Assays If you are notified that your TaqMan SNP Genotyping Assay has failed Applied Biosystems manufacturing quality control, you can use this bulletin to find a replacement assay. To replace the failed assay, first search the dbsnp database of the National Center for Biotechnology Information (NCBI) web site for alternate SNPs that are near the target SNP of the failed assay. Next, use one of three Applied Biosystems tools to find replacement TaqMan SNP Genotyping Assays based on the list of reference SNP cluster (rs) IDs that you obtained from the NCBI web site. The following figure summarizes the process. Begin Locate the SNP Associated with the Failed Assay Search the NCBI dbsnp Database for Alternate SNPs Search for Alternate TaqMan SNP Genotyping Assays Order the Replacement TaqMan SNP Genotyping Assay Using the Applied Biosystems Web Site Search using the: Applied Biosystems Web Site or SNPbrowser Software or Mouse SNPbrowser Software Finish

2 Replacing TaqMan SNP Genotyping Assays that Fail Applied Biosystems Manufacturing Quality Control Search the NCBI dbsnp Database for Alternate SNPs Locate the SNP Associated with the Failed Assay Determine the location of the target SNP of the failed assay, then define the search parameters to find alternate SNPs upstream or downstream from the target SNP. 1. Obtain the rs ID of the failed assay from the Applied Biosystems that was sent to you regarding the manufacturing failure. For example: Affected Part Number Affected Assay ID Reference SNP Cluster (rs) ID C _10 rs Go to 3. Search the dbsnp database of the NCBI web site for the rs ID of the failed assay: a. In the Search field of the Entrez SNP page of the NCBI web site, enter the rs ID of the failed assay, then click Go. 3a b. When the NCBI web site displays the results of the search, click the link for the rs ID of the failed assay. c. In the Reference SNP (refsnp) Cluster Report page, scroll to the Integrated Maps section, then record the chromosome number and chromosome position (bp) of the SNP target of the failed assay. 3c 3c 2 TaqMan SNP Genotyping Assays User Bulletin

3 Search the NCBI dbsnp Database for Alternate SNPs 4. Choose a range of base pairs that are upstream and downstream from the target SNP to search for alternate SNPs. Record the base pair positions for the upper and lower limits of the search. Original 5000 bp upstream Target SNP 5000 bp downstream Upstream Position bp Position bp Downstream Position bp 5. Go to Search the NCBI dbsnp Database for Alternate SNPs below. Search the NCBI dbsnp Database for Alternate SNPs After defining the range of base pairs for the search, use the NCBI web site to find alternate SNPs that are upstream or downstream from the target SNP. 1. Go to 2. In the dbsnp database interface of the NCBI web site, search for the alternate SNPs: a. In the Entrez SNP page, select the Limits tab. b. In the Organism selection box, select the desired species. c. In the Chromosome selection box, select the chromosome number you determined in step 3c on page 2. d. In the Chromosome Range selection box, enter the upstream (From) and downstream (To) base pair limits determined in step 4 above. e. Click Go. 2a 2e 2c 2d 2b TaqMan SNP Genotyping Assays User Bulletin 3

4 Replacing TaqMan SNP Genotyping Assays that Fail Applied Biosystems Manufacturing Quality Control 3. When the NCBI web site displays the results of the search, either record the rs IDs of the alternate SNPs, or export the results of the search as a text file (TXT) as explained below: a. Select File in the Send to drop-down list. b. When prompted, select Open with, select Notepad or Microsoft Excel in the drop-down list, then click OK. 3a 3b 3b The exported text file contains rs IDs and additional information for the alternate SNPs. You can edit the file using the Microsoft Excel or Microsoft Notepad software. 4. Go to Search for Alternate TaqMan SNP Genotyping Assays on page 5. 4 TaqMan SNP Genotyping Assays User Bulletin

5 Search for Alternate TaqMan SNP Genotyping Assays Search for Alternate TaqMan SNP Genotyping Assays Options for Searching After obtaining the list of SNPs from the NCBI web site, you can search for alternate TaqMan SNP Genotyping Assays using the Applied Biosystems software tools shown in the following table. Tool Applied Biosystems Web Site SNPbrowser Software Mouse SNPbrowser Software Description Provides an extensive search capability to access more than 4.5 million TaqMan SNP Genotyping Assays, including 3.5 million HapMap SNPs, 70,000 csnps, and 160,000 validated assays. The web site supports batch queries of the assay database by keyword (such as gene symbol, gene name, public accession number, biological process, or molecular function) or by standard identifiers (such as assay IDs, RefSeq accession numbers, GenBank GI numbers, LocusLink IDs, and gene symbols). Aids in the selection of TaqMan SNP Genotyping Assays for association and fine mapping studies. The software can display SNPs in their genomic context along with linkage disequilibrium maps and putative haplotype blocks derived from the analysis of over 3 million SNPs that are genotyped in several populations by the International HapMap project and Applied Biosystems. Note: For more information, see the Applied Biosystems product bulletin, SNPbrowser Software v3.5: A Free Software Tool for the Knowledge-Driven Selection of SNP Genotyping Assays. Aids in the selection of SNP sets to distinguish mouse strains of interest, with specific applications in genotype/phenotype mapping and strain verification. The mouse SNP database underlying the software includes approximately 10,000 evenly spaced SNP loci genotyped across 48 inbred mouse strains. Note: For more information, see the Applied Biosystems product bulletin, Mouse SNPbrowser Software v1.0: A free tool for efficient and easy selection of Mouse SNP Genotyping Assays. See Page Free tool available from the Applied Biosystems web site. TaqMan SNP Genotyping Assays User Bulletin 5

6 Replacing TaqMan SNP Genotyping Assays that Fail Applied Biosystems Manufacturing Quality Control Find and Order Alternate Assays Using the Web Site 1. Log in to the web site, then open the TaqMan SNP Genotyping Assays page: a. Go to b. In the Home page of the Applied Biosystems web site, click Store Log In (or click Log In), then log in using your customer account information. c. After logging in, select TaqMan SNP Genotyping Assays in the Products drop-down list. 1b 1b 1c 2. In the TaqMan SNP Genotyping Assays page, select the Assay Search tab. 3. In the Assay Search tab: a. Click Batch ID Search. b. In the Option 1 Enter IDs list, enter the rs IDs of the alternate SNPs that you obtained in step 3 on page 4. c. Click Search. 2 3a 3b 3c 6 TaqMan SNP Genotyping Assays User Bulletin

7 Search for Alternate TaqMan SNP Genotyping Assays 4. Order the replacement assay: a. Select an alternate assay in the list of available assays. To view details about a replacement assay, click the assay ID for the assay of interest. b. Click Add to Basket. c. Order the assay as instructed by the web site. 4b 4a Find and Order Alternate Assays Using the SNPbrowser Software 1. If necessary, download and install the SNPbrowser Software: a. Go to b. In the Home page of the Applied Biosystems web site, click Support. c. In the Support page, select SNPbrowser Software Version 3.5 in the Software Downloads, Patches & Updates drop-down list, then click Continue. d. Click Download Free Software. e. Complete the registration as directed by the web site. f. When you receive an from Applied Biosystems, follow the instructions to download and install the SNPbrowser Software. 2. In the desktop, select Start Program Files SNPbrowser Software SNPbrowser Software. 3. In the SNPbrowser Software window: a. In the View Chr drop-down list, select the chromosome number determined in step 3c on page 2. b. In the Search ( ) drop-down list, select Base Range. Note: You can also search for alternate SNPs by dbsnp ID or Entrez Gene ID. c. In the Left Base Position and Right Base Position fields, enter the upstream and downstream base positions determined in step 4 on page 3. TaqMan SNP Genotyping Assays User Bulletin 7

8 Replacing TaqMan SNP Genotyping Assays that Fail Applied Biosystems Manufacturing Quality Control d. Click Search ( ). e. Click Zoom In/Zoom Out ( / ) to display reference SNP clusters within the target region. f. When you identify a reference SNP cluster of interest, right-click the rs ID, then select Add rs<number> to Order List. If you cannot find a suitable pre-developed TaqMan SNP Genotyping Assay that targets a SNP near to the SNP of the failed assay, choose a SNP in the NCBI dbsnp database and obtain its associated sequence. Submit the sequence for custom TaqMan SNP Genotyping Assay design according to the instructions on the Applied Biosystems web site. g. After you add one or more assays to the order, click Order SNPs ( ) to view the desired TaqMan SNP Genotyping Assay(s). 3e 3c 3d 3g 3a 3b 3f 4. In the Current TaqMan Assay Order dialog box, click Order TaqMan Assays On-Line. 5. Log in to the Applied Biosystems store, then order the selected TaqMan SNP Genotyping Assay(s) as instructed. 8 TaqMan SNP Genotyping Assays User Bulletin

9 Search for Alternate TaqMan SNP Genotyping Assays Find and Order Alternate Assays Using the Mouse SNPbrowser Software 1. If necessary, download and install the Mouse SNPbrowser Software: a. Go to b. In the Home page of the Applied Biosystems web site, click Support. c. In the Support page, select Mouse SNPbrowser Software in the Software Downloads, Patches & Updates drop-down list, then click Continue. d. In the Mouse SNPbrowser Software page, click Download Free Software. e. Complete the registration as directed by the web site. f. When you receive an from Applied Biosystems, follow the instructions to download and install the Mouse SNPbrowser Software. 2. Select Start Program Files Mouse SNPbrowser Software Mouse SNPbrowser Software. 3. In the Mouse SNPbrowser Software window, click SNP Wizard ( ) In the Wizard Functions dialog box: a. Select Genetic Mapping. b. Click Next ( ). 4a 4b 5. In the Mapping Form dialog box: a. Select the strains of interest in the Strain 1 and Strain 2 drop-down lists. b. Select Candidate Region, then in the: Chr drop-down list Select the chromosome number determined in step 3c on page 2. Units drop-down list Select Mb. Start/End fields Enter the upstream and downstream base positions determined in step 4 on page 3. c. Specify the resolution level (for example, 1 Mb). TaqMan SNP Genotyping Assays User Bulletin 9

10 Replacing TaqMan SNP Genotyping Assays that Fail Applied Biosystems Manufacturing Quality Control d. Click Submit ( ). 5a 5b 5c 5d 6. In the Mouse SNPbrowser Software window: a. Click Zoom In ( ). b. Click the highlighted region of the chromosome map to display the reference SNP clusters within the region. c. When the reference SNP clusters are displayed, click Export Selected SNPs in View ( ). d. When prompted to save the file, navigate to the desired location, enter a name for the text file, then click Save. The exported text file contains a list of the alternate SNPs that you identified using the Mouse SNPbrowser Software. 6a 6b 6c 10 TaqMan SNP Genotyping Assays User Bulletin

11 Search for Alternate TaqMan SNP Genotyping Assays 7. Log in to the Applied Biosystems Store: a. Go to b. In the Home page of the Applied Biosystems web site, click Store Log In (or click Log In). c. Enter your customer account information (username and password), then click Login to log into the Applied Biosystems Store. IMPORTANT! You must log in to the Applied Biosystems Store before you order replacement assays using the Mouse SNPbrowser Software. 7b 7b 7c 8. In the Mouse SNPbrowser Software, order the replacement assay: a. Click Add Selected SNPs to Cart. b. Click SNP Cart ( ) to open the SNP Cart Editor. c. Click Order TaqMan Assays Online to transfer the order to the Applied Biosystems web site. 8c 9. In the Applied Biosystems web site, complete the order: a. In the TaqMan SNP Genotyping Assays page, select an alternate assay. To view details about the replacement assay, click the assay ID for the assay of interest. b. Click Add to Basket. c. Order the assay as instructed by the web site. TaqMan SNP Genotyping Assays User Bulletin 11

12 Copyright 2008, Applied Biosystems. All rights reserved. For Research Use Only. Not for use in diagnostic procedures. Information in this document is subject to change without notice. Applied Biosystems assumes no responsibility for any errors that may appear in this document. APPLIED BIOSYSTEMS DISCLAIMS ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT, EXPRESSED OR IMPLIED, INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE. IN NO EVENT SHALL APPLIED BIOSYSTEMS BE LIABLE, WHETHER IN CONTRACT, TORT, WARRANTY, OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING BUT NOT LIMITED TO THE USE THEREOF. TRADEMARKS: Applera, Applied Biosystems, and AB (Design) are registered trademarks, and SNPbrowser is a trademark of Applera Corporation or its subsidiaries in the U.S. and/or certain other countries. TaqMan is a registered trademark of Roche Molecular Systems, Inc. All other trademarks are the sole property of their respective owners. Worldwide Sales and Support Applied Biosystems vast distribution and service network, composed of highly trained support and applications personnel, reaches 150 countries on six continents. For sales office locations and technical support, please call our local office or refer to our Web site at Applera is committed to providing the world s leading technology and information for life scientists. Applera Corporation consists of the Applied Biosystems and Celera Genomics businesses. Headquarters 850 Lincoln Centre Drive Foster City, CA USA Phone: Toll Free (In North America): Fax: / Part Number Rev. A Stock Number 135UB01-01