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Genome-Wide Human SNP Nsp/Sty Assay 5.0 User Manual

1. llus 104 Reagents Required vk kk a kk KK KK KK eee 105 Gels and Related Materials Required WWW RR KRN 105 Important Information About This Stage 0 20 20 eee 106 Prepare the Reagents Consumables and Other Components 107 Prepare the Samples for Fragmentation 108 What To Do Next D ay seein cGy ee eis Eh ma 110 Check the Fragmentation Reaction 0 0 0 00000 111 Stage l0 ka Dell Giese one ttt rettet emet haa Abd CER Mna 112 Abo t this Stage 2 5 CoL Ie due EE LV VPE PCI 112 Location and Duration 0 0 eee 112 Input Required from Previous Stage KRI eee 112 Equipment and Consumables Required llus 112 Reagents Required i ka kk kK KK KK KK KK eee 113 Important Information About This Stage 000 KK 114 Prepare the Reagents Consumables and Other Components 114 Prepare the Labeling Master Mix KK KIRI eee eee eee 115 Add the Labeling Master Mix to the Samples 115 What bo Do Next s xwa w l oc a enc bee eee aed 116 Stage 11 Target Hybridization Ak kk kk KK KK KI IIIIII KII eee 117 AbOurtHIS Stage x ete Xwa a ka A Bee be Fa RE es 117 bocatonmrand Dau Sa kul XA os Mato e ahs nan Bae R 117 Input Required from Previous Stage 0 000 eee 117 Equipment and Consumables Required 00 118 Reagents Required iius e ey ab beb mod M Oa 119 Important Information Abou
2. 148 QC and Genotyping Analysis Workflow 00 000000 149 Set up the OC Analysis XL KK KK KK KK KK KK KK KIR KK 149 Set Up the Genotyping Analysis KK KIRI KIY 153 Output File Formats eara renr v ba aed BEP peed 4484 158 qe Tep Pk EO rg cde v De rep PR BILE DEPO DO QS d 158 Clustering Report File Ak A AA KK KK eee 158 Genotype Calls File kk kk kk KK eae 160 Genotype Confidences Files WAKI ee 161 Probeset genotype log kk kk eee 162 Probeset q6 log gt ca ahen t e M oec eater A ek aes 163 SNP Intensity Summary File llle 164 contents vii Chapter 7 Chapter 8 Appendix A Assessing Data Quality kk kk kk eee 165 Galli Rate N N DR E RR 166 Oligonucleotide Controls 4 4 i 2k S pa kk WW sk a an aa Zu 168 B2 Oligo Performance Wk kk kk KK eee 168 Downstream Analysis Considerations 0 0 000002 170 Troubleshooting nasara a eR en ph ERR EE 173 Assay Recommendations else 173 Troubleshooting the Genome Wide Human SNP 5 0 Nsp Sty Assay 176 OD Troubleshooting Guidelines sells 179 When to Contact Technical Support 0 0 0 0 182 MAMEN S 4 lorke bk eee A ale bik w me 00 d b1 se EE W Be ida 182 Vacuum Manifold and Fluidics Station Care and Maintenance 183 Cleaning the Vacuum Manifold AA KK K cee eee 183 General Fluidics Station Care AW kk kk KK RR RR R RR R RR YR RIK KIRI 183 Fluidics Station
3. 70 Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Dilute the Samples IMPORTANT It is crucial to dilute the ligated DNA with AccuGENE water prior to PCR To dilute the samples 8 Place the AccuGENE water on ice 20 minutes prior to use 1 When the GW5 0 Ligate program is finished remove the plate and spin it down at 2000 rpm for 30 sec 2 Place the plate in a cooling chamber on ice 3 Dilute each reaction as follows A Pour 10 mL AccuGENE water into the solution basin B Using a 12 channel P200 pipette add 75 uL of the water to each reaction The total volume in each well is 100 uL Nsp Ligated DNA 25 uL AccuGENE water 75 uL Total 100 uL Seal the plate tightly with adhesive film 5 Vortex the center of the plate at high speed for 3 sec 6 Spin down the plate at 2000 rpm for 30 sec What To Do Next Do one of the following If following the recommended workflow Figure 4 1 on page 23 proceed immediately to Stage 6 Nsp PCR on page 71 Store the plate in a cooling chamber on ice for up to 60 minutes If not proceeding directly to the next step store the plate at 20 C chapter 4 Affymetrix Genome Wide Human SNP Nsp Sty 5 0 Assay 48 Sample Protocol 71 Stage 6 Nsp PCR About this Stage During this stage you will 1 Transfer equal amounts of each Nsp ligated sample into four fresh 96 well plates 2 Prepare the Nsp PCR Master Mix and add it to e
4. C Output summaries file Figure 6 8 Example of Window When Advanced Options are Used 2 Optional To reset the parameters back to their default values click Default chapter 6 Data Analysis 155 3 In the Output Options box specify the output as follows To analyze a very large number of samples gt 500 at a time Select the Output Tables option Deselect the Output CHP Files and Output text files options Results are written to a single file of genotype calls Output CHP Files AGCC Format The genotyping results are stored in a binary CHP file Selection of AGCC format will store the results in a format compatible with Affymetrix GeneChip Command Console software in a sub folder named cc chp See http www affymetrix com products software specific command console software affx for more information Selection of GCOS format will store the genotyping results in a binary format compatible with GCOS in a sub folder named chp x NOTE BRLMM P results can only be stored in text files or AGCC format CHP files Output text files Output genotype calls and confidences in a directory named txt under the specified output directory This option creates a single txt file per CEL file analyzed The txt file has 11 lines of header followed by tab delimited text with one line per SNP and three columns Column 1 SNP ID Column 2 Genotype call 1 NoCall 02AA 1 AB 2 BB Column 3 Genotype
5. Prepare Your Work Area To prepare the work area 1 2 Place a double cooling chamber and a cooler on ice Figure 4 3 on page 37 Label the following tubes then place in the cooling chamber One strip of 12 tubes labeled Dig A 2 0 mL Eppendorf tube labeled Dig MM Place the AccuGENE water on ice Prepare the plate with genomic DNA as follows A Vortex the center of the plate at high speed for 3 sec B Spin down the plate at 2000 rpm for 30 sec C Place back in the cooling chamber on ice Prepare the reagents except for the enzyme as follows A Vortex 3 times 1 sec each time B Pulse spin for 3 sec C Place in the cooling chamber Preheat the Thermal Cycler Lid Power on the thermal cycler to preheat the lid Leave the block at room temperature chapter 4 Affymetrix Genome Wide Human SNP Nsp Sty 5 0 Assay 48 Sample Protocol 37 Set of strip tubes AccuGene water NEBuffer 3 BSA and Eppendorf tube Plate of genomic DNA labeled Sty Double cooling chamber Figure 4 3 Work Area Prepared for Processing Samples with Sty Digest Mix Sty Enzyme Not Pictured Still at 20 C Prepare the Sty Digestion Master Mix Keeping all reagents and tubes on ice prepare the Digestion Master Mix as follows 1 To the 2 0 mL Eppendorf tube add the volumes of the following reagents as shown in Table 4 8 AccuGENE water NE Buffer 3 BSA 2 Remove the Sty I enzyme from the freeze
6. mus mH E Affymetrix Genome Wide Human SNP Nsp Sty Assay 5 0 P N 702419 Rev 2 For research use only Not for use in diagnostic procedures Trademarks 2 Affymetrixe AX GeneChips HuSNPs GenFlexe Flying Objective CustomExpresse CustomSeq NetAffx Tools to Take You As Far As Your Visione The Way Ahead Powered by Affymetrix GeneChip compatible and Command Console are trademarks of Affymetrix Inc All other trademarks are the property of their respective owners Limited License Subject to the Affymetrix terms and conditions that govern your use of Affymetrix products Affymetrix grants you a non exclusive non transferable non sublicensable license to use this Affymetrix product only in accordance with the manual and written instructions provided by Affymetrix You understand and agree that except as expressly set forth in the Affymetrix terms and conditions that no right or license to any patent or other intellectual property owned or licensable by Affymetrix is conveyed or implied by this Affymetrix product In particular no right or license is conveyed or implied to use this Affymetrix product in combination with a product not provided licensed or specifically recommended by Affymetrix for such use Patents Arrays Products may be covered by one or more of the following patents and or sold under license from Oxford Gene Technology U S Patent Nos 5 445 934 5 700 637 5 744
7. Main Lab appendix A Reagents Equipment and Consumables 199 Table A 5 Continued Other Equipment Required to Run the Genome Wide Human SNP 5 0 Nsp Sty Assay Equipment Quantity Manufacturer Part Number Laboratory Distributor Location Pipette 12 channel P100 2 Rainin P N L12 100 Pre PCR and Main Lab Pipette 12 channel P200 2 Rainin P N L12 200 Pre PCR and Main Lab Pipette 12 or 8 channel P1200 1 Rainin P N Main Lab Plate Centrifuge multipurpose 1 Eppendorf 5804 or 5810 Pre PCR must be deep well in Main Lab Plate Centrifuge multipurpose deep well 1 Eppendorf 5804 or 5810 Main Lab must accommodate plates 54mm height 160g weight Plate holders 9 USA Scientific 2300 9602 7 Main Lab Spectrophotometer high throughput 1 Molecular SpectraMax Main Lab microplate spectrophotometer Devices Plus384 Thermal Cyclers see Table A 6 on page 200 Vortexer for plates and tubes 2 VWR 58816 12 Pre PCR and must have plate pad Main Lab Thermal Cyclers PCR Plates and Plate Seals Quantity Required Five thermal cyclers are required for this protocol One in the PCR Staging Room Four in the Main Lab Vendor and Part Number Information This protocol has been optimized using the following thermal cyclers PCR plate and adhesive film IMPORTANT Use only the PCR plate adhesive film and thermal cyclers listed in Table A 6 Using other PCR plates and film that are incompatibl
8. Preheat the Hybridization Ovens To preheat the hybridization ovens 1 Turn each oven on and set the temperature to 50 C 2 Setthe rpm to 60 3 Turn the rotation on and allow to preheat for 1 hour before loading arrays IMPORTANT An accurate hybridization temperature is critical for this assay Therefore we recommend that your hybridization ovens be serviced at least once per year to ensure that they are operating within the manufacturer s specifications 122 Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Thaw Reagents If the labeled samples from the previous stage were frozen 1 9 RON Thaw the plate on the bench top Vortex the center of the plate at high speed for 3 sec Spin down the plate at 2000 rpm for 30 sec Place in a cooling chamber on ice If hybridizing samples using Method 1 or 2 the labeled samples must be placed in a Bio Rad unskirted 96 well plate P N MLP 9601 For Method 2 the used wells on the plate are cut into 2 strips of 24 wells each Preheat the Thermal Cycler Lid Power on the thermal cycler to preheat the lid Leave the block at room temperature Prepare the Arrays To prepare the arrays 1 2 Unwrap the arrays and place on the bench top septa side up Mark each array with a meaningful designation e g a number to ensure that you know which sample is loaded onto each array Allow the arrays to warm to room temperature by leaving on the bench to
9. a brimm confidences txt Notepad Joe File Edit Format View Help chiptype program name BRLMM Analysis Tool j 2 0 program version 1 D alg name brlmm alg version 1 D analysis guid 0000001507 1163518841 0000012119 0000027790 0000017066 exec guid 0000065295 116 3518806 0000016062 00000083531 0000014973 het mult 1 iterations 0 iter thresh 0 3 K 4 transform CCS prior weight 40 prior mincall 2 lowprecision 0 prior si ze 10000 chrX file C Documents and Settings jburriiMy Documents Data genotype LibraryFiles Mapping250K_Nsp chn max score 0 5 dm thresh 0 17 dm het mult 0 fe at effect file target sketch file qmethod spec plier optmethod 1 prior fi le chrX force 0 force 0 analysis brlmm cel count 6 cvs id BRLMM Analysis T ool 2 0 model file time str Tue Nov 14 07 40 06 2006 analysis text quant norm sketch 50000 pm only brimm transform ccs K 4 MS 0 5 guid 0000057699 1163519120 0000012036 0000002313 0000018402 exec_guid 0000065295 1163513306 0000016062 0000002531 0000014973 exec_version BRLMM Analysis Tool 2 0 1 0 create_date Tue Nov 14 07 40 06 2006 Scmd chip_type Mapping250K_Nsp lib_set_name C Documents and Settings jburriiMy Documents Data genotype LibraryFiles Mapping250K_Nsp cdf lib_set_version C Documents and Settings jburri My Documents Datalgenotype LibraryFiles Mapping250K_Nsp cdf probeset_id NA10351 CEL NA10855 CEL N 10863 CEL NA11831 CEL NA11832 CEL N 12056 CEL AFFX 2315060 0 00001 0 002704 0 010249 0
10. Oncogene 2004 23 2716 26 65 Herr A Grutzmann R Matthaei A Artelt J Schrock E Rump A Pilarsky C High resolution analysis of chromosomal imbalances using the Affymetrix 10K SNP genotyping chip Genomics 2005 85 392 400 Lieberfarb ME Lin M Lechpammer M Li C Tanenbaum DM Febbo PG Wright RL Shim J Kantoff PW Loda M et al Genome wide loss of heterozygosity analysis from laser capture microdissected prostate cancer using single nucleotide polymorphic allele SNP arrays and a novel bioinformatics platform darraysNP Cancer Res 2003 63 4781 5 Ishikawa S Komura D Tsuji S Nishimura K Yamamoto S Panda B Huang J Fukayama M Jones KW Aburatani H Allelic dosage analysis with genotyping microarrays Biochem Biophys Res Commun 2005 333 1309 1314 Zhao X Weir BA LaFramboise T Lin M Beroukhim R Garraway L Beheshti J Lee JC Naoki K Richards WG et al Homozygous deletions and chromosome amplifications in human lung carcinomas revealed by single nucleotide polymorphism array analysis Cancer Res 2005 65 5561 70 Komura D Nishimura K Ishikawa S Panda B Huang J Nakamura H Ihara S Hirose M Jones KW Aburatani H Noise reduction from genotyping microarrays using probe level information n Silico Biol 2006 6 79 92 Koed K Wiuf C Christensen LL Wikman FP Zieger K Moller K von der Maase H Orntoft TF High density single nucleotide polymorphism array defi
11. Pre PCR Clean Area Hands on time 30 minutes GW5 0 Digest thermal cycler program time 2 5 hours Input Required From Previous Stage The input required is shown below Quantity 48 samples Genomic DNA prepared as instructed under Genomic DNA Plate Preparation on page 29 5 uL at 50 ng uL in each well 34 Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Equipment and Consumables Required The following equipment and consumables are required for this stage Refer to Appendix A for vendor and part number information IMPORTANT Use only the PCR plate adhesive film and thermal cyclers listed in Table 4 1 on page 27 Table 4 6 Equipment and Consumables Required for Stage 1 Sty Restriction Enzyme Digestion Quantity Item 1 Cooler chilled to 20 C 1 Cooling chamber double chilled to 4 C do not freeze 1 Ice bucket filled with ice 1 Marker fine point permanent 1 Microcentrifuge 1 Pipette single channel P100 1 Pipette single channel P200 1 Pipette single channel P1000 1 Pipette 12 channel P20 As needed Pipette tips for pipettes listed above full racks 1 Plate centrifuge 1 Plate seal 1 Thermal cycler 1 strip Tubes 12 strip 0 2 mL 1 Tube Eppendorf 2 0 mL 1 Vortexer chapter 4 Affymetrix Genome Wide Human SNP Nsp Sty 5 0 Assay 48 Sample Protocol 35 Reagents Required The following reagents are required for this s
12. e Prepare Sty PCR Master Mix immediately prior to use and prepare in Pre PCR Clean room To help ensure the correct distribution of fragments be sure to add the correct amount of primer to the master mix Mix the master mix well to ensure the even distribution of primers e To ensure consistent results take 3 uL aliquots from each PCR to run on gels About Controls A PCR negative control can be included in the experiment to assess the presence of contamination Refer to Chapter 3 and Chapter 7 for more information Prepare the Reagents Consumables and Other Components Thaw Reagents and Ligated Samples To thaw the reagents and ligated samples 1 Allow the following reagents to thaw on ice TITANIUM Taq PCR Buffer dNTPs PCR Primer 002 IMPORTANT Leave the TITANIUM Taq DNA Polymerase at 20 C until ready to use 2 Ifthe Sty ligated samples are frozen allow to thaw in a cooling chamber on ice chapter 4 Affymetrix Genome Wide Human SNP Nsp Sty 5 0 Assay 48 Sample Protocol 51 Prepare Your Work Area Pre PCR Clean Area To prepare the work area 1 Place two double cooling chambers and one cooler on ice 2 Label the following then place in a cooling chamber Three 96 well reaction plates labeled P P2 P3 see Figure 4 4 on page 52 One 50 mL Falcon tube labeled PCR MM 3 Place on ice AccuGENE water GC Melt Solution basin 4 Prepare the Sty ligated samples as follows A Vortex
13. EA O Toole JF Nurnberg G Kennedy MA Becker C Hennies HC Helou J Attanasio M Fausett BV et al The centrosomal protein nephrocystin 6 is mutated in Joubert syndrome and activates transcription factor ATF4 Nat Genet 2006 38 674 81 Zhang C Cawley S Liu G Cao M Gorrell H Kennedy GC A genome wide linkage analysis of alcoholism on microsatellite and single nucleotide polymorphism data using alcohol dependence phenotypes and electroencephalogram measures BMC Genet 2005 6 Suppl 1 S17 Vance C Al Chalabi A Ruddy D Smith BN Hu X Sreedharan J Siddique T Schelhaas HJ Kusters B Troost D et al Familial amyotrophic lateral sclerosis with frontotemporal dementia is linked to a locus on chromosome 9p13 2 21 3 Brain 2006 129 868 76 Almeida AM Murakami Y Layton DM Hillmen P Sellick GS Maeda Y Richards S Patterson S Kotsianidis I Mollica L et al Hypomorphic promoter mutation in PIGM causes inherited glycosylphosphatidylinositol deficiency Nat Med 2006 12 846 51 Gutierrez Roelens I Sluysmans T Jorissen M Amyere M Vikkula M Localization of candidate regions for a novel gene for Kartagener syndrome Eur J Hum Genet 2006 14 809 15 Hu N Wang C Hu Y Yang HH Giffen C Tang ZZ Han XY Goldstein AM Emmert Buck MR Buetow KH et al Genome wide association study in esophageal cancer using GeneChip mapping 10K array Cancer Res 2005 65 2542 6 Affymetrix Ge
14. Genome Wide Human SNP Nsp Sty 5 0 Assay 48 Sample Protocol 41 Equipment and Consumables Required The following equipment and consumables are required for this stage Refer to Appendix A for vendor and part number information Table 4 10 Equipment and Consumables Required for Stage 2 Sty Ligation Quantity Item 1 Cooler chilled to 20 C 1 Cooling chamber double chilled to 4 C do not freeze 1 Ice bucket filled with ice 1 Marker fine point permanent 1 Microcentrifuge 1 Pipette single channel P100 1 Pipette single channel P1000 1 Pipette 12 channel P20 1 Pipette 12 channel P200 As needed Pipette tips for pipettes listed above full racks 1 Plate centrifuge 2 Plate seal 1 Solution basin 55 mL 1 Thermal cycler 1 strip Tubes 12 strip 0 2 mL 1 Tube Eppendorf 2 0 mL 1 Vortexer IMPORTANT Use only the PCR plate adhesive film and thermal cyclers listed in Table 4 1 on page 27 42 Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Reagents Required The following reagents are required for this stage Refer to Appendix A for vendor and part number information The amounts listed are sufficient to process 48 samples Table 4 11 Reagents Required for Stage 2 Sty Ligation Quantity Reagent 1 vial T4 DNA Ligase 400 U uL NEB 1 vial T4 DNA Ligase Buffer 10X 1 vial Adaptor Sty 50 uM 10 mL AccuGENE water molecular
15. H Di X Liu G Hubbell E Law J Berntsen T Chadha M Hui H et al Genotyping over 100 000 SNPs on a pair of oligonucleotide arrays Nat Methods 2004 1 109 111 Uimari P Kontkanen O Visscher PM Pirskanen M Fuentes R Salonen JT Genome wide linkage disequilibrium from 100 000 SNPs in the East Finland founder population Twin Res Hum Genet 2005 8 185 97 Klein RJ Zeiss C Chew EY Tsai JY Sackler RS Haynes C Henning AK Sangiovanni JP Mane SM Mayne ST et al Complement factor H polymorphism in age related macular degeneration Science 2005 308 385 9 Serono Identifies 80 Genes Involved in Multiple Sclerosis Using 100 000 SNPs In Affymetrix Microarray Bulletin 2005 Issue 1 1 4 www microarraybulletin com Arking DE Pfeufer A Post W Kao WH Newton Cheh C Ikeda M West K Kashuk C Akyol M Perz S et al A common genetic variant in the NOSI regulator NOSIAP modulates cardiac repolarization Nat Genet 2006 38 644 5 Papassotiropoulos A Stephan DA Huentelman MJ Hoerndli FJ Craig DW Pearson JV Huynh KD Brunner F Corneveaux J Osborne D et al Common Kibra alleles are associated with human memory performance Science 2006 314 475 8 chapter 1 Overview 9 53 54 55 56 57 58 59 60 61 62 63 Shriver MD Kennedy GC Parra EJ Lawson HA Sonpar V Huang J Akey JM Jones KW The genomic distribution of population su
16. Jen M Chen X Frazer KA Common deletions and SNPs are in linkage disequilibrium in the human genome Nat Genet 2006 38 82 5 Chapter LABORATORY SETUP AND RECOMMENDATIONS General Workflow The table below indicates the laboratory areas in which the various stages of the Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay should be carried out Pre PCR Clean Room PCR Staging Room and Main Lab Guidelines for each area are provided in this chapter Table 2 1 Overview of the Areas Required to Perform the Genome Wide Human SNP 5 0 Nsp Sty Assay Area Template Genomic DNA PCR Product Pre PCR Clean Room Assay Steps Reagent Preparation PCR Staging Room Assay Steps Digestion Ligation PCR set up only Main Lab Assay Steps PCR thermal cycling PCR cleanup Fragmentation Labeling Hybridization Washing and staining Scanning Ss v O vy vo 14 Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Contamination Prevention Care should be taken to minimize possible sources of contamination that would reduce genotyping accuracy call rate and consequently genetic power To reduce the possibility of cross contamination Affymetrix recommends maintaining a single direction workflow NOTE The most likely potential source of contamination for the Genome Wide Human SNP 5 0 Nsp Sty Assay is previously amplified PCR product Each room should contain ded
17. PD Dunning AM Ponder BA Easton DF Association studies for finding cancer susceptibility genetic variants Nat Rev Cancer 2004 4 850 60 Patil N Berno AJ Hinds DA Barrett WA Doshi JM Hacker CR Kautzer CR Lee DH Marjoribanks C McDonough DP et al Blocks of limited haplotype diversity revealed by high resolution scanning of human chromosome 21 Science 2001 294 1719 23 Crawford DC Carlson CS Rieder MJ Carrington DP Yi Q Smith JD Eberle MA Kruglyak L Nickerson DA Haplotype diversity across 100 candidate genes for inflammation lipid metabolism and blood pressure regulation in two populations 4m J Hum Genet 2004 74 610 22 Dawson E Abecasis GR Bumpstead S Chen Y Hunt S Beare DM Pabial J Dibling T Tinsley E Kirby S et al A first generation linkage disequilibrium map of human chromosome 22 Nature 2002 418 544 8 Phillips MS Lawrence R Sachidanandam R Morris AP Balding DJ Donaldson MA Studebaker JF Ankener WM Alfisi SV Kuo FS et al Chromosome wide distribution of haplotype blocks and the role of recombination hot spots Nat Genet 2003 33 382 7 Kennedy GC Matsuzaki H Dong S Liu WM Huang J Liu G Su X Cao M Chen W Zhang J et al Large scale genotyping of complex DNA Nat Biotechnol 2003 21 1233 7 Matsuzaki H Loi H Dong S Tsai Y Y Fang J Law J Di X Liu W M Yang G Liu G et al Parallel genotyping of over 10 000 SNPs
18. Transfer 2 uL to the corresponding well s on the OD plate e Proceed to fragmentation with 45 uL in each well Insufficient purified PCR product for fragmentation Volume in a particular well s on the elution catch plate is 45 uL Do the following in this order Measure the actual volume using a pipettor Add Buffer EB to a final volume of 47 yL Mix by pipetting up and down Transfer 2 uL to the corresponding well s in the OD plate e Proceed to fragmentation with 45 uL in each well 178Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Problem Likely Cause Fragmented PCR product is not the correct size Solution PCR product is still visible Failed or incomplete fragmentation in 200 1 100 bp size region due to reduced DNase activity Check that you have selected the correct activity of DNase from Table 4 46 on page 108 to add to fragmentation reaction See Dilute the Fragmentation Reagent on page 108 Ensure fragmentation reagent DNase is kept at 20 C Do not reuse diluted working stock CEL file can not be generated GCOS is unable to align grid Unable to place a grid on the dat file due to the absence of B2 signal Hybridization controls including oligo B2 must be added to hybridization cocktail for grid alignment dat image is dim Insufficient signal intensity or staining failure Make fresh stain buffers
19. keeping the basin on ice Table 4 17 Sty PCR Master Mix for 48 Samples Reagent For 1 Reaction 3 PCR Plates 48 Samples Each Plate 15 extra AccuGENE water 39 5 uL 6 541 mL TITANIUM Taq PCR Buffer 10X 10 uL 1 656 mL GC Melt BM 20 uL 3 312mL dNTP 2 5 mM each 14 uL 2 318 mL PCR Primer 002 100 uM 4 5 uL 0 745 mL TITANIUM Taq DNA Polymerase 50X 2 uL 0 331 mL do not add until ready to aliquot master mix to ligated samples Total 90 uL 14 903 mL 54 Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Add Sty PCR Master Mix to Samples Location PCR Staging Area Procedure To add Sty PCR Master Mix to samples 1 Using a 12 channel P200 pipette add 90 uL Sty PCR Master Mix to each sample To avoid contamination change pipette tips after each dispense The total volume in each well is 100 uL Seal each reaction plate tightly with adhesive film Vortex the center of each reaction plate at high speed for 3 sec Spin down the plates at 2000 rpm for 30 sec ao RON Keep the reaction plates in cooling chambers on ice until loaded onto the thermal cyclers Load Sty PCR Plates Onto Thermal Cyclers IMPORTANT PCR protocols for the MJ Tetrad PTC 225 and Applied Biosystems thermal cyclers are different See Table 4 18 and Table 4 19 below Location Main Lab Procedure To load the plates and run the GW5 0 PCR program 1 Transfer the plates to the Main Lab 2
20. systematic or sporadic errors that occur due to stochastic sample or experimental factors To filter out errors and exclude these SNPs in downstream analysis a two tiered filtering process is recommended In the first filter samples are included only if the QC call rate is greater than 86 when using high quality DNA see Chapter 3 Genomic DNA General Requirements The QC call rate is based on generating calls using the DM algorithm This algorithm can run on a single array using a set of about 3000 SNPs enriched for SNPs that are challenging to call These QC call rates are well correlated with the higher call rates and concordance achieved when calls are subsequently made with BRLMM P The genotypes for passing samples are generated using the BRLMM P algorithm In general clustering larger batches of samples will improve the performance of the algorithm Prior to downstream analysis it is prudent to apply some SNP filtering criteria to remove SNPs that are not performing ideally in the data set in question The subject of SNP filtering is an area of current research and best practices are still being developed by the community Some common filters used will Remove SNPs with a significantly low per SNP call rate Remove SNPs out of HW equilibrium in controls Remove SNPs with significantly different call rates in cases and controls Remove SNPs with Mendelian errors Studies on multiple data sets have shown that SNPs with a lower per
21. 000123 0 003339 0 000347 AFFX 2315061 0 00021 0 001688 0 000047 0 002802 0 002316 0 001255 AFFX 2315062 0 009728 0 000612 0 001143 0 000531 0 006553 0 000708 AFFX 2315057 0 015506 0 006161 0 012064 0 012611 0 001695 0 001313 AFFX 2315058 0 009664 0 003937 0 015404 0 005133 0 000512 0 000204 AFFX 2315059 0 005509 0 015714 0 001237 0 000582 0 00021 0 002272 Figure 6 14 Example of Genotype Confidences File 162Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Probeset genotype log The probeset genotype log file contains the output of the algorithm engine for the analysis run a probeset genotype log Notepad Leg File Edit Format View Help Wed Nov 15 13 36 41 2006 cvsid BRLMM Analysis Tool 20 Wed Nov 15 13 36 41 2006 version 1 0 Wed Nov 15 13 36 41 2006 command line Wed Nov 15 13 36 41 2006 exec guid 0000024479 1 163626601 0000023416 0000029294 0000003957 Wed Nov 15 13 36 41 2006 time Wed Nov 15 13 36 41 2006 Wed Nov 15 13 36 41 2006 free mem 593477222 Wed Nov 15 13 36 41 2006 Beginning analysis of 5 cel files Wed Nov 15 13 36 41 2006 Opening layout file C Documents and Settings jburri My Documents Datalgenotype LibraryFiles Mapping250K_Nsp cdf Wed Nov 15 13 37 19 2006 Loaded 262338 probe sets Wed Nov 15 13 37 25 2006 Loaded 262338 probesets Wed Nov 15 13 37 26 2006 Made ChipStream of type quant norm Wed Nov 15 13 37 26 2006 chiptype program name BRLMM Analysis Tool
22. 2006 4 54 05 PM Batch 20061218_165125 Probe Array Type GenomeWideSNP 5 NAO6S91_HW_301106_Vn _DO1_r1 CEL Gender Report File Name C Documents and Settings dbaile D esktop 20061 218_165125 qc report txt QC call rate Nsp QC call rate Nsp Sty overlap QC call rate Sty Analysis method1 analysis_name QC call rate all analysis dm group_name all_qc as percentage 1 dm cutoff 0 33 dm hetMult 1 25 precision 4 QC call rate all female 93 83 96 49 89 21 94 31 female 93 57 95 5 88 89 93 65 N406993_HW_301106_Vn _BO1_11 CEL male 31 52 95 07 89 05 92 92 NAO6993_HW_301106_Vn _B11_11 CEL male 97 34 98 34 95 01 97 55 N D6833 Hw 301 106 Vnv C11 rl CEL male 95 37 96 61 90 98 95 14 NA06333 Hw 301106 Vnv D04 rl CEL male 92 8 94 27 88 41 92 69 NA4O7019_HW_301106_Vn _BO5_r1 CEL Figure 6 18 Example of QC Report female 94 6 96 12 88 57 94 18 166Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Table 6 2 OC Call Rate Report Metrics Defined Metric Description Chip CEL file name Gender Called gender of the sample OC Call Rate Nsp OC call rate for SNPs on NSP fragments OC Call Rate Nsp Sty Overlap QC call rate for SNPs on both NSP and STY fragments QC Call Rate Sty OC call rate for SNPs on STY fragments OC Call Ra
23. 209 GWDb O EID uet hi tete ot entes iter ker e rb dad A dib W iT 209 Appendix C Kel DEN er ov atem Pe cree e e Rd ti 211 Before Using E GelsS iu oce yes E Rb RE A KI KK KIR 211 When Using the E Gel 48296 li eee 211 When Using the E Gel 48496 kk kk ee 211 Modifications for Stage 3 Sty PCR ee 212 Gels and Related Materials Required WW RR 000 uae 212 R NNO Gels cuiu s oes sce Dee ata ud tue Rat gs 212 Modifications for Stage 6 Nsp PCR kk kk RR ne 214 Gels and Related Materials Required WWW RR RR RR 214 RUNNING GES ua ict te s eae sessi eaten LE Eb ded 214 Before Running Gels JWA a kk kK KK KK KK KK RR ee eee 214 Modifications for Stage 9 Fragmentation RR RR RIK 216 Gels and Related Materials Required 0000 cae 216 Check the Fragmentation Reaction AKIL 216 Chapter OVERVIEW About This Manual This manual is a guide for technical personnel conducting the Affymetrix Genome Wide Human SNP Nsp Sty 5 0 Assay Genome Wide SNP 5 0 Assay experiments in the laboratory It contains Protocols for sample preparation and 48 sample processing Instructions for washing staining and scanning arrays Instructions for generating genotype calls Troubleshooting information A description of each chapter follows Chapter 1 Overview Provides a scientific overview of the concept behind the Genome Wide SNP 5 0 Assay including the biochemical process data generation potential app
24. 29 Genomic DNA Plate Preparation About this Stage The human genomic DNA you will process using the Genome Wide SNP 5 0 Assay should meet the general requirements listed in Chapter 3 Genomic DNA General Requirements During this stage you will prepare the genomic DNA by 1 Determining the concentration of each sample 2 Diluting each sample to 50 ng uL using reduced EDTA TE buffer 3 Aliquoting 5 uL of each sample to the corresponding wells of two 96 well plates Location and Duration PCR Staging Room Hands on time time will vary can be up to 4 hours Input Required This protocol is written for processing two replicates of 48 genomic DNA samples including controls Table 4 3 Input Required for Genomic DNA Plate Preparation Quantity Item Genomic DNA samples that meet the general requirements listed in Chapter 3 Genomic DNA General Requirements 30 Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Equipment and Consumables Required The following equipment and consumables are required for this stage Refer to Appendix A for vendor and part number information Table 4 4 Equipment and Consumables Required for Genomic DNA Plate Preparation Quantity Item enough for three 96 well plates Cooling chambers chilled to 4 C do not freeze 1 Ice bucket filled with ice 1 1 1 Plate centrifuge Pipette single channel P20 Pipette 12 channel P200 1 Pipette s
25. 5 0 Assay 48 Sample Protocol 115 Prepare the Labeling Master Mix Preparation Keep all reagents and tubes on ice while preparing the Labeling Master Mix To prepare the Labeling Master Mix 1 NON PR WN Add the following to the 15 mL centrifuge tube on ice using the volumes shown in Table 4 50 on page 115 5X TdT Buffer DNA Labeling Reagent Remove the TdT enzyme from the freezer and immediately place in the cooler Pulse spin the enzyme for 3 sec then immediately place back in the cooler Add the TdT enzyme to the master mix Vortex the master mix at high speed 3 times 1 sec each time Pulse spin for 3 sec Immediately proceed to the next set of steps Add the Labeling Master Mix to the Samples Table 4 50 Labeling Master Mix Reagent 1 Sample 48 Samples 15 extra TdT Buffer 5X 14 uL 772 8 uL DNA Labeling Reagent 30 mM 2 uL 110 4 uL TdT enzyme 30 U uL 3 5 uL 193 2 uL Total 19 5 uL 1076 4 pL Add the Labeling Master Mix to the Samples To add the Labeling Master Mix to the samples Keep samples in the cooling chamber and all tubes on ice when making additions 1 Aliquot 89 uL of Labeling Master Mix to each tube of the strip tubes 2 Add the Labeling Master Mix as follows A Using a 12 channel P20 pipette aliquot 19 5 uL of Labeling Master Mix to each sample 116Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual B Pipette up and down one time to ensure that
26. As needed Plate seal 1 Solution basin 55 mL 4 Thermal cycler 1 Tube Falcon 50 mL 1 Vortexer IMPORTANT Use only the PCR plate adhesive film and thermal cyclers listed in Table 4 1 on page 27 chapter 4 Affymetrix Genome Wide Human SNP Nsp Sty 5 0 Assay 48 Sample Protocol 73 Reagents Required The following reagents are required for this stage Refer to Appendix A for vendor and part number information The amounts listed are sufficient to process 48 samples Table 4 29 Reagents Required for Stage 6 Nsp PCR Quantity Reagent 15 mL AccuGENE water molecular biology grade 1 vial PCR Primer 002 100 uM The following reagents from the Clontech TITANIUM DNA Amplification Kit dNTPs 2 5 mM each e GC Melt 5M TITANIUM Taq DNA Polymerase 50X e TITANIUM Taq PCR Buffer 10X Gels and Related Materials Required Verifying the PCR reaction is required for this stage You can use the following gels and related materials or E Gels as described in Appendix C E gels on page 211 The amounts listed are sufficient to process 48 Sty samples Refer to Appendix A for vendor and part number information Table 4 30 Gels and Related Materials Required for Stage 6 Nsp PCR Quantity Reagent 190 uL DNA Marker 19 Gels 2 TBE As needed Gel loading solution 4 Plates 96 well reaction 74 Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay M
27. Bleach Protocol 0 KK KK RR eee 185 The Bleach Cyele s s sea ettet era e E bb derba dote 185 Thes RInSe CVCl8u uibs orc erecta iw iet 190 Reagents Equipment and Consumables 193 About this Appendix llle 193 FACOG EMIS s teeters uiua Aw aceto acetone erige T rer ipie heat aves niae 194 Affymetrix Reagents Required WAK kk RR RR RR RR RIK 194 New England Biolabs Reagents Required 00005 195 Other Reagents Required 0 0 KK KERR KK KE KK YI 196 Equipment and Software Required WAV KK RIIJ 197 Affymetrix Equipment and Software Required 197 Other Equipment Required WA kk KK eee 198 Thermal Cyclers PCR Plates and Plate Seals 199 Consumables Required so ov wae ae Meee ace Zh eda ee ala RES 201 viii Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Arrays Required nnau anaa kk kK KK KK KK eee 201 Gels and Gel Related Materials Required 000 201 Other Consumables Required kk KK KERR RR RR KK 202 Supplier Coritact EISt 3 s a a sa awa oh edad Wi he ERE RES v sd 204 Appendix B Thermal Cycler ProgramS SS kk kK KK KK RR KK KK 207 GW5 0 DIOGSt gt ice eosin Sx AAS oe Bn anal RE ahs sales 207 GWS EIgate sins axa re mo bab hee i rere tete terse mata doc sem ectetur Maze 207 e WENA MO RA DD DMME E DR 208 GWS O Fragment ios petanan bed dedi nune tue uo v e debi Cah 209 GWBb 0 Eabel 5 Lies eu Y aet der ge te MIR A
28. Ensure that the thermal cycler lids are preheated The block should be at room temperature 3 Load each reaction plate onto a thermal cycler 4 Run the GW5 0 PCR program The program varies depending upon the thermal cyclers you are using See Table 4 18 for Applied Biosystems thermal cyclers and Table 4 19 for Bio Rad thermal cyclers chapter 4 Affymetrix Genome Wide Human SNP Nsp Sty 5 0 Assay 48 Sample Protocol 55 IMPORTANT If using GeneAmp PCR System 9700 thermal cyclers be sure the blocks are silver or gold plated silver Do NOT use thermal cyclers with aluminum blocks It is not easy to visually distinguish between silver and aluminum blocks Table 4 18 GW5 0 PCR Thermal Cycler Program for the GeneAmp PCR System 9700 silver or gold plated silver blocks GW5 0 PCR Program for GeneAmp PCR System 9700 Temperature Time Cycles 94 C 3 minutes 1X 94 C 30 sec 60 C 45 sec 30X 68 C 15 sec 68 C 7 minutes 1X 4 C HOLD Can be held overnight Volume 100 uL Specify Maximum mode Table 4 19 GW5 0 PCR Thermal Cycler Program for the MJ Tetrad PTC 225 GW5 0 PCR Program for MJ Tetrad PTC 225 Temperature Time Cycles 94 C 3 minutes 1X 94 C 30 sec 60 C 30 sec 30X 68 C 15 sec 68 C 7 minutes 1X 4 C HOLD Can be held overnight Volume 100 uL Use Heated Lid and Calculated Temperature 56 Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Runnin
29. Lau CC Allelic imbalance analysis by high density single nucleotide polymorphic allele SNP array with whole genome amplified DNA Nucleic Acids Res 2004 32 e69 Zhao X Li C Paez JG Chin K Janne PA Chen TH Girard L Minna J Christiani D Leo C et al An integrated view of copy number and allelic alterations in the cancer genome using single nucleotide polymorphism arrays Cancer Res 2004 64 3060 71 Zhou X Mok SC Chen Z Li Y Wong DT Concurrent analysis of loss of heterozygosity LOH and copy number abnormality CNA for oral premalignancy progression using the Affymetrix 10K SNP mapping array Hum Genet 2004 115 327 30 Rauch A Ruschendorf F Huang J Trautmann U Becker C Thiel C Jones KW Reis A Nurnberg P Molecular karyotyping using an SNP array for genomewide genotyping J Med Genet 2004 41 916 22 Bignell GR Huang J Greshock J Watt S Butler A West S Grigorova M Jones KW Wei W Stratton MR et al High resolution analysis of DNA copy number using oligonucleotide microarrays Genome Res 2004 14 287 95 10 Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual 64 65 66 67 68 69 70 7 72 73 Janne PA Li C Zhao X Girard L Chen TH Minna J Christiani DC Johnson BE Meyerson M High resolution single nucleotide polymorphism array and clustering analysis of loss of heterozygosity in human lung cancer cell lines
30. M NaCl 18 5 mL Tween 20 10 0 1 mL Water 73 1 mL Total 100 mL Add 1 mL of Array Holding Buffer to each microcentrifuge tube One tube is needed per module used lt NOTE A vial containing Array Holding Buffer must be placed in sample holder 3 for each module used 140Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Washing and Staining Arrays Fluidics Station 450 Protocol Table 5 7 Fluidics Station 450 Protocol Antibody Amplification for Mapping Targets 49 Format Standard GenomeWideSNP5v1_450 Post Hyb Wash 1 6 cycles of 5 mixes cycle with Wash Buffer A at 25 C Post Hyb Wash 2 24 cycles of 5 mixes cycle with Wash Buffer B at 45 C Stain Stain the array for 10 minutes in SAPE solution at 25 C Post Stain Wash 6 cycles of 5 mixes cycle with Wash Buffer A at 25 C 2nd Stain Stain the array for 10 minutes in Antibody Stain Solution at 25 C 3rd Stain Stain the array for 10 minutes in SAPE solution at 25 C Final Wash 10 cycles of 6 mixes cycle with Wash Buffer A at 30 C The final holding temperature is 25 C Filling Array Fill the array with Array Holding Buffer Wash Buffer A non stringent wash buffer Wash Buffer B stringent wash buffer IMPORTANT The wash and stain buffers are different from the GeneChip expression buffers Washing and Staining Arrays To wash and stain the arrays 1 In the Fluidics Station dialog box on the workstat
31. PCR Master Mix to Samples Location PCR Staging Area Procedure To add Nsp PCR Master Mix to samples 1 a Pw N Using a 12 channel P200 pipette add 90 uL Nsp PCR Master Mix to each sample To avoid contamination change pipette tips after each dispense The total volume in each well is 100 uL Seal each reaction plate tightly with adhesive film Vortex the center of each reaction plate at high speed for 3 sec Spin down the plates at 2000 rpm for 30 sec Keep the reaction plates in cooling chambers on ice until loaded onto the thermal cyclers 78 Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Load Nsp PCR Plates Onto Thermal Cyclers IMPORTANT PCR protocols for the MJ Tetrad PTC 225 and Applied Biosystems thermal cyclers are different Thermal cycler program parameters are on page 79 Location Main Lab Procedure To load the plates and run the GW5 0 PCR program 1 Transfer the plates to the Main Lab 2 Ensure that the thermal cycler lids are preheated The block should be at room temperature 3 Load each reaction plate onto a thermal cycler 4 Run the GW5 0 PCR program The program varies depending upon the thermal cyclers you are using See Table 4 32 and Table 4 33 on page 79 program parameters IMPORTANT If using GeneAmp PCR System 9700 thermal cyclers be sure the blocks are silver or gold plated silver Do NOT use thermal cyclers with aluminum blocks It is not easy to vi
32. Protocol Area Thermal Cyclers Plate Film Validated for Use Applied Biosystems units e 2720 Thermal Cycler Pre PCR e GeneAmp PCR System Multiplate 96 Well MicroAmp Clear 9700 Unskirted PCR Plates Adhesive Films Bio Rad P N MLP 9601 Applied Biosystems Bio Rad units P N 4306311 MJ Tetrad PTC 225 DNA Engine Tetrad 2 Applied Biosystems GeneAmp PCR System 9700 silver block or gold Multiplate 96 Well MicroAmp Clear PCR and Post PCR plated silver block Unskirted PCR Plates Adhesive Films Bio Rad P N MLP 9601 Applied Biosystems Bio Rad units P N 4306311 MJ Tetrad PTC 225 DNA Engine Tetrad 2 28 Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Program Your Thermal Cyclers The thermal cycler programs listed below are used during this protocol Before you begin processing samples enter and store these programs on the appropriate thermal cyclers in the PCR Staging Room and the Main Lab Thermal cycler program details are listed in Appendix B Thermal Cycler Programs Table 4 2 Thermal Cycler Programs Required for the 48 Sample Protocol Figure 4 1 on page 23 Program Name of Thermal Cyclers Required Laboratory GW5 0 Digest 1 PCR Staging Room GW5 0 Ligate 1 PCR Staging Room GW5 0 PCR 4 Main Lab GW5 0 Fragment 1 Main Lab GWS5 0 Label 1 Main Lab GW5 0 Hyb 1 Main Lab chapter 4 Affymetrix Genome Wide Human SNP Nsp Sty 5 0 Assay 48 Sample Protocol
33. Reactions to a Filter Plate To transfer the reactions to a filter plate 1 Place the filter plate on the Millipore vacuum manifold 2 Using a 12 channel P1200 pipette transfer each reaction from the pooling plate to the corresponding row and well of the filter plate chapter 4 Affymetrix Genome Wide Human SNP Nsp Sty 5 0 Assay 48 Sample Protocol 89 3 IMPORTANT You will need to pipette twice to transfer all of the solution from each well to the filter plate The solution is viscous and sticky so check to ensure that all of it has been transferred Tightly seal the unused wells with a MicroAmp Clear Adhesive Film To ensure a tight seal cover 1 2 to 1 3 of the wells in row D as well Unused wells must be sealed to ensure proper vacuum pressure a O O S O S S a E e E ERR HEV MER ER DAN MERE HED EREN E Plate seal covering empty F wells rows E through H and 1 2 to 1 3 of the wells in row D Figure 4 8 Sealing Empty Wells on the Filter Plate Purify the Reactions To purify the reactions 1 ao RON Turn on the vacuum to 20 to 24 in Hg and check the seals Do not exceed 24 in Hg Adjust the leak valve if necessary Ensure that the unused wells are completely sealed Cover the plate to protect it from environmental contaminants Allow the liquid to filter 60 to 90 minutes When all of the wells appear dry A Turn off the vacuum and inspect each well using a flashlight The surfa
34. Reagents Affymetrix Reagents Required The Affymetrix Genome Wide Human SNP Nsp Sty Assay Kit 5 0 is required to perform this protocol The kit is available in two sizes 100 reaction size P N 901015 30 reaction size P N 901013 Table A 1 Affymetrix Genome Wide Human SNP Nsp Sty Assay Kit 5 0 Kit Contents Part Number Reference Genomic DNA 103 50 ng uL use as a positive control Included Box 1 e Adaptor Nsp l 50 uM 901015 e PCR Primer 002 100 uM 100 reactions Box 2 or e Adaptor Sty l 50 uM 4 E 901013 PCR Primer 002 100 uM 30 reactions Box 3 Oligonucleotide Control Reagent 0100 GeneChip DNA Labeling Reagent 30 mM Terminal Deoxynucleotidyl Transferase 30 U uL 5X Terminal Deoxynucleotidyl Transferase Buffer 10X Fragmentation Buffer e GeneChip Fragmentation Reagent see label on tube for U uL concentration appendix A Reagents Equipment and Consumables 195 New England Biolabs Reagents Required Table A 2 New England Biolabs Reagents Required Reagent Description Part Number Nsp I 125 uL vial 10 000 U mL containing R0602L Bovine Serum Albumin BSA NEB P N B9001S NEBuffer 2 NEB P N B7002S The BSA and NEBuffer can be ordered separately using these part numbers Sty I 300 uL vial 10 000 U mL containing RO500S Bovine Serum Albumin BSA NEB P N B9001S e NEBuffer NEB P N B7003S The BSA and
35. To ensure proper functioning of the instrument perform periodic maintenance When not using the instrument leave the sample needles in the lowered position Each needle should extend into an empty vial This will protect them from accidental damage Always use deionized water to prevent contamination of the lines Change buffers with freshly prepared buffer at each system startup 184 Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual The fluidics station should be positioned on a sturdy level bench away from extremes in temperature and away from moving air WARNING Before performing any maintenance turn off power to the fluidics station to avoid injury in case of a pump or electrical malfunction chapter 8 Vacuum Manifold and Fluidics Station Care and Maintenance 185 Fluidics Station Bleach Protocol Affymetrix recommends a weekly cleaning protocol for the fluidics station This protocol uses commonly purchased sodium hypochlorite bleach This protocol is designed to eliminate any residual SAPE antibody complex that may be present in the fluidics station tubing and needles The protocol runs a bleach solution through the system followed by a rinse cycle with deionized DI water This protocol takes approximately one hour and forty minutes to complete Affymetrix recommends running this protocol weekly regardless of the frequency of use The current version of the protocol can be found at www affymetrix com
36. What To Do Next i e See nett ake ou ile ah oe c EE A AR 1 39 Stage 2 Sty Ligao ss comu xu AA S E Bec ob LL A 40 Abo t thiS Stage ze a E 2 edo e Md rb ec eX RR ER don bus edad 40 Location and Duration 2 2 z s ze ee RR fure kan Maka WW 40 Input Required From Previous Stage XWA KK ee 40 Equipment and Consumables Required ills 41 Reagents Required vk kk kk kK KK KK KK es 42 Important Information About This Procedure 04 42 Prepare the Reagents Consumables and Other Components 42 Prepare the Sty Ligation Master MIX 0 0 00000 KII 44 Add Sty Ligation Master Mix to Reactions 0 44 Dilute the Samples 0 0 kk KK KK KK KK eee eee 46 What TODO NEXT nir Ade 25 ou ter estt eee Rene ede utut bu aree ve ved 46 Stage 3t SV PO Buses 4 di dilan nil deb dieit de bete E dizl iy pagai 47 About this Stage s eue K ka l Rodeo e p o Md 47 Location and Duration kk kk kk kk KK KK KIR KIRI KI KIR es 47 Input Required from Previous Stage KK RR RR RR 47 Equipment and Materials Required WAIKIKI 48 contents iii Reagents Required kk kk kk eee eee 49 Gels and Related Materials Required WA RR RR 49 Important Information About This Stage 0 0 0 0 20 50 Prepare the Reagents Consumables and Other Components 50 Aliquot Sty Ligated DNA to the PCR Plates 51 Prepare the Sty PCR Master Mix X KK KI KIRI KI eee
37. all of the mix is added to the samples The total volume in each well is now 73 uL Fragmented DNA less 1 5 uL for gel analysis 53 5 uL Labeling Mix 19 5 uL Total 73 pL 3 Seal the plate tightly with adhesive film IMPORTANT Check to ensure that the plate is tightly sealed particularly around the wells on the edge of the plate The plate must be tightly sealed to minimize evaporation while on the thermal cycler Vortex the center of the plate at high speed for 3 sec 5 Spin down the plate at 2000 rpm for 30 sec Place the plate on the pre heated thermal cycler block and run the GW5 0 Label program Table 4 51 GW5 0 Label Thermal Cycler Program GW5 0 Label Program Temperature 37 C 4 hours 95 C 15 minutes 4 C Hold 7 When the GW5 0 Label program is finished A Remove the plate from the thermal cycler B Spin down the plate at 2000 rpm for 30 sec What To Do Next Do one of the following Proceed to the next stage If not proceeding directly to the next stage freeze the samples at 20 C chapter 4 Affymetrix Genome Wide Human SNP Nsp Sty 5 0 Assay 48 Sample Protocol 117 Stage 11 Target Hybridization About this Stage During this stage each reaction is loaded onto a Genome Wide Human SNP Array 5 0 Two methods for performing this stage are presented Method 1 Using a GeneAmp PCR System 9700 Requires the use of a GeneAmp PCR System 97
38. biology grade Important Information About This Procedure To help ensure the best results carefully read the information below before you begin this stage of the protocol Prepare the Reagents Consumables and Other Components IMPORTANT Aliquot the T4 DNA Ligase Buffer 10X after thawing for the first time to avoid multiple freeze thaw cycles See vendor instructions e Be sure to use the Sty adaptor Thaw the Reagents and Sty Digestion Stage Plate To thaw the reagents and Sty Digestion Stage Plate 1 Allow the following reagents to thaw on ice Adaptor Sty I T4 DNA Ligase Buffer 10X Requires approximately 20 minutes to thaw 2 Ifthe Sty digested samples were frozen allow them to thaw in a cooling chamber on ice Hi IMPORTANT Leave the T4 DNA Ligase at 20 C until ready to use chapter 4 Affymetrix Genome Wide Human SNP Nsp Sty 5 0 Assay 48 Sample Protocol 43 Prepare Your Work Area To prepare the work area 1 Place a double cooling chamber and a cooler on ice Figure 4 2 on page 26 2 Label the following tubes then place in the cooling chamber One strip of 12 tubes labeled Lig A 2 0 mL Eppendorf tube labeled Lig MM Solution basin 3 Prepare the digested samples as follows A Vortex the center of the plate at high speed for 3 sec B Spin down the plate at 2000 rpm for 30 sec C Place back in the cooling chamber on ice 4 To prepare the reagents A Vortex at
39. call confidence Output tables Two tab delimited text matrices named brImm calls txt and brlmm confidences txt containing the genotype calls and the confidences respectively are created The format for each file is a few comment lines prefixed with a header line probeset id tab CEL file 1 lt TAB gt CEL file 2 and then a line per SNP SNP ID TAB genotype call or confidence scores CEL file 1 lt TAB gt genotype call or confidence scores CEL file 2 Output summaries file A tab delimited text file named brImm p normalized summary txt containing the allele signal estimates for each allele The format for the file is a few comment lines prefixed with followed by a header line probeset id tab CEL file 1 lt TAB gt CEL file 2 and then one line per allele of each SNP probeset id allele lt TAB gt allele intensity CEL file 1 lt TAB gt allele intensity CEL file 2 Regardless of which output files are selected a report file named lt filename gt report txt is generated The content of this file includes the call rate per CEL file analyzed and additional metrics See Output File Formats on page 158 for more information on the output formats available 4 Click Next to start the analysis The Progress Page window opens automatically Figure 6 9 Status messages and a progress meter are displayed 156Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual BRLMM Analysis Tool P
40. for error To familiarize yourself with the use of multi channel pipettes we strongly recommend practicing several times before processing actual samples You can use water to get a feel for aspirating and dispensing solutions to multiple wells simultaneously Post PCR stages 7 through 11 are best performed by the more experienced operators in your laboratory These operators should be proficient in The use of multi channel pipettes High throughput sample processing When processing multiple full plates we recommend that the same operator not perform too many stages in a given day Dedicating small teams to different stages of the protocol has proven to be a highly effective method of managing this workflow For example the full process can be sub divided into four teams with each team being responsible for the following stages Team 1 Pre PCR digestion and ligation Team 2 PCR PCR and PCR product purification and quantitation Team 3 Post PCR fragmentation and labeling Team 4 Array processing hybridization fluidics and scanning Your technical support representative can provide additional guidance on how best to organize lab personnel for this protocol chapter 4 Affymetrix Genome Wide Human SNP Nsp Sty 5 0 Assay 48 Sample Protocol 25 Before You Begin Master Mix Preparation Carefully follow each master mix recipe Use pipettes that have been calibrated to 5 When molecular biology grade wa
41. has not been optimized for use with this instrument In addition the NanoDrop quantifies a single sample at a time and is not amenable to 96 well plate processing Prepare the Reagents Equipment and Consumables Turn on the Spectrophotometer Plate Reader Turn on the spectrophotometer now and allow it to warm for 10 minutes before use Prepare Your Work Area To prepare the work area 1 Place the following on the bench top Optical plate Solution basin AccuGENE water 2 Label the optical plate OP 3 Prepare the purified eluted PCR product plate as follows A If the plate was frozen allow it to thaw in a cooling chamber on ice B Spin down the plate at 2000 rpm for 30 sec C Place the plate on the bench top 98 Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Prepare Diluted Aliquots of Purified Sample IMPORTANT One row of wells on the optical plate are used as blanks and contain AccuGENE water only The 12 channel P20 pipette must be accurate to within 5 To prepare diluted aliquots of the purified samples 1 2 Pour 15 mL of room temperature AccuGENE water into the solution basin Using a 12 channel P200 pipette aliquot 198 uL of water to each well in rows A through E of the optical plate Using a 12 channel P20 pipette A Transfer 2 uL of each purified PCR product from rows A through D of the purified sample plate to the corresponding rows and wells ofthe optical plate se
42. high speed 3 times 1 sec each time except for the enzyme B Pulse spin for 3 sec C Place in the cooling chamber IMPORTANT T4 DNA Ligase Buffer 10X contains ATP and should be thawed on ice Vortex the buffer as long as necessary before use to ensure precipitate is re suspended and that the buffer is clear Avoid multiple freeze thaw cycles per vendor instructions Preheat the Thermal Cycler Lid Power on the thermal cycler to preheat the lid Leave the block at room temperature The lid must be preheated before samples are loaded 44 Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Prepare the Sty Ligation Master Mix Keeping all reagents and tubes on ice prepare the Sty Ligation Master Mix as follows 1 g NOP Ww To the 2 0 mL Eppendorf tube add the following reagents based on the volumes shown in Table 4 12 Adaptor Sty I T4 DNA Ligase Buffer 10X Remove the T4 DNA Ligase from the freezer and immediately place in the cooler on ice Pulse spin the T4 DNA Ligase for 3 sec Immediately add the T4 DNA Ligase to the master mix then place back in the cooler Vortex the master mix at high speed 3 times sec each time Pulse spin for 3 sec Place the master mix on ice Proceed immediately to Add Sty Ligation Master Mix to Reactions Table 4 12 Sty Ligation Master Mix Reagent 1 Sample 48 Samples 25 extra T4 Ligase Buffer 10X 2 5 uL 150 uL Adaptor Sty 50 uM 0 75
43. products without purification were immediately used in the subsequent protocol steps using 250 ng amplified DNA for each Nsp I and Sty I restriction digestion This procedure gave BRLMM P call rates averaging 97 7 0 3 with an average concordance of 99 2 0 2 Other pre amplification methods or pre digestion with restriction enzymes other than Nsp I or Sty I have not been tested by Affymetrix If other methods are desired we recommend conducting experiments to evaluate their performance with the Genome Wide Human SNP 5 0 Nsp Sty Assay Sources of Human Genomic DNA Genomic The following sources of human genomic DNA have been successfully tested in the laboratories at Affymetrix for DNA that meets the requirements described in the section General Requirements on page 17 blood cell line Success with other types of samples such as formalin fixed paraffin embedded tissue will depend on quality degree of degradation degree of inhibitors present etc quantity of genomic DNA extracted and purity of these types of samples as described under General Requirements on page 17 DNA Extraction Purification Methods Genomic DNA extraction and purification methods that meet the general requirements outlined above should yield successful results Methods that include boiling or strong denaturants are not acceptable because the DNA would be rendered single stranded Genomic DNA extracted using the following methods have been tested
44. replace it with a fresh bottle Not planning to use the system for an Remove the DI water and perform a dry extended period of time longer than protocol shutdown This will remove most of the one week water from the system and prevent unwanted microbial growth in the supply lines Also remove the pump tubing from the peristaltic pump rollers 192 Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual About this Appendix This appendix includes the vendor and part number information for the reagents equipment and consumables that have been validated for use with the Genome Wide Human SNP 5 0 Nsp Sty Assay IMPORTANT Use only the PCR plate adhesive film and thermal cyclers listed in Table A 6 Using other PCR plates and film that are incompatible with the thermal cycler can result in crushed tubes loss of sample or poor results The following lists of reagents equipment and consumables are included in this appendix Affymetrix Reagents Required on page 194 New England Biolabs Reagents Required on page 195 Other Reagents Required on page 196 Affymetrix Equipment and Software Required on page 197 Other Equipment Required on page 198 Thermal Cyclers PCR Plates and Plate Seals on page 199 Arrays Required on page 201 Gels and Gel Related Materials Required on page 201 Other Consumables Required on page 202 194 Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual
45. same row of each PCR plate to the corresponding row and wells of the pooling plate F Repeat steps C D and E until all of the reactions from each PCR plate are pooled on the pooling plate 8 When finished look at the wells of each PCR plate to ensure that all of the product has been transferred and pooled Table 4 36 Pooled Sty and Nsp PCR Products Sty PCR plates 3 100 uL from each well 300 uL well Nsp PCR Plate 4 100 uL from each well 400 uL well Total Volume Each Well of Pooling Plate 700 yL well 87 chapter 4 Affymetrix Genome Wide Human SNP Nsp Sty 5 0 Assay 48 Sample Protocol 35 Ge Ga Go ce HD S D amp 38 38 E E COE 38 E d 38 HOE A oE GOOG KEE QD GD GD 35 GE Gh G G9 35 ED GE GG c Ge GE Es ES d DE 88 8 DE K DE DE E A d DE StyP1 Ge G G9 Ge GO GE EP GE G gt Ge D E DE EE DE CO de GE TO dE d RE Gb GO G RD GEO 6 GO GO ED Gp SD G GD Ee Ge G G G G GE G GD Ep Ge S5 Ge GG Gib GS G5 MD GO G GO G Pooling Plate Deep Well DE d ED d 9 DE e DE BE Sty P3 Nsp P1 Nsp P2 Nsp A of plates Sty P
46. set 52 The same characteristics that make SNPs useful markers for genetic studies also make SNPs powerful markers for additional biological applications such as the analysis of population and admixture structure 53 56 and DNA copy number changes The latter include but are not limited to loss of heterozygosity LOH deletions uniparental disomy UPD and gene amplifications 57 80 The integration of DNA copy number changes with gene expression profiles provides a powerful paradigm for elucidating gene function elegantly illustrated for example by the demonstration that MITF is an oncogene amplified in malignant melanoma 81 In the last several years there has been an increasing appreciation of the extent of structural variation present among normal individuals 82 88 Copy number variations CNVs can encompass a wide range of molecular alterations including duplications losses and inversions can span sizes from 5kb to 50kb intermediate sized and 50kb to 3Mb large scale and are distinct from the genetic sequence diversity represented by SNPs Although there are for example several clear examples of how CNVs can influence susceptibility to HIV infection 89 modulate drug responses 90 or contribute to genomic micro deletion and duplication syndromes 91 a comprehensive biological understanding of the roles of CNVs is not yet currently available but will be important in the context of both the normal and disease states To
47. should be checked chapter 7 Troubleshooting 181 Table 7 3 PROBLEM OD260 OD280 ratio is not between 1 8 and 2 0 Possible causes include The PCR product may be not be sufficiently purified Ensure the vacuum manifold is working properly An error may have been made while taking the OD readings The PCR product may not have been adequately washed Check the 7596 EtOH wash solution Table 7 4 PROBLEM The OD320 measurement is significantly larger than zero 0 0 005 Possible causes include e Magnetic beads may have been carried over into purified sample Precipitate may be present in the eluted samples There may be defects in the OD plate Air bubbles in the OD plate or in solutions 182 Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual When to Contact Technical Support Instruments Under any of the following conditions unplug the instrument from the power source and contact Affymetrix Technical Support when the power cord is damaged or frayed if any liquid has penetrated the instrument if after service or calibration the instrument does not perform to specifications If the instrument must be returned for repair call Affymetrix Technical Support NOTE Make sure you have the model and serial number Affymetrix Inc 3420 Central Expressway E mail support affymetrix com Santa Clara CA 95051 Tel 1 888 362 2447 1 888 DNA CHIP USA Fax 1 408 731 54
48. silver blocks on the GeneAmp PCR System 9700 other blocks are not capable of maximum mode which will affect ramp times Use the recommended plate seal Make sure the seal is tight and that no significant evaporation occurs during the PCR NOTE The Genome Wide SNP 5 0 Assay reaction amplifies a size range of fragments that represents 30 of the genome The Genome Wide Human SNP Array 5 0 are designed to detect the SNPs that are amplified in this complex fragment population Subtle changes in the PCR conditions may not affect the PCR yield but may shift the amplified size range up or down very slightly This can lead to reduced amplification of SNPs that are assayed on the array subsequently leading to lower call rates Troubleshooting Possible Problems with the Elution or OD Readings possible causes include The purified PCR product was eluted in a volume greater than 55 uL The purified PCR product was not mixed adequately before making the 1 100 dilution The diluted PCR product was not mixed adequately before taking the OD reading The water blank reading was not subtracted from each sample OD reading The spectrophotometer plate reader may require calibration Pipettes may require calibration There may be air bubbles or dust in the OD plate There may be defects in the plastic of the plate The settings on the spectrophotometer plate reader or the software may be incorrect OD calculations may be incorrect and
49. support technical fluidics_scripts affx The Bleach Cycle To avoid carryover or cross contamination from the bleach protocol Affymetrix recommends the use of dedicated bottles for bleach and DI water Additional bottles can be obtained from Affymetrix Table 8 1 Affymetrix Recommended Bottles Part Number Description 400118 Media Bottle SO 500 mL 400119 Media Bottle SO 1000 mL 1 Disengage the washblock for each module by pressing down on the cartridge lever Remove any probe array cartridge Figure 8 1 on page 186 2 Prepare 500 mL of 0 525 sodium hypochlorite solution using deionized water You can follow these directions to make 500 mL of bleach In a 1 liter plastic or glass graduated cylinder combine 43 75 mL of commercial bleach such as Clorox bleach which is 6 sodium hypochlorite with 456 25 mL of DI H O mix well Pour the solution into a 500 mL plastic bottle and place the plastic bottle on fluidics station IMPORTANT The shelf life of this solution is 24 hours After this period you must prepare fresh solution Each fluidics station with 4 modules requires 500 mL of 0 525 sodium hypochlorite solution 186Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Remove cartridges if any GoneChip Washblocks disengaged with cartridge lever down Figure 8 1 Disengaged washblocks showing cartridge levers in the down position Remove any cartridges 3 As
50. to process 48 samples Table 4 53 Reagents Required for Stage 11 Target Hybridization Quantity Reagent 5mL Denhardt s Solution 50X 1 5 mL DMSO 100 0 5 mL EDTA 0 5 M 1mL Herring Sperm DNA HSDNA 10 mg mL 500 uL Human Cot 1 DNA 1 mg mL 80g MES Hydrate SigmaUltra 200 g MES Sodium Salt 16 mL Tetramethyl Ammonium Chloride TMACL 5M 10 mL Tween 20 10 250 uL Oligo Control Reagent OCR 0100 120Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Important Information About This Stage To help ensure the best results carefully read the information below before you begin this stage of the protocol IMPORTANT This procedure requires two operators working simultaneously when loading samples onto arrays and placing arrays in the hybridization ovens It is critical that the samples remain on a thermal cycler at 49 C after denaturation and while being loaded onto arrays If you have a GeneAmp PCR System 9700 located adjacent to the hybridization ovens we recommend using method 1 Otherwise you must use method 2 see About this Stage on page 117 About DMSO When adding to the Hybridization Master Mix pipette DMSO into the middle of the tube Do not touch the sides of the tube as the DMSO can leach particles out of the plastic which in turn may cause high background DMSO is light sensitive and must be stored in a dark glass bottle Do not store in a plastic contain
51. using a one primer assay on a high density oligonucleotide array Genome Res 2004 14 414 25 Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual 2 22 23 24 25 26 27 28 29 30 Sellick GS Longman C Tolmie J Newbury Ecob R Geenhalgh L Hughes S Whiteford M Garrett C Houlston RS Genomewide linkage searches for Mendelian disease loci can be efficiently conducted using high density SNP genotyping arrays Nucleic Acids Res 2004 32 e164 John S Shephard N Liu G Zeggini E Cao M Chen W Vasavda N Mills T Barton A Hinks A et al Whole genome scan in a complex disease using 11 245 single nucleotide polymorphisms comparison with microsatellites 4m J Hum Genet 2004 75 54 64 Schaid DJ Guenther JC Christensen GB Hebbring S Rosenow C Hilker CA McDonnell SK Cunningham JM Slager SL Blute ML et al Comparison of microsatellites versus single nucleotide polymorphisms in a genome linkage screen for prostate cancer susceptibility Loci 4m J Hum Genet 2004 75 948 65 Sellick GS Garrett C Houlston RS A novel gene for neonatal diabetes maps to chromosome 10p12 1 p13 Diabetes 2003 52 2636 8 Middleton FA Pato MT Gentile KL Morley CP Zhao X Eisener AF Brown A Petryshen TL Kirby AN Medeiros H et al Genomewide linkage analysis of bipolar disorder by use of a high density single nucleotide polymorphism SNP genotyping as
52. wee ont t Figure 4 5 Example of PCR products run on 2 TBE agarose gel at 120V for 1 hour Average product distribution is between 250 to 1100 bp What To Do Next Do one of the following f following the recommended workflow Figure 4 1 on page 23 seal the Sty PCR product plates and store them at 20 C Proceed to the next stage within 60 minutes 58 Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Stage 4 Nsp Restriction Enzyme Digestion About this Stage During this stage the genomic DNA is digested by the Nsp I enzyme You will 1 Prepare a Nsp Digestion Master Mix 2 Add the master mix to one set of 48 samples 3 Place the samples onto a thermal cycler and run the GW5 0 Digest program Location and Duration Pre PCR Clean Area Hands on time 30 minutes GW5 0 Digest thermal cycler program time 2 5 hours Input Required From Previous Stage The input required is shown below Quantity 48 samples Genomic DNA prepared as instructed under Genomic DNA Plate Preparation on page 29 5 uL at 50 ng uL in each well chapter 4 Affymetrix Genome Wide Human SNP Nsp Sty 5 0 Assay 48 Sample Protocol 59 Equipment and Consumables Required The following equipment and consumables are required for this stage Refer to Appendix A for vendor and part number information IMPORTANT Use only the PCR plate adhesive film and thermal cyclers listed in Table 4 1 o
53. 00 located adjacent to the hybridization ovens Samples are on a 96 well reaction plate See Method 1 Using a GeneAmp PCR System 9700 on page 124 Method 2 Using an Applied Biosystems 2720 Thermal Cycler or an MJ Tetrad PTC 225 Thermal Cycler Requires the use of an Applied Biosystems 2720 Thermal Cycler or an MJ Tetrad PTC 225 Thermal Cycler located adjacent to the hybridization ovens Samples are on a 96 well reaction plate See Method 2 Using an Applied Biosystems 2720 MJ Tetrad PTC 225 or MJ Tetrad 2 Thermal Cycler on page 127 First you will prepare a Hybridization Master Mix and add the mix to each sample Then you will denature the samples on a thermal cycler After denaturation you will load each sample onto a Genome Wide Human SNP Array 5 0 one sample per array The arrays are then placed into a hybridization oven that has been preheated to 50 C Samples are left to hybridize for 16 to 18 hours NOTE Two operators are required for all of the methods Location and Duration Main Lab Hands on time 45 minutes Hybridization time 16 to 18 hours Input Required from Previous Stage The input required from Stage 10 Labeling is Quantity Item 1 Plate of labeled DNA 118Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Equipment and Consumables Required The following equipment and consumables are required for this stage Refer to Appendix A for vendor and part number info
54. 006 Computing prior with 10000 initial snps Figure 6 15 Example of the probeset genotype log chapter 6 Data Analysis 163 Probeset qc log This file contains the output of the QC algorithm engine 3 probeset qc log Notepad n t3 File Edit Format View Help Wed Nov 15 10 40 30 2006 Loading parameters Wed Nov 15 10 40 33 2006 Initializing OC Process Wed Nov 15 10 40 34 2006 Checking methods Wed Nov 15 10 40 34 2006 Checking SNP lists Wed Nov 15 10 40 37 2006 Processing CEL FilesWed Nov 15 10 40 37 2006 Processing 1 of 6 C Documents and SettingsXburr My DocumentsiData genotype dataWA10851 FinNsp vRl 579813 A1 1 SC2 CEL Wed Nov 15 10 40 37 2006 Processing probesets Done Med Nov 15 10 41 24 2006 Processing 2 of 6 C Documents and Settings jburri My DocumentsiData genotype data NA10855_FinNsp_vR1_579548_A4 1 SCI CEL Wed Nov 15 10 41 24 2006 Processin probesets EIE EINE RITE DUE ETT TRIER TCU DUREE ND TURN SR BETTE SET SIENTES REI IIT Figure 6 16 Example of the probeset qc log 164 Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual SNP Intensity Summary File The SNP intensity summary file is a tab delimited text file containing the allele signal estimates for each allele The file name includes the extension plier summary txt The file format is a few comment lines prefixed with followed by a header line probeset_id lt tab gt CE
55. 096 of the genome The Genome Wide Human SNP Array 5 0 is designed to detect the SNPs that are amplified in this complex fragment population Subtle changes in the PCR conditions may not affect the PCR yield but may shift the amplified size range up or down very slightly This can lead to reduced amplification of SNPs that are assayed on the array set subsequently leading to lower call rates Troubleshooting Possible Problems with the Elution or OD Readings possible causes include The purified PCR product was eluted in a volume greater than 55 uL The purified PCR product was not mixed adequately before making the 1 100 dilution The diluted PCR product was not mixed adequately before taking the OD reading 102 Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Table 4 40 Continued PROBLEM Sample OD is Less Than 1 0 5 ug uL The water blank reading was not subtracted from each sample OD reading The spectrophotometer plate reader may require calibration Pipettes may require calibration There may be air bubbles or dust in the OD plate There may be defects in the plastic of the plate The settings on the spectrophotometer plate reader or the software may be incorrect OD calculations may be incorrect and should be checked Table 4 41 PROBLEM OD260 0D280 ratio is not between 1 8 and 2 0 Possible causes include The PCR product may be not be sufficiently purified Ensure th
56. 1 Sty P2 P3 and Nsp P4 to N o Lj c IY E N E gt eL 3 2 ed o gt fe N o oc QO iq ae ke EE m1 8 fe a ll LL L OI e AELEKEE eee ss ase 83 9 98 06 6 EEEE AAEL e e SAMLI EEEE Geoeoeeeoee CICICTISIS PIEKEN EEKE Geseeees e e a e e eeeee 3 08 0 9 Goeeeseee ees SUI ITII 9069008 eese EEEE eee 90 97 0 66 e CT A a amp 2 z 2 Ge Ga o O HO GEO GE GE d Ge qe m d E di z E I CIO LI q DE d 9 t1 corresponding wells of row A on the pooling plate 2 GO GRO GO 9 GO 0 90 GEO Ge E GO GO Gs 9 Gs G 2 Ge G G3 G GI GI GO Ge 385 Go G3 GO GO GI GO 98 GE 80 Ee DE 8E d d di d d COLO d Ge amp E OE d GE GEO RD GEO E 6 GEO Ge EEL 5 GE d ED GP EP ELE ese 39 d 62 H006 EKI tJ I I1 90068 amp 85 GS Nsp P3 GS GEP RD GO GO GO RD ES d QE E ee amp E BE Z e CI d RE CIS CI 0 G GNO ND QD ae Figure 4 7 Pooling Sty and Nsp PCR Products on a Deep Well Pooling Plate 88 Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Purify the Pooled PCR products Add Magnetic Beads and Incubate During incubatio
57. 1000 Rainin Pipetman _ or equivalent Sterile barrier pipette tips and non barrier _ _ pipette tips Tygon Tubing 0 04 inner diameter Cole Parmer H 06418 04 Tough Spots Label Dots USA Scientific 9185 0000 132 Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Reagents Required The following reagents are required for washing and staining arrays These reagents are recommendations and have been tested and evaluated by Affymetrix scientists Information and part numbers listed are based on U S catalog information Table 5 2 Reagents Required for Washing and Staining Arrays Reagent Vendor Part Number AccuGENE Molecular Biology Grade Water Cambrex 51200 1L Distilled water Invitrogen 15230147 20X SSPE 3 M NaCl 0 2 M NaH2P04 0 02 M Cambrex 51214 EDTA Anti streptavidin antibody goat biotinylated Vector Laboratories BA 0500 reconstitute according to product instructions R Phycoerythrin Streptavidin Molecular Probes S 866 10 Surfact Amps 20 Tween 20 Pierce Chemical 28320 Bleach 5 2596 Sodium Hypochlorite VWR Scientific 21899 504 or equivalent Denhardt s Solution 50X concentrate Sigma Aldrich D2532 MES hydrate Sigma Aldrich M5287 MES Sodium Salt Sigma Aldrich M5057 5 M NaCI RNase free DNase free Ambion 9760G chapter 5 Washing Staining and Scanning Arrays 133 Reagent Preparation Wash A Non Stringent Wash Bu
58. 2 0 program version 1 0 alg name brlmm alg version 1 0 analysis guid 0000065535 1163626646 0000013249 0000014956 0000032007 exec guid 0000024479 116 3626601 0000023416 0000029294 0000003957 het mult 1 iterations 0 iter thresh 0 3 K 4 transform CCS prior weight 40 prior mincall 2 lowprecision 0 prior siz e 10000 chrX file C Documents and Settings jburriiMy Documents Data genotype LibraryFiles Mapping250K_Nsp chrx max score 0 5 dm thresh 0 17 dm het mult 0 feat effect file target sketch file qmethod spec plier optmethod l prior fil e chrX force 0 force 0 analysis brlmm cel count 5 cvs id BRLMM Analysis T ool 2 0 model file time str Wed Nov 15 13 36 41 2006 analysis text quant norm sketch 50000 pm only brimm transform cces K 4 MS 0 5 Wed Nov 15 13 37 26 2006 Using maximum score threshold of 0 5 Wed Nov 15 13 37 26 2006 Appears to be 239 MB free 1023 MB total Wed Nov 15 13 37 26 2006 Appears to be 215 MB available 1023 MB total Wed Nov 15 13 37 26 2006 Appears that 643 MB is swappable upping usable RAM to 705MB Wed Nov 15 13 37 26 2006 Block size is 262338 maximum probe sets are 272333 Wed Nov 15 13 37 26 2006 Using block size of 262338 Wed Nov 15 13 37 31 2006 Doing 1 iteration Wed Nov 15 13 37 31 2006 Loading probeset info for iteration 1 Wed Nov 15 13 38 05 2006 Loaded 262338 probe sets Wed Nov 15 13 38 14 2006 Reading cel files Wed Nov 15 13 38 14 2006 Reading 5 cel files Wed Nov 15 13 40 50 2
59. 20 C To prevent loss of enzyme activity Immediately place the enzyme in a cooler chilled to 20 C when removed from the freezer Immediately return the enzyme to 20 C after use 174 Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Take care when pipetting enzymes stored in glycerol which is viscous Do not store at 80 C Because Fragmentation Reagent DNAse I activity can decline over time after dilution on ice add it to the samples as quickly as possible Preparing master mixes with a 15 excess ensures consistency in reagent preparation by minimizing pipetting errors and reducing handling time of temperature sensitive reagents The success of this assay depends on the accurate pipetting and subsequent thorough mixing of small volumes of reagents The PCR reaction for this assay has been validated using one of the specified thermal cyclers These thermal cyclers were chosen because of their ramping times We highly recommend the PCR thermal cyclers be calibrated regularly Take care programming your thermal cycler and use the thin walled reaction tubes recommended Thicker walled tubes may result in reduced PCR efficiency and lower yields It is essential to run gels to monitor both the PCR reaction and the fragmentation reaction For the PCR reaction individual PCR products are run on a gel Product bands should be visible in the 200 to 1100 bp size range See Chapter 4 Affvmetrix Genome Wide H
60. 2604 69295 2348 179938 2367 76627 2302 34769 AFFX 2315058 B 721 77691 648 99054 767 64720 649 56213 703 84390 138122761 AFFX 2315059 A 855 44867 742 88895 805 35785 123639382 243 45437 803 13166 AFFX 2315059 B 674 91792 531 57283 594 25065 226 62522 919 56777 650 43842 AFFX 2315063 A 1122 28376 991 69568 730 61224 680 24644 684 66161 722 90574 AFFX 2315063 B 214 84977 210 00090 861 74949 725 38501 805 78214 889 36937 AFFX 2315064 A 1236 16984 742 51223 1152 89295 781 77060 210 61361 1151 75697 NA11832 CEL 2220 56106 2095 71355 1289 33619 111465777 NA12056 CEI 1980 24866 2440 03358 1620 44308 Figure 6 17 Example of the SNP Intensity Summary File chapter 6 Data Analysis 165 Assessing Data Quality This section is designed to assist you with establishing guidelines for evaluating results generated from genotyping experiments To assess data quality and to identify outlier samples the BAT 2 0 QC report Figure 6 18 has a number of metrics that should be evaluated for each array These metrics are defined in Table 6 2 on page 166 It is important to check these metrics and to create a running log for each project The Reference Genomic DNA 103 included in the Genome Wide Human SNP Nsp Sty Assay Kit 5 0 can serve as a positive control to ensure that all of the steps of the assay are being performed correctly Evaluation of a particular sample should be based on QC report performance metrics Date 12 18
61. 3 with the modifications listed below Gels and Related Materials Required Reference Table 4 45 on page 105 The amounts listed are sufficient to process 48 samples Table C 3 E Gels and Related Materials Required e TTE TEE Reagent 60 uL 25 bp DNA Ladder diluted 1 15 with pre diluted 5xSB Loading Medium See Before Using E Gels on page 211 As needed 5xSB Loading Medium diluted See Before Using E Gels on page 211 1 E Gel 48 496 agarose gel Invitrogen P N G8008 04 Check the Fragmentation Reaction Reference the instructions on page 111 To ensure that fragmentation was successful 1 When the GW5 0 Fragment program is finished A Remove the plate from the thermal cycler B Spin down the plate at 2000 rpm for 30 sec and place in a cooling chamber on ice 2 Dilute 1 5 uL of each fragmented PCR product with 13 5 uL of diluted 5xSB Loading Medium 3 Run on E Gel 48 4 agarose gels with the 25 bp DNA Ladder for 22 min The colorless 25 bp DNA ladder is diluted 1 15 with diluted 5xSB Loading Medium Use 15 uL diluted ladder for each marker lane 4 Inspect the gel and compare it against the example shown in Figure C 2 on page 217 appendix C E gels 217 125 50 25 Figure C 2 Typical Example of Fragmented PCR Products Run on an E Gel 48 4 Agarose Gel for 22 min 218 Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual
62. 305 5 945 334 6 054 270 6 140 044 6 261 776 6 291 183 6 346 413 6 399 365 6 420 169 6 551 817 6 610 482 6 733 977 and EP 619 321 373 203 and other U S or foreign patents Copyright 2006 2007 Affymetrix Inc All rights reserved Chapter 1 Chapter 2 Chapter 3 Chapter 4 CONTENTS OWEIVIEW c 2 205 Sa goes i EE Ib Ue e Seas tA tee oe teense 1 About This Manual ess 1 About Whole Genome Sampling Analysis 00200000 05 2 References ees 4 Laboratory Setup and Recommendations T3 General Workflow RR RR RR 13 Contamination Prevention lees 14 Pre PCR Clean Room eee ees 14 PGR Staging ROOM d dd ci b W DA N Wan n iei di boo eet et 15 Main Lab awd wastes bd d bd r rd 15 Safety Precautions kk kk kk kk kK KK KK KK KK eee 15 Genomic DNA General Requirements a 17 General Requirements sisse 17 Sources of Human Genomic DNA kk KK KIR KIRI KRI RR eA 18 Genomic DNA Extraction Purification Methods 18 DNA Glearup x ans sud ue E DRE XAN Qi derana E RE bd PAUSE aU 19 References ers 19 Affymetrix Genome Wide Human SNP Nsp Sty 5 0 Assay 48 Sample Protocol 00000eeeeeee 21 About This Protocol gt lt k bd eode dor d 0 244 03 4k dard 2aeba 21 Workflow Recommendations KK RR RR RR RR RIK 23 Before You Begin kk kk kk heo iR KK KK eere de
63. 41 Affymetrix UK Ltd Voyager Mercury Park Wycombe Lane Wooburn Green High Wycombe HP10 OHH United Kingdom E mail supporteurope affymetrix com UK and Others Tel 44 0 1628 552550 France Tel 0800919505 Germany Tel 01803001334 Fax 44 0 1628 552585 Affymetrix Japan K K Mita NN Bldg 16 Floor 4 1 23 Shiba Minato ku Tokyo 108 0014 Japan Tel 03 5730 8200 Fax 03 5730 8201 sym E E aaa ae ie ree V Chapter VACUUM MANIFOLD AND FLUIDICS STATION CARE AND MAINTENANCE This chapter includes guidelines and instructions on Cleaning the vacuum manifold General care of the fluidics station A cleaning bleach protocol that should be run once per week Cleaning the Vacuum Manifold Salt buildup occurs with repeated use of the vacuum manifold The vacuum can be compromised and sample contamination may occur when too much salt is present Regular cleaning of the vacuum manifold is recommended To clean the vacuum manifold 1 2 3 4 Disassemble the vacuum manifold base cover cover gasket Soak the parts in warm water for 5 minutes Thoroughly rinse and dry each part Reassemble the vacuum manifold General Fluidics Station Care Use a surge protector on the power line to the fluidics station Always run a Shutdown protocol when the instrument will be off or unused overnight or longer This will prevent salt crystals from forming within the fluidics system
64. 52 Add Sty PCR Master Mix to Samples RR RR RR RR YI 54 Load Sty PCR Plates Onto Thermal Cyclers 005 54 RUNNING G Si bd dee aken e t tare ay W NE NE Bed ka XE TER eU 56 What Lo Do Next 2s 2 ose al le AW RE EF a lel liv A 57 Stage 4 Nsp Restriction Enzyme Digestion 000 58 About this Stage cssc ce kK GOON Aa f a e ewe tet als 58 l ocationiand Duratioti setaa d tpi kr RIBUS 4 Ea A pihaa Sardet 58 Input Required From Previous Stage KK KK KRI RR KK KK 58 Equipment and Consumables Required lll 59 Reagents Regulred ix use tete ge erede e n POR as 60 Important Information About This Stage 00 0 0 60 Prepare the Reagents Equipment and Consumables 61 Prepare the Nsp Digestion Master Mix KK KRI K K I 62 Add Nsp Digestion Master Mixto Samples 63 What To Do Next 2 2 5 ci det A ALA A RLW me RR RD 63 Stage 5 NSpEIGatlOn anda erp ert e e Rr decet dol s n rds 64 About this Stage nost es vr Dee Rea Tora gore Ed 64 location and DUFAtION 5st Albay bte eO e Xt rid ge4 ydp 64 Input Required From Previous Stage ee eee 64 Equipment and Consumables Required lll 65 Reagerits Regulr d eru e et EE Ea vr tps 66 Important Information About This Procedure sss 66 Prepare the Reagents Consumables and Other Components 66 Prepare the Nsp Ligation Master Mi
65. 69 30 901070 100 901071 Gels and Gel Related Materials Required Use either standard gels Table A 8 or E Gels Table A 9 on page 202 Table A 8 Standard Gels and Related Materials Item Vendor Part Number Gel Reliant Gel System precast agarose gel Cambrex 54939 2 SeaKem 2 SeaKem Gold TBE or 54929 4 BMA 4 NuSieve 3 1 Plus TBE Buffer 8 bp 1 kb 2 x 12 wells ethidium bromide All Purpose Hi Lo DNA Marker Bionexus BN2050 Gel Loading Buffer Sigma Aldrich G2526 202 Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Table A 9 E Gels and Related Materials Item Vendor Part Number Mother E Base EB M03 Daughter E Base EB D03 E Gel 48 296 agarose gel 8 pack nvitrogen G8008 02 E Gel 48 4 agarose gel 8 pack G8008 04 25 bp DNA Ladder 10597 011 used with E Gel 48 4 5X SB loading medium Faster Better Media SB5N 8 used with E Gel 48 4 All Purpose Hi Lo DNA Marker Bionexus BN2050 used with E Gel 48 2 Gel Loading Buffer Sigma Aldrich G2526 used with E Gel 48 2 Other Consumables Required Table A 10 Other Consumables Required for the Genome Wide Human SNP 5 0 Nsp Sty Assay Item Manufacturer Part Number Laboratory Location Distributor Pipette tips Rainin GP L10F Pre PCR and Main Lab As needed for pipettes listed in Table GP L200F A 5 GP L1000F RT L10F RT L200F RT L1000F GP refill RT with rack Pl
66. Appendix A Reagents Equipment and Consumables 212 Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Modifications for Stage 3 Sty PCR Follow the Stage 3 instructions listed in Stage 3 Sty PCR on page 47 with the modifications listed below Gels and Related Materials Required Reference Table 4 16 on page 49 The amounts listed are sufficient to process 48 Sty samples Table C 1 E Gels and Related Materials Required for Stage 3 Sty PCR e TET iia Reagent 180 uL All Purpose Hi Lo DNA Marker diluted 1 3 with H20 See When Using the E Gel 48 2 on page 211 As needed Gel loading buffer diluted 1 20 or 1 30 with H20 See When Using the E Gel 48 2 6 on page 211 3 E Gel 48 2 agarose gel 3 Plates 96 well reaction Running Gels Before Running Gels To ensure consistent results take 3 uL aliquot from each PCR WARNING Wear the appropriate personal protective equipment when handling ethidium bromide Run the Gels When the GW5 0 PCR program is finished 1 Remove each plate from the thermal cycler Spin down plates at 2000 rpm for 30 sec Place plates in cooling chambers on ice or keep at 4 C Label three fresh 96 well reaction plates P Gel P2Gel and P3Gel Aliquot 12 uL of diluted gel loading buffer to each well in rows A through D of the fresh labeled PXGel plates ao FPF WD appendix C E gels 213 6 Using a 12 channel P20 pipette transfer 3 uL of each PCR pro
67. Fluidics Station The Fluidics Station 450 is used to wash and stain the arrays it is operated using GeneChip Operating Software Set Up the Fluidics Station To set up the Fluidics Station 1 Turn on the Fluidics Station using the toggle switch on the lower left side of the machine 2 Select Run Fluidics from the menu bar in GCOS The Fluidics Station dialog box appears with a drop down list for selecting the experiment name for each of the fluidics station modules A second drop down list is accessed for choosing the Protocol for each of the fluidics station modules Use the radio buttons to access each module x NOTE Refer to the GeneChip Fluidics Station User s Guide for instructions on connecting and addressing multiple fluidics stations Prime the Fluidics Station Priming ensures the lines ofthe fluidics station are filled with the appropriate buffers and the fluidics station is ready to run fluidics station protocols Priming should be done when the fluidics station is first started when wash solutions are changed before washing if a shutdown has been performed if the LCD window instructs the user to prime chapter 5 Washing Staining and Scanning Arrays 137 To prime the Fluidics Station 1 To prime the fluidics station select Protocol in the Fluidics Station dialog box 2 Choose Prime_450 for the respective modules in the Protocol drop down list 3 Change the intake buffer rese
68. Incorrect wash buffers used on fluidics station Prime the fluidics station with the correct buffers prior to running the assay Incorrect wash buffers will disrupt hybridization of the labeled fragmented DNA Low SNP call rates Gel images and spectrophotometric quantitation indicate successful PCR reaction Over fragmentation of DNA sample due to incorrect dilution of Fragmentation Reagent DNase stock Check that you have selected the correct activity of DNase from Table 4 46 on page 108 to add to fragmentation reaction See Dilute the Fragmentation Reagent on page 108 Work quickly and on ice transfer reaction tubes to pre heated thermal cycler 37 C Mix thoroughly Extremely low call rate Sample hybridization is absent on cel and dat images but B2 grid is bright Labeling reaction suboptimal Use a new vial of Terminal Dideoxynucleotidyl Transferase Verify the labeling reagents and repeat labeling Positive control has good call rates but samples are lower than expected Genomic DNA not optimal Ensure DNA samples are of high quality i e run in a 1 to 2 gel and compare to Reference 103 DNA control Use positive control sample as a reference guide for assay procedures Prepare master mixes for samples and controls Very low call rates Mixed up Nsp and Sty enzymes during the digestion or ligation stages Repeat the experiment making sure the correct reagents are u
69. L file 1 lt TAB gt CEL file 2 and then one line per allele of each SNP probeset_id allele TAB summarized intensity CEL file 1 lt TAB gt summarized intensity CEL file 2 a brimm plier summary bt Notepad File Edit Format View Help guid 0000001507 1163518341 00000121 19 0000027790 000001 7066 exec_guid 0000065295 1163518806 0000016062 0000002531 0000014973 W oexec version BRLMM Analysis T ool 2 0 1 0 create_date Tue Nov 14 07 40 06 2006 cmd chip_type Mapping250K_Nsp lib_set_name C Documents and Settings jburriiMy Documents Datalgenotype LibraryFiles Mapping250K_Nsp cdf lib_set_version C Documents and Settings jburriiMy Documents Data genotype LibraryFiles Mapping250K_Nsp cdf WHIBBH HIN HHH HHHH H HI HH HHHH HHHH HH HIBIN HH HHHH HH HHH HH HIN N HH HIBBN U probeset_id NA10851 CEL NA10855 CEL NA10863 CEL NA11831 CEL AFFX 2315060 A4 2048 483894 2727 69136 2785 73681 2848 22498 AFFX 2315060 B 217231143 974 41519 1122 98332 1026 84506 2350 96801 AFFX 2315061 A 1277000911 1226 54423 2020 11283 335 55897 1372 58106 AFFX 2315061 B 117725276 1093 98693 314 51128 199293178 1245 42528 AFFX 2315062 A 564 00682 606 13917 333 79829 32797921 1009 31506 590 78353 AFFX 2315062 B 780 73475 741 96052 104925004 1064 15977 414 68513 739 34616 AFFX 2315057 A 365 32217 378 63277 768 94517 33473452 574 43554 369 94687 AFFX 2315057 B 1026 36470 953 20782 386 90473 889 57536 618 73956 958 93899 AFFX 2315058 A
70. Lab Tube Eppendorf 2 0 mL VWR 20901 540 Pre PCR Tube Falcon 50 mL VWR 21008 940 Pre PCR 204 Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Supplier Contact List Table A 11 Supplier Contact List Affymetrix www affymetrix com Agencourt Bioscience Corp agencourt com Applied Biosystems www appliedbiosystems com Bionexus Inc www bionexus net Bio Rad bio rad com Boekel Scientific www boekelsci com Cambrex www cambrex com CLP Direct clpdirect com Clontech www clontech com Diversified Biotech divbio com E amp K Scientific eandkscientific com Eppendorf eppendorf com Faster Better Media fasterbettermedia com Fisher Scientific www thermofisher com Greiner Bio One www gbo com ISC Bioexpress iscbioexpress com Invitrogen Life Technologies invitrogen com Labcor labcorproducts com Millipore millipore com Molecular Devices moleculardevices com New England Biolabs www neb com Pierce Biotechnology piercenet com part of Thermo Fisher Scientific Promega www promega com Rainin www rainin com Seahorse Bioscience www seahorselabware com Table A 11 Continued Supplier Contact List Supplier appendix A Reagents Equipment and Consumables 205 Web Site Address Sigma Aldrich www sigma aldrich com Stratagene stratagene com Takara Bio Inc www takara bio com Teknova teknova com USA Scien
71. NEBuffer can be ordered separately using these part numbers T4 DNA Ligase 250 uL vial Contains Mo202L T4 DNA Ligase T4 DNA Ligase Buffer NEB P N B202S 196 Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Other Reagents Required Table A 3 Other Reagents Required for the Genome Wide Human SNP 5 0 Nsp Sty Assay Reagent Vendor Description Part Number TITANIUM DNA Clontech Contains 639240 Amplification Kit e 50X TITANIUM Taq DNA Polymerase 300 reactions 10X TITANIUM Taq PCR Buffer enough for 100 e GC Melt samples or dNTPs 639243 400 reactions TITANIUM Taq DNA Clontech Contains P N 639209 Polymerase 50X and e 50X Clontech TITANIUM Taq DNA also in kit TITANIUM Taq PCR Polymerase P N 639240 or Buffer 10X Clontech TITANIUM Taq PCR Buffer 639243 above GC Melt Clontech 5M 639238 also in kit P N 639240 or 639243 above Beads Magnetic Agencourt AMPure 130 60 mL 000130 Buffer EB 250 mL Qiagen 250 ml Elution Buffer 19086 Included in the Clontech TITANIUM DNA Amplification Kit listed above Chloride TMACL 5M dNTPs Takara 4030 mixture of dATP dCTP dGTP dTTP at Fisher 2 5 mM each TAK 4030 Scientific Denhardt s Solution Sigma Aldrich D2532 DMSO Sigma Aldrich D5879 Ethanol Sigma Aldrich ACS reagent S 99 5 200 proof absolute 459844 Herring Sperm DNA Promega D1815 HSDNA Human Cot 1 DNA Invitrogen 15279 011 ME
72. OD readings Possible problems with input genomic DNA that would lead to reduced yield include The presence of inhibitors heme EDTA etc Severely degraded genomic DNA Inaccurate concentration of genomic DNA Check the OD reading for the PCR products derived from RefDNA 103 as a control for these issues To prevent problems with the PCR reaction that would lead to reduced yield Use the recommended reagents and vendors including AccuGENE water for all PCR mix components 180Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Table 7 2 Continued PROBLEM Sample OD is Less Than 1 0 5 ug L Thoroughly mix all components before making the PCR Master Mix Pipette all reagents carefully particularly the PCR Primer when making the master mix Check all volume calculations for making the master mix Store all components and mixes on ice when working at the bench Do not allow reagents to sit at room temperature for extended periods of time Be sure to use the recommended PCR plates Plates from other vendors may not fit correctly in the thermal cycler block Differences in plastic thickness and fit with the thermal cycler may lead to variance in temperatures and ramp times Be sure to use the correct cycling mode when programming the thermal cycler maximum mode on the GeneAmp PCR System 9700 calculated mode on the MJ Tetrad PTC 225 or Tetrad 2 Be sure to use silver or gold plated
73. PCR has been confirmed proceed to Stage 7 PCR Product Pooling and Purification on page 82 f not proceeding directly to the next stage seal the plates with PCR product and store at 20 C 82 Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Stage 7 PCR Product Pooling and Purification About this Stage During this stage you will Pool the Sty and Nsp PCR reactions to a single deep well pooling plate for a total of 700 pL well Add beads to each pool and incubate Transfer each pool to a filter plate and dry down on a vacuum manifold Wash the PCR products with EtOH and dry down Elute the PCR products using Buffer EB Vacuum and spin transfer the PCR products to a new 96 well plate Location and Duration Main Lab Hands on time 1 hour Initial dry down 60 to 90 minutes EtOH wash approximately 10 to 20 minutes Elution 15 to 30 minutes Total time for this stage approximately 3 5 hours Input Required from Previous Stage The input required from Stage 3 Sty PCR is Quantity Item 3 plates Sty PCR product 4 plates Nsp PCR product chapter 4 Affymetrix Genome Wide Human SNP Nsp Sty 5 0 Assay 48 Sample Protocol 83 Equipment and Consumables Required Refer to Appendix A for vendor and part number information Table 4 34 Equipment and Consumables Required for Stage 7 PCR Product Pooling and Purification 1 Collar Multiscree
74. S Hydrate SigmaUltra Sigma Aldrich M5287 MES Sodium Salt Sigma Aldrich M5057 Reduced EDTA TE Buffer TEKnova 10 mM Tris HCL 0 1 mM EDTA pH 8 0 T0223 Tetramethyl Ammonium Sigma Aldrich 5M T3411 appendix A Reagents Equipment and Consumables 197 Table A 3 Continued Other Reagents Required for the Genome Wide Human SNP 5 0 Nsp Sty Assay Reagent Vendor Description Part Number Tween 20 10 Pierce 10 diluted to 3 in molecular biology 28320 Surfact grade water AmpsO Water AccuGENE Cambrex AccuGENE Molecular Biology Grade 51200 Water 1 L dNTPs from Invitrogen P N R72501 have been tested on a limited basis with similar results You should test in your own lab prior to full scale production Equipment and Software Required This protocol has been optimized using the following equipment and software Affymetrix Equipment and Software Required Table A 4 Affymetrix Equipment and Software Required Item Part Number GeneChip Fluidics Station 450 00 0079 GeneChip Hybridization Oven 640 800139 GeneChip Scanner 3000 7G 00 0205 GeneChip Operating Software version 1 4 690031 BRLMM Analysis Tool 2 0 Denotes critical reagents equipment or supplies Formulations or vendors not listed here have not been tested and verified at Affymetrix In some cases lower performance has been demonstrated by reagents from non qualified vendors 198 Affymetrix Genome Wide Human SNP 5 0 Ns
75. SNP call rate tend to have a higher error rate and disproportionately contribute to the overall error rate in the experiment SNP Cluster Visualization Applying per SNP filters helps remove the majority of problematic SNPs However no filtering scheme is perfect Even with stringent filtering a small proportion of poorly performing SNPs will remain chapter 6 Data Analysis 171 Moreover the poorly performing SNPs will often be the ones most likely to perform differently between cases and controls The list of most significantly associated SNPs is often enriched for such problematic SNPs The SNP filtering process greatly reduced the occurrence of these false positives But given their tendency to end up on the list of associated SNPs it is likely that some will remain Before carrying forth SNPs to subsequent phases of analysis visual inspection of the SNPs in the clustering space used by BRLMM P is strongly recommended Visual inspection typically helps in identifying problematic cases BAT 2 0 has an option to export a tab delimited text file of the SNP allele signals which can be used as an input to standard analysis programs such as Matlab R and Splus to plot SNP clusters Increasing or Decreasing Accuracy and Call Rate Adjust the default QC call rate or BRLMM P thresholds to increase or decrease accuracy and call rate Affymetrix genotyping software provides flexible options to enable a trade off between call rate and gen
76. ab PCR Plates placed on thermal cyclers Hands on time 1 hour e GWS5 0 PCR thermal cycler program time 1 5 hours samples can be held overnight at 4 C Input Required from Previous Stage The input required from Stage 2 Sty Ligation is Quantity Item 48 Diluted Sty ligated samples 48 Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Equipment and Materials Required The following equipment and materials are required to perform this stage Refer to Appendix A for vendor and part number information Table 4 14 Equipment and Consumables Required for Stage 3 Sty PCR Quantity Item 1 Cooler chilled to 20 C Enough for upto Cooling chambers chilled to 4 C do not freeze five 96 well plates 1 Ice bucket filled with ice 1 Marker fine point permanent 1 Microcentrifuge 1 Pipette single channel P20 1 Pipette single channel P100 1 Pipette single channel P200 1 Pipette single channel P1000 1 Pipette 12 channel P20 1 Pipette 12 channel P200 As needed Pipette tips for pipettes listed above full racks 3 Plates 96 well reaction 1 Plate centrifuge As needed Plate seal 1 Solution basin 55 mL 3 Thermal cycler 1 Tube Falcon 50 mL 1 Vortexer IMPORTANT Use only the PCR plate adhesive film and thermal cyclers listed in Table 4 1 on page 27 chapter 4 Affymetrix Genome Wide Human SNP Nsp Sty 5 0 Assay 48 Sample Pr
77. ach sample 3 Place each plate on a thermal cycler and run the GW 5 0 PCR program 4 Confirm the PCR by running 3 uL of each PCR product on a 2 TBE gel or an E Gel 48 2 agarose gel Location and Duration Pre PCR Clean Area Nsp PCR Master Mix preparation PCR Staging Area PCR set up Main Lab PCR Plates placed on thermal cyclers Hands on time 1 hour e GWS5 0 PCR thermal cycler program time 1 5 hours samples can be held overnight at 4 C Input Required from Previous Stage The input required from Stage 5 Nsp Ligation is Quantity Item 48 Diluted Nsp ligated samples 72 Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Equipment and Materials Required The following equipment and materials are required to perform this stage Refer to Appendix A for vendor and part number information Table 4 28 Equipment and Consumables Required for Stage 6 Nsp PCR Quantity Item 1 Cooler chilled to 20 C Enough for five Cooling chambers chilled to 4 C do not freeze 96 well plates 1 Ice bucket filled with ice 1 Marker fine point permanent 1 Microcentrifuge 1 Pipette single channel P20 1 Pipette single channel P100 1 Pipette single channel P200 1 Pipette single channel P1000 1 Pipette 12 channel P20 1 Pipette 12 channel P200 As needed Pipette tips for pipettes listed above full racks 4 Plates 96 well reaction 1 Plate centrifuge
78. all of which can adversely modulate enzyme activity 22 Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Successful sample processing can be achieved by incorporating the following principles Use only fresh reagents from the recommended vendors to help eliminate changes in pH or the salt concentration of buffers Properly store all enzyme reagents Storage methods can profoundly impact activity When using reagents at the lab bench Ensure that enzymes are kept at 20 C until needed Keep all master mixes and working solutions in chilled cooling chambers Properly chill essential equipment such as centrifuges cooling chambers and reagent coolers before use Since enzyme activity is a function of temperature ensure that all temperature transitions are rapid and or well controlled to help maintain consistency across samples Keep dedicated equipment in each of the areas used for this protocol including pipettors ice buckets coolers etc To avoid contamination do not move equipment from one area to another Along with the enzymatic stages lab instrumentation plays an important role in WGSA To aid in maintaining consistency across samples and operators all equipment should be well maintained and calibrated including All of the thermal cyclers PCR Staging Room and Main Lab GeneChip Hybridization Oven 640 GeneChip Fluidics Station 450 GeneChip Scanner 3000 7G The UV spectrophotomet
79. alysis text quant norm sketch 50000 pm only brlmm transform ccs K 4 MS 0 5 guid 0000048939 1163565324 0000028217 0000017501 0000024119 exec_guid 0000065415 1163565045 0000021742 000001 183 0000002130 exec_version BRLMM Analysis T ool 2 0 1 0 create_date Tue Nov 14 20 30 45 2006 chip_type Mapping250K_Nsp lib_set_name C Documents and Settings jburriiMy Documents Data genotype LibraryFiles Mapping250K_Nsp cdf lib_set_version C Documents and Settings jburri My Documents Data genotype LibraryFiles Mapping250K_Nsp cdf NA10351 CEL N 10855 CEL NA10863 CEL NA11831 CEL N 11832CEL NA12056 CEL 1 1 e O O KA O MI NM O KA 0K 0 M69 oo oowoo b O O MIM t2 O B m Nn e MD O e O NR O KRA KA KAMA KS Figure 6 13 Example of Genotype Calls File for the BRLMM P Algorithm chapter 6 Data Analysis 161 Genotype Confidences Files The genotype confidences file is a text file containing the confidences for each genotype call for the analysis run This file has the extension confidences txt and consists of a comment section followed by the tab delimited confidences The comment section is identified by the prefix and contains information about the analysis run including the program the algorithm library files and command line used to generate the file The tab delimited section contains a header row indicating which column contains the probeset_ids and the confidences for each genotype call
80. and spin it down at 2000 rpm for 30 sec 2 Place the plate in a cooling chamber on ice 3 Dilute each reaction as follows A Pour 10 mL AccuGENE water into the solution basin B Using a 12 channel P200 pipette add 75 uL of the water to each reaction The total volume in each well is 100 uL Sty Ligated DNA 25 uL AccuGENE water 75 uL Total 100 uL Seal the plate tightly with adhesive film 5 Vortex the center of the plate at high speed for 3 sec 6 Spin down the plate at 2000 rpm for 30 sec What To Do Next Do one of the following If following the recommended workflow Figure 4 1 on page 23 proceed immediately to Stage 3 Sty PCR on page 47 Store the plate in a cooling chamber on ice for up to 60 minutes If not proceeding directly to the next step store the plate at 20 C chapter 4 Affymetrix Genome Wide Human SNP Nsp Sty 5 0 Assay 48 Sample Protocol 47 Stage 3 Sty PCR About this Stage During this stage you will 1 Transfer equal amounts of each Sty ligated sample into three fresh 96 well plates Figure 4 4 on page 52 2 Prepare the Sty PCR Master Mix and add it to each sample 3 Place each plate on a thermal cycler and run the GW 5 0 PCR program 4 Confirm the PCR by running 3 uL of each PCR product on a 2 TBE gel or an E Gel 48 2 agarose gel Location and Duration Pre PCR Clean Area Sty PCR Master Mix preparation PCR Staging Area PCR set up Main L
81. anual Important Information About This Stage To help ensure the best results carefully read the information below before you begin this stage of the protocol IMPORTANT e Make sure the Nsp ligated DNA was diluted to 100 pL with AccuGENE water Set up the PCRs in PCR Staging Area e Prepare Nsp PCR Master Mix immediately prior to use and prepare in Pre PCR Clean room To help ensure the correct distribution of fragments be sure to add the correct amount of primer to the master mix Mix the master mix well to ensure the even distribution of primers e To ensure consistent results take 3 uL aliquots from each PCR to run on gels About Controls A PCR negative control can be included in the experiment to assess the presence of contamination Refer to Chapter 3 and Chapter 7 for more information Prepare the Reagents Consumables and Other Components Thaw Reagents and Nsp Ligated Samples To thaw the reagents and Nsp ligated samples 1 Allow the following reagents to thaw on ice TITANIUM Taq PCR Buffer dNTPs PCR Primer 002 IMPORTANT Leave the TITANIUM Taq DNA Polymerase at 20 C until ready to use 2 Ifthe Nsp ligated samples are frozen allow to thaw in a cooling chamber on ice chapter 4 Affymetrix Genome Wide Human SNP Nsp Sty 5 0 Assay 48 Sample Protocol 75 Prepare Your Work Area Pre PCR Clean Area To prepare the work area 1 Place enough cooling chambers for 5 plates an
82. at Affymetrix 1 SDS ProK digestion phenol chloroform extraction Microcon or Centricon Millipore ultrapurification and concentration 2 QIAGEN QIAamp DNA Blood Maxi Kit chapter 3 Genomic DNA General Requirements 19 DNA Cleanup If a genomic DNA preparation is suspected to contain inhibitors the following cleanup procedure can be used 1 Add 0 5 volumes of 7 5 M NH OAc 2 5 volumes of absolute ethanol stored at 20 C and 0 5 uL of glycogen 5 mg mL to 250 ng genomic DNA Vortex and incubate at 20 C for 1 hour Centrifuge at 12 000 x g in a microcentrifuge at room temperature for 20 minutes Remove supernatant and wash pellet with 0 5 mL of 80 ethanol Centrifuge at 12 000 x g at room temperature for 5 minutes Remove the 80 ethanol and repeat the 80 ethanol wash one more time Re suspend the pellet in reduced EDTA TE buffer 10 mM Tris pH 8 0 0 1 mM EDTA pH 8 0 No SI PR WD References Feigelson H S Rodriguez C Robertson A S Jacobs E J Calle E E Reid Y A Thun M J Determinants of DNA yield and quality from buccal cell samples collected with mouthwash Cancer Epidemiol Biomarkers Prev 10 9 1005 8 2001 Heath Ellen M Morken Nathaniel W Campbell Kristen A Tkach Dennis Boyd Erin A Strom Daniel A Use of Buccal Cells Collected in Mouthwash as a Source of DNA for Clinical Testing Arch Pathol Lab Med 125 127 133 2001 King I B Satia Abouta J Tho
83. ate seals see Table A 6 on page 200 Pre PCR and Main Lab Plates 96 well PCR see Table A 6 on page 200 Pre PCR and Main Lab Microplate 96 well conical bottom In the U S A only EK 21101 Main Lab Elution Catch Plate E amp K Scientific All other countries 651101 Greiner Bio One appendix A Reagents Equipment and Consumables 203 Table A 10 Continued Other Consumables Required for the Genome Wide Human SNP 5 0 Nsp Sty Assay Item Manufacturer Part Number Laboratory Location Distributor Plate 2ml 96 Well Format Filterplate In the U S A only XP0228 Main Lab PES 0 45 um Hydrophilic Long Drip E amp K Scientific Director All other countries Seahorse Bioscience In the U S A only EK 22280 Deep Well Storage Plate 2 4 mL E amp K Scientific Main Lab Pooling Plate All other countries 780280 Greiner Bio One Plates 96 well UV Star 370 uL well E amp K Scientific 25801 Main Lab Solution Basin 100 mL sterile Labcor 730 014 Main Lab multichannel Solution Basin 55 mL sterile Labcor 730 004 Pre PCR and Main Lab multichannel Solution Basin lid 55 mL Labcor 730 021 Pre PCR and Main Lab Diversified Biotech SPOT 1000 Tough Spots Main Lab USA Scientific 9185 1000 CLP Direct 3426 12 Tubes strip of 12 thin wall 0 2 mL Pre PCR and Main Lab ISC BioExpress T 3114 1 Tube centrifuge 15 mL VWR 20171 020 Main Lab Tube centrifuge 50 mL VWR 21008 178 Main
84. avelength for the GeneChip Scanner 3000 7G are preset and cannot be changed 5 Open the sample door of the scanner and insert the array into the holder The door of the GeneChip Scanner 3000 7G closes automatically Lu IMPORTANT Do not force the array into the holder 6 Click OK in the Start Scanner dialog box The scanner begins scanning the array When Scan in Progress is selected from the View menu the array image appears on the screen as the scan progresses chapter 5 Washing Staining and Scanning Arrays 145 Shutting Down the Fluidics Station To shut down the Fluidics Station 1 Gently lift up the cartridge lever to engage close the washblock After removing an array from the holder the LCD window displays the message ENGAGE WASHBLOCK The instrument automatically performs a Cleanout procedure The LCD window indicates the progress of this procedure When REMOVE VIALS is displayed in the LCD remove the vials from the sample holders The REMOVE VIALS message indicates the Cleanout procedure is complete If no other processing is to be performed place the wash lines into a bottle filled with deionized water Choose Shutdown_450 for all modules from the drop down Protocol list Click the Run button for all modules The Shutdown protocol is critical to instrument reliability Refer to the instrument User s Guide for more information When the Shutdown protocol is complete turn the instrument of
85. ays Operator 1 Tasks 1 When the plate reaches 49 C open the lid on the thermal cycler 2 Remove the film from the first row 3 Using a single channel P200 pipette remove 200 uL of denatured sample from the first well Immediately inject the sample into an array 5 Pass the array to Operator 2 NOTE The tasks for Operator 2 are listed below Remove 200 uL of denatured sample and immediately inject it into an array Pass the array to Operator 2 Repeat this process one sample at a time until all 24 samples are loaded onto arrays se oN 9 Cover the wells with a fresh strip of adhesive film and place in the cooling chamber on ice 10 Remove the next strip of 24 wells and place it on the thermal cycler 11 Run the GW5 0 Hyb program 12 Repeat steps 1 through 11 until all of the samples have been loaded onto arrays Operator 2 Tasks 1 Cover the septa on each array with a Tough Spot Figure 4 15 2 When 4 arrays are loaded and the septa are covered A Load the arrays into an oven tray evenly spaced B Immediately place the tray into the hybridization oven Do not allow loaded arrays to sit at room temperature for more than approximately 1 minute Ensure that the oven is balanced as the trays are loaded and ensure that the trays are rotating at 60 rpm at all times Because you are loading 4 arrays per tray each hybridization oven will have a total of 32 arrays chapter 4 Affymetrix Genome Wide Hu
86. beat de 25 Master Mix Preparation kk kk kk kk kK KK KK KK KK KR K KK KIR KI KIRI KI 25 Reagent Handling and Storage WWW ee 25 Preparing the Work Area for Each Stage 0 02005 26 Thermal Cyclers Plates and Plate Seals 0005 27 Program Your Thermal Cyclers JW kk kk KK KK KK RR RR RR KI gt 28 ii Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Genomic DNA Plate Preparation A KK KK RR KIRR ee 29 About this Stage 4 kk kk kk kK KK es 29 locations diburatioti gt 4 tetas D Mu ott eL o Re Mekk thn 29 INOUtREGUIFCE xa ax 2 6 6 theo beet ETSI E E Edo ay tiM 29 Equipment and Consumables Required lll 30 Reagents Required Jk kk kk kK KK KK KK eee eee 31 Preparing the Genomic DNA Plate Ak KK KK RR eee eee 31 Aliquoting Prepared Genomic DNA WR RR RR RR KI 32 WhacT Do NEX n c hate Pid DUE NEW dad pu ee ee 32 Stage 1 Sty Restriction Enzyme Digestion K S 33 About this Stage srs rreri ridenti onte es 33 Location and Duration kk kk kk kK KK KK KK es 33 Input Required From Previous Stage KK KK RR KIRR 33 Equipment and Consumables Required VV III 34 Reagents Required vk kk kk KK KK KK KK eee eee 35 Important Information About This Stage RR RR eee 35 Prepare the Reagents Equipment and Consumables 36 Prepare the Sty Digestion Master MIX 0 000000 eee 37 Add Sty Digestion Master Mix to Samples 38
87. bstructure in four populations using 8 525 autosomal SNPs Hum Genomics 2004 1 274 86 Shriver MD Mei R Parra EJ Sonpar V Halder I Tishkoff SA Schurr TG Zhadanov SI Osipova LP Brutsaert TD et al Large scale SNP analysis reveals clustered and continuous patterns of human genetic variation Hum Genomics 2005 2 81 89 Bonnen PE Pe er I Plenge RM Salit J Lowe JK Shapero MH Lifton RP Breslow JL Daly MJ Reich DE et al Evaluating potential for whole genome studies in Kosrae an isolated population in Micronesia Nat Genet 2006 38 214 7 Gonzalez Burchard E Borrell LN Choudhry S Naqvi M Tsai HJ Rodriguez Santana JR Chapela R Rogers SD Mei R Rodriguez Cintron W et al Latino populations a unique opportunity for the study of race genetics and social environment in epidemiological research 4m J Public Health 2005 95 2161 8 Huang J Wei W Zhang J Liu G Bignell GR Stratton MR Futreal PA Wooster R Jones KW Shapero MH Whole genome DNA copy number changes identified by high density oligonucleotide arrays Hum Genomics 2004 1 287 99 Nannya Y Sanada M Nakazaki K Hosoya N Wang L Hangaishi A Kurokawa M Chiba S Bailey DK Kennedy GC et al A robust algorithm for copy number detection using high density oligonucleotide single nucleotide polymorphism genotyping arrays Cancer Res 2005 65 6071 9 Wong KK Tsang YT Shen J Cheng RS Chang YM Man TK
88. ce changes from shiny reflective to matte non reflective when dry There should be no standing liquid in any of the wells being used B If all of the wells are not dry place the plate back on the manifold and resume filtering IMPORTANT Continue filtering and inspecting the wells with a flashlight until completely dry There should be no standing liquid in any of the wells being used 90 Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual e sg N 10 11 12 Using a 12 channel P1200 set to 900 uL add 1 8 mL of 75 EtOH to each reaction Turn the vacuum back on to 20 to 24 in Hg and cover the plate Allow the wells to filter to dryness for 10 to 20 minutes When all of the wells appear dry A Turn off the vacuum and inspect each well using a flashlight The surface changes from shiny reflective to matte non reflective when dry There should be no standing liquid in any of the wells being used B If all of the wells are not dry place the plate back on the manifold and resume filtering IMPORTANT Continue filtering and inspecting the wells with a flashlight until completely dry There should be no standing liquid in any of the wells being used When all wells are completely dry remove the filter plate from the manifold and tap it several times on towels Kimwipes to blot off any excess EtOH from the bottom of the plate Return the filter plate to the manifold turn on the vacuum
89. centration greater than 6 ug pL a problem exists with either the elution of PCR products or the OD reading The limit on PCR yield is approximately 6 ug uL as observed in practice and as predicted by the mass of dNTPs in the reaction Possible causes include The purified PCR product was eluted in a volume less than 55 uL The purified PCR product was not mixed adequately before making the 1 100 dilution The diluted PCR product was not mixed adequately before taking the OD reading The water blank reading was not subtracted from each sample OD reading The spectrophotometer plate reader may require calibration Pipettes may require calibration There may be air bubbles or dust in the OD plate There may be defects in the plastic of the plate The settings on the spectrophotometer plate reader or the software may be incorrect OD calculations may be incorrect and should be checked chapter 4 Affymetrix Genome Wide Human SNP Nsp Sty 5 0 Assay 48 Sample Protocol 101 Table 4 40 PROBLEM Sample OD is Less Than 1 0 5 ug uL If the sample OD is less than 1 0 calculated concentration less than 5 ug uL a problem may exist with either the genomic DNA the PCR reaction the elution of purified PCR products or the OD readings Possible problems with input genomic DNA that would lead to reduced yield include The presence of inhibitors heme EDTA etc Severely degraded genomic DNA Inaccurate con
90. centration of genomic DNA Check the OD reading for the PCR products derived from RefDNA 103 as a control for these issues To prevent problems with the PCR reaction that would lead to reduced yield Use the recommended reagents and vendors including AccuGENE water for all PCR mix components Thoroughly mix all components before making the PCR Master Mix Pipette all reagents carefully particularly the PCR Primer when making the master mix Check all volume calculations for making the master mix Store all components and mixes on ice when working at the bench Do not allow reagents to sit at room temperature for extended periods of time Be sure to use the recommended PCR plates Plates from other vendors may not fit correctly in the thermal cycler block Differences in plastic thickness and fit with the thermal cycler may lead to variance in temperatures and ramp times Be sure to use the correct cycling mode when programming the thermal cycler maximum mode on the GeneAmp PCR System 9700 calculated mode on the MJ Tetrad PTC 225 or Tetrad 2 Be sure to use silver or gold plated silver blocks on the GeneAmp PCR System 9700 other blocks are not capable of maximum mode which will affect ramp times Use the recommended plate seal Make sure the seal is tight and that no significant evaporation occurs during the PCR NOTE The Genome Wide SNP 5 0 Assay reaction amplifies a size range of fragments that represents 3
91. ciation studies Nature 2004 429 446 52 6 Wang DG Fan JB Siao CJ Berno A Young P Sapolsky R Ghandour G Perkins N Winchester E Spencer J et al Large scale identification mapping and genotyping of single nucleotide polymorphisms in the human genome Science 1998 280 1077 82 7 Kruglyak L Nickerson DA Variation is the spice of life Nat Genet 2001 27 234 6 chapter 1 Overview 5 10 11 12 13 14 15 16 17 18 19 20 Gibbs RA Belmont JW Hardenbol P Willis TD Yu F Yang H Ch ang L Y Huang W Liu B Shen Y et al The International HapMap Project Nature 2003 426 789 96 Sachidanandam R Weissman D Schmidt SC Kakol JM Stein LD Marth G Sherry S Mullikin JC Mortimore BJ Willey DL et al A map of human genome sequence variation containing 1 42 million single nucleotide polymorphisms Nature 2001 409 928 33 Syvanen AC Toward genome wide SNP genotyping Nat Genet 2005 37 SuppL S5 10 Ardlie KG Kruglyak L Seielstad M Patterns of linkage disequilibrium in the human genome Nat Rev Genet 2002 3 299 309 Hinds DA Stuve LL Nilsen GB Halperin E Eskin E Ballinger DG Frazer KA Cox DR Whole genome patterns of common DNA variation in three human populations Science 2005 307 1072 9 Hirschhorn JN Daly MJ Genome wide association studies for common diseases and complex traits Nat Rev Genet 2005 6 95 108 Pharoah
92. cient to process 48 samples Table 4 44 Reagents Required for Stage 9 Fragmentation Quantity Reagent 1 vial Fragmentation Buffer 10X 1 vial Fragmentation Reagent DNase l 1mL AccuGENE water molecular biology grade Gels and Related Materials Required Verifying the PCR reaction is required for this stage You can use the following gels and related materials or E Gels as described in Appendix C E gels on page 211 The amounts listed are sufficient to process 48 Sty samples Refer to Appendix A for vendor and part number information Table 4 45 Gels and Related Materials Required Quantity Reagent 5 4 TBE Gel 10 DNA Markers 5 uL each As needed Gel loading solution 106Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Important Information About This Stage To help ensure the best results carefully read the information below before you begin this stage of the protocol IMPORTANT The degree of fragmentation is critical Perform this stage carefully to ensure uniform reproducible fragmentation e Use only the AccuGENE water listed in Appendix A Using in house ddH O or other water can negatively affect your results The reaction in Stage 9 Fragmentation is particularly sensitive to pH and metal ion contamination e All additions dilutions and mixing must be performed on ice Be sure to allow all reagents to reach equilibrium before adding new fluid About t
93. col Lu IMPORTANT Bring the Buffer EB and 75 EtOH to room temperature prior to use The storage temperature for the magnetic beads is 4 C refrigerator To avoid cross contamination pipette very carefully when pooling the PCR reactions into the deep well plate Maintain the vacuum between 20 24 in Hg do not exceed 24 in Hg Inspect the vacuum manifold for salt buildup after each use and clean regularly Refer to Chapter 8 for cleaning instructions After the EtOH wash the wells must be completely dry before eluting the samples with Buffer EB Any extra EtOH carried with the EB Buffer to the next stage can result in poor fragmentation Immediately after the EtOH wash remove the plate stack from the manifold and blot the bottom of the plate to remove any excess EtOH chapter 4 Affymetrix Genome Wide Human SNP Nsp Sty 5 0 Assay 48 Sample Protocol 85 Prepare the 75 EtOH Dilute ACS grade ethanol to 75 using AccuGENE water Prepare the Reagents Allow the Buffer EB and 75 EtOH to warm room temperature prior to use Prepare the Vacuum Manifold To prepare the manifold 1 Connect the manifold and regulator to a suitable vacuum source able to maintain 20 to 24 in Hg Leave the vacuum turned off at this time Lower the vacuum flask trap below the level of the manifold Inspect the manifold for salt and other contaminants and clean if necessary IMPORTANT Inspect the vacuum manifold for salt bui
94. cooler Vortex the master mix at high speed 3 times sec each time Pulse spin for 3 sec Place the master mix on ice Proceed immediately to Add Nsp Ligation Master Mix to Reactions Table 4 26 Nsp Ligation Master Mix Reagent 1 Sample 48 Samples 25 extra T4 DNA Ligase Buffer 10X 2 5 uL 150 uL Adaptor Nsp 50 uM 0 75 uL 45 uL T4 DNA Ligase 400 U uL 2uL 120 uL Total 5 25 uL 315 uL Add Nsp Ligation Master Mix to Reactions To add Nsp Ligation Master Mix to samples 1 Using a single channel P100 pipette aliquot 25 uL of Nsp Ligation Master Mix to each tube of the strip tubes on ice Using a 12 channel P20 pipette aliquot 5 25 uL of Nsp Ligation Master Mix to each reaction on the Nsp Digestion Stage Plate The total volume in each well is now 25 uL chapter 4 Affymetrix Genome Wide Human SNP Nsp Sty 5 0 Assay 48 Sample Protocol 69 Nsp Digested DNA 19 75 uL Nsp Ligation Master Mix 5 25 uL Total 25 uL Contains ATP and DTT Keep on ice Seal the plate tightly with adhesive film Vortex the center of the plate at high speed for 3 sec Spin down the plate at 2000 rpm for 30 sec Ensure that the thermal cycler lid is preheated Load the plate onto the thermal cycler and run the GW5 0 Ligate program NOP Ww Table 4 27 GW5 0 Ligate Thermal Cycler Program GW5 0 Ligate Program Temperature Time 16 C 180 minutes 70 C 20 minutes 4 C Hold
95. d apply a new spot Ensure that the spots lie flat chapter 5 Washing Staining and Scanning Arrays 143 Figure 5 2 Applying Tough Spots to Arrays Scanning the Array x NOTE Customers using the Autoloader should refer to the Autoloader User s Guide To scan arrays 1 Select Run Scanner from the menu bar Alternatively click the Start Scan icon in the tool bar The Scanner dialog box appears with a drop down list of all unscanned experiments Select the experiment name that corresponds to the array being scanned A previously run experiment can also be selected by using the Include Scanned Experiments option box After selecting this option previously scanned experiments appear in the drop down list lt NOTE If the experiment name is not seen in the dialog box open the workflow monitor right click your experiment and select Advance to Scan Refer to the GeneChip Operating Software User s Guide for further information Click the Load Eject button and place the array in the scanner Only one scan is required for the GeneChip Scanner 3000 7G 144 Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual WARNING Do not attempt to manually open or close the GeneChip Scanner 3000 7G scanner door as this may damage the scanner 4 Once the experiment has been selected click the Start button A dialog box prompts to load the array into the scanner Pixel resolution and w
96. d one cooler on ice 2 Label the following then place in a cooling chamber Four 96 well reaction plates labeled P P2 P3 P4 One 50 mL Falcon tube labeled PCR MM 3 Place on ice AccuGENE water GC Melt Solution basin 4 Prepare the Nsp ligated samples as follows A Vortex the center of the plate at high speed for 3 sec B Spin down the plate at 2000 rpm for 30 sec C Label the plate Lig D Place back in the cooling chamber on ice 5 To prepare the reagents A Vortex at high speed 3 times 1 sec each time except for the enzyme B Pulse spin for 3 sec C Place in a cooling chamber Preheat the Thermal Cycler Lids Main Lab Have someone in the Main Lab power on the thermal cyclers to be used for PCR to preheat the lids The lids must be preheated before loading samples leave the blocks at room temperature If you are preparing the plates for PCR it is best not to go from the Pre PCR Room or Staging Area to the Main Lab and then back again Aliquot Nsp Ligated DNA to the PCR Plates To aliquot Nsp ligated DNA to the PCR plates 1 Working one row at a time and using a 12 channel P20 pipette transfer 10 uL of each Nsp ligated sample to the corresponding well of each PCR plate P1 P2 P3 and P4 2 Seal each plate with adhesive film and leave in cooling chambers on ice 76 Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Prepare the Nsp PCR Master Mix Location Pre PCR Clean Room Pre
97. de Human SNP 5 0 Nsp Sty Assay Genome Wide SNP 5 0 Assay is designed for processing 48 samples The protocol is presented in the following stages Genomic DNA Plate Preparation Stage 1 Sty Restriction Enzyme Digestion Stage 2 Sty Ligation Stage 3 Sty PCR Stage 4 Nsp Restriction Enzyme Digestion Stage 5 Nsp Ligation Stage 6 Nsp PCR Stage 7 PCR Product Pooling and Purification Stage 8 Quantitation Stage 9 Fragmentation Stage 10 Labeling Stage 11 Target Hybridization Key points regarding the various molecular biology steps that comprise whole genome sampling analysis WGSA are included in the protocol and guidelines Successful performance of the various molecular biology steps in this protocol requires accuracy and attention to detail Many of these stages involve specific yet distinct enzymatic reactions For example in stage 1 genomic DNA is digested with the restriction enzyme Sty I In stage 2 it is ligated to a common adaptor with T4 DNA ligase Following ligation the template undergoes PCR using TITANIUM Taq DNA polymerase Once the product has been purified stage 7 it is then fragmented in stage 9 with Fragmentation Reagent DNAse I and end labeled using terminal deoxynucleotidyl transferase stage 10 The stages involving enzymatic reactions are the most critical of the assay Thus it is important to carefully monitor and control any variables such as pH salt concentrations time and temperature
98. disease studies Hum Mol Genet 2006 15 Suppl 1 R57 66 Gonzalez E Kulkarni H Bolivar H Mangano A Sanchez R Catano G Nibbs RJ Freedman BI Quinones MP Bamshad MJ et al The influence of CCL3L1 gene containing segmental duplications on HIV 1 AIDS susceptibility Science 2005 307 1434 40 Ouahchi K Lindeman N Lee C Copy number variants and pharmacogenomics Pharmacogenomics 2006 7 25 9 Inoue K Lupski JR Molecular mechanisms for genomic disorders Annu Rev Genomics Hum Genet 2002 3 199 242 Redon R Ishikawa S Fitch KR Feuk L Perry GH Andrews TD Fiegler H Shapero MH Carson AR Chen W et al Global variation in copy number in the human genome Nature 2006 In Press Komura D Shen F Ishikawa S Fitch KR Chen W Zhang J Liu G Ihara S Nakamura H Hurles ME et al Genome wide detection of human copy number variations using high density DNA oligonucleotide arrays Gen Res 2006 In Press McCarrol SA Hadnott TN Perry GH Sabeti PC Zody MC Barrett JC Dallaire S Gabriel SB Lee C Daly MJ et al Common deletion polymorphisms in the human genome Nat Genet 2006 38 86 92 Locke DP Sharp AJ McCarroll SA McGrath SD Newman TL Cheng Z Schwartz S Albertson DG Pinkel D Altshuler DM et al Linkage Disequilibrium and Heritability of Copy Number Polymorphisms within Duplicated Regions of the Human Genome Am J Hum Genet 2006 79 275 90 Hinds DA Kloek AP
99. dization efficiency Li Ld L Ld Lj Li ha LI LI UNS GE Z 2 EE 1 e P M Figure 6 21 Array name image has been rotated for display chapter 6 Data Analysis 169 Figure 6 22 on page 169 is the scanned image of the Genome Wide Human SNP Array 5 0 Notice how the array appears to be divided into four quadrants The genotyping probes are tiled within each quadrant Non polymorphic probes are tiled in the bands that form the quadrant boundaries Non polymorphic probes Non polymorphic probes Figure 6 22 Scanned Image of the Genome Wide Human SNP Array 5 0 170Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Downstream Analysis Considerations Association studies are designed to identify SNPs with subtle allele frequency differences between different populations Genotyping errors differences in sample collection and processing and population differences are among the many things that can contribute to false positives or false negatives Efforts should be made to minimize or account for technical or experimental differences For example randomization of cases and controls prior to genotyping can reduce or eliminate any possible effects from running cases and controls under different conditions Data Filtering For many genotyping applications poorly performing SNPs can lead to an increase in false positives and a decrease in power Such under performing SNPs can be caused by
100. don D Chaplin T Foot NJ Skoulakis S Raghavan M Harwood CA Proby CM Philpott MP Young BD et al Genomewide single nucleotide polymorphism microarray mapping in basal cell carcinomas unveils uniparental disomy as a key somatic event Cancer Res 2005 65 8597 603 Baron CA Tepper CG Liu SY Davis RR Wang NJ Schanen NC Gregg JP Genomic and functional profiling of duplicated chromosome 15 cell lines reveal regulatory alterations in UBE3A associated ubiquitin proteasome pathway processes Hum Mol Genet 2006 15 853 69 Lu YJ Yang J Noel E Skoulakis S Chaplin T Raghavan M Purkis T McIntyre A Kudahetti SC Naase M et al Association between large scale genomic homozygosity without chromosomal loss and nonseminomatous germ cell tumor development Cancer Res 2005 65 9137 41 Fitzgibbon J Smith LL Raghavan M Smith ML Debernardi S Skoulakis S Lillington D Lister TA Young BD Association between acquired uniparental disomy and homozygous gene mutation in acute myeloid leukemias Cancer Res 2005 65 9152 4 Calhoun ES Hucl T Gallmeier E West KM Arking DE Maitra A Iacobuzio Donahue CA Chakravarti A Hruban RH Kern SE Identifying Allelic Loss and Homozygous Deletions in Pancreatic Cancer without Matched Normals Using High Density Single Nucleotide Polymorphism Arrays Cancer Res 2006 66 7920 7928 Garraway LA Widlund HR Rubin MA Getz G Berger AJ Ramaswamy S Bero
101. duct from the 3 Sty PCR plates to the corresponding plate row and wells of the PXGel plates Example 3 uL of each PCR product from each well of row A on plate P1 is transferred to the corresponding wells of row A on plate P1Gel 7 Seal the PXGel plates Vortex the center of each PXGel plate then spin them down at 2000 rpm for 30 sec 9 Load the total volume of 15 uL from each well of each PXGel plate onto E Gel 48 2 agarose gels 10 Run the gels for 22 min 11 Verify that the PCR product distribution is between 250 bp to 1100 bp Figure C 1 1000 500 200 Figure C 1 Example of PCR Products Run on E Gel 48 2 Agarose Gel for 22 min Average Product Distribution is Between 250 to 1100 bp 214 Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Modifications for Stage 6 Nsp PCR Follow the Stage 3 instructions in Stage 6 Nsp PCR on page 71 with the modifications listed below Gels and Related Materials Required Reference Table 4 30 on page 73 The amounts listed are sufficient to process 48 samples Table C 2 E Gels and Related Materials Required for Stage 6 Nsp PCR OTETULLA Y Reagent 240 uL All Purpose Hi Lo DNA Marker diluted 1 3 with H20 See When Using the E Gel 48 2 on page 211 As needed Gel loading buffer diluted 1 20 or 1 30 with H20 See When Using the E Gel 48 2 on page 211 4 E Gel 48 2 agarose gel 4 Plates 96 well reaction Running Gels Reference the inst
102. e Figure 4 13 on page 99 Row E remains water only and will serve as a blank x NOTE If a particular well s contain less than 2 uL of purified PCR product see page 177 of Chapter 7 Troubleshooting for instructions Pipette up and down 2 times after each transfer to ensure that all of the product is dispensed Examine the pipette tips and aliquots before and after each dispense to ensure that exactly 2 uL has been transferred The result is a 100 fold dilution Set a 12 channel P200 pipette to 180 uL Mix each sample by pipetting up and down 3 times Be careful not to scratch the bottom of the plate or to introduce air bubbles chapter 4 Affymetrix Genome Wide Human SNP Nsp Sty 5 0 Assay 48 Sample Protocol 99 e amp amp amp amp amp amp e 000008 e 6 6 amp d d e 9 gt 6 6666666606006 Wi W ON w w WW WW w Wo SW WS Plate of Purified Samples Optical Plate W water only blanks Figure 4 13 Loading the Optical Plate with Purified Sample and Water Blanks Quantitate the Diluted PCR Product To quantitate the diluted PCR product 1 Measure the OD of each PCR product at 260 280 and 320 nm OD280 and OD320 are used as process controls Their use is described under Process Control Metrics on page 100 2 Dete
103. e with the thermal cycler can result in crushed tubes loss of sample or poor results 200 Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Table A 6 Thermal Cyclers PCR Plates and Plate Seals Optimized for Use With the Genome Wide Human SNP 5 0 Nsp Sty Assay Area PCR Staging Room Any 2 of these thermal cyclers can be used for the pre PCR steps of the protocol Thermal Cyclers Validated Plate for Use Applied Biosystems units 2720 Thermal Cycler GeneAmp PCR System 9700 Bio Rad units MJ Tetrad PTC 225 DNA Engine Tetrad 2 PTC0240 TETRAD2 Multiplate 96 Well Unskirted PCR Plates Bio Rad P N MLP 9601 Cover MicroAmp Clear Adhesive Films Applied Biosystems P N 4306311 Main Lab Any 7 of these thermal cyclers can be used for the PCR and post PCR steps of the protocol Applied Biosystems GeneAmp PCR System 9700 by silver block or gold plated silver block Bio Rad units MJ Tetrad PTC 225 DNA Engine Tetrad 2 PTC0240 TETRAD2 Multiplate 96 Well Unskirted PCR Plates Bio Rad P N MLP 9601 MicroAmp Clear Adhesive Films Applied Biosystems P N 4306311 appendix A Reagents Equipment and Consumables 201 Consumables Required Arrays Required This protocol requires the use of Affymetrix Genome Wide Human SNP Arrays 5 0 Table A 7 Affymetrix Genome Wide Human SNP Array 5 0 Arrays Pack Part Number 5 9010
104. e hybridization ovens Because the lids on these thermal cyclers do not slide back you will process 24 samples at a time Add Hybridization Master Mix and Denature To add Hybridization Master Mix and denature the samples 1 Pour 11 mL Hybridization Master Mix into a solution basin 2 Using a 12 channel P200 pipette add 190 uL of Hybridization Master Mix to each sample on the Label Plate Total volume in each well is 263 uL 3 Sealthe plate tightly with adhesive film Lu IMPORTANT It is critical to seal the plate tightly Vortex the center of the plate for 3 minutes Cut the used wells into 2 strips of two rows each Put each strip of 24 samples into a plate holder Spin down the strips at 2000 rpm for 30 sec 9 N s Cut the adhesive film between each row of samples Do not remove the film 9 Place one set of 24 wells onto the thermal cycler and close the lid 10 Keep the remaining sets of wells in a cooling chamber on ice 11 Run the GW5 0 Hyb program Table 4 56 GW5 0 Hyb Thermal Cycler Program GW5 0 Hyb Program Temperature 95 C 10 minutes 49 C Hold 128Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Load the Samples onto Arrays This procedure requires 2 operators working simultaneously Operator loads the samples onto the arrays Operator 2 covers the septa with Tough Spots and loads the arrays into the hybridization ovens To load the samples onto arr
105. e vacuum manifold is working properly An error may have been made while taking the OD readings The PCR product may not have been adequately washed Check the 7596 EtOH wash solution Table 4 42 PROBLEM The OD320 measurement is significantly larger than zero 0 0 005 Possible causes include Magnetic beads may have been carried over into purified sample Precipitate may be present in the eluted samples There may be defects in the OD plate Air bubbles in the OD plate or in solutions What To Do Next Do one of the following Proceed immediately to the next step If not proceeding immediately to the next step A Seal the plate with the eluted samples B Store the plate at 20 C chapter 4 Affymetrix Genome Wide Human SNP Nsp Sty 5 0 Assay 48 Sample Protocol 103 Stage 9 Fragmentation About this Stage During this stage the purified PCR products will be fragmented using Fragmentation Reagent You will first dilute the Fragmentation Reagent by adding the appropriate amount of Fragmentation Buffer and AccuGENE water You will then quickly add the diluted reagent to each reaction place the plate onto a thermal cycler and run the GW5 0 Fragment program Once the program is finished you will check the results of this stage by running 1 5 uL of each reaction on a 4 TBE gel or an E Gel 46 4 agarose gel Location and Duration Main Lab Hands on time 30 minutes e GW5 0 Fra
106. eagents are required for this stage Refer to Appendix A for vendor and part number information The amounts listed are sufficient to process 48 samples Table 4 25 Reagents Required for Stage 5 Nsp Ligation Quantity Reagent 1 vial T4 DNA Ligase 400 U uL NEB 1 vial T4 DNA Ligase Buffer 10X 1 vial Adaptor Nsp 50 uM 10 mL AccuGENE water molecular biology grade Important Information About This Procedure To help ensure the best results carefully read the information below before you begin this stage of the protocol Prepare the Reagents Consumables and Other Components IMPORTANT e Aliquot the T4 DNA Ligase Buffer 10X after thawing for the first time to avoid multiple freeze thaw cycles See vendor instructions Be sure to use the Nsp adaptor Thaw the Reagents and Nsp Digestion Stage Plate To thaw the reagents and Nsp Digestion Stage Plate 1 Allow the following reagents to thaw on ice Adaptor NspI T4 DNA Ligase Buffer 10X Takes approximately 20 minutes to thaw 2 Ifthe Nsp digested samples were frozen allow them to thaw in a cooling chamber on ice Eu IMPORTANT Leave the T4 DNA Ligase at 20 C until ready to use chapter 4 Affymetrix Genome Wide Human SNP Nsp Sty 5 0 Assay 48 Sample Protocol 67 Prepare Your Work Area To prepare the work area 1 Place a double cooling chamber and a cooler on ice 2 Label the following tubes then place in the c
107. eheat the Thermal Cycler Lid Power on the thermal cycler to preheat the lid Leave the block at room temperature 62 Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Prepare the Nsp Digestion Master Mix Keeping all reagents and tubes on ice prepare the Nsp Digestion Master Mix as follows 1 To the 2 0 mL Eppendorf tube add the appropriate volumes of the following reagents based on Table 4 22 AccuGENE water NE Buffer 2 BSA 2 Remove the Nsp I enzyme from the freezer and immediately place in a cooler w Pulse spin the enzyme for 3 sec 2 Immediately add the enzyme to the master mix then place remaining enzyme back in the cooler Vortex the master mix at high speed 3 times 1 sec each time Pulse spin for 3 sec Place in the cooling chamber Return any remaining enzyme to the freezer e g N 9 g Proceed immediately to 44d Nsp Digestion Master Mix to Samples on page 63 Table 4 22 Nsp Digestion Master Mix Reagent 1 Sample 48 Samples 15 extra AccuGENE Water 11 55 uL 637 6 uL NE Buffer 2 10X 2 uL 110 4 uL BSA 100X 10 mg mL 0 2 uL 11 uL Nsp I 10 U uL 1 uL 55 2 uL Total 14 75 uL 814 2 pL chapter 4 Affymetrix Genome Wide Human SNP Nsp Sty 5 0 Assay 48 Sample Protocol 63 Add Nsp Digestion Master Mix to Samples To add Nsp Digestion Master Mix to samples 1 Using a single channel P200 pipette aliquot 67 uL of Nsp Digestion Master Mix to each tube of t
108. eotides Check dNTP stock concentration and vendor Used Nsp adaptor for Sty digest or vice versa Repeat Ligation step with correct adaptors chapter 7 Troubleshooting 177 Problem Likely Cause Faint absent bands on PCR gel continued Solution Samples affected but positive controls OK Non optimal reaction conditions Use master mixes and include a positive control to eliminate reagents and assay problems as detailed above Insufficient starting material 250 ng genomic DNA should be used Confirm concentration using calibrated spectrophotometer Sample DNA contains enzymatic or chemical inhibitors Ensure genomic DNA is purified and diluted in Low EDTA 0 1mM TE buffer Use recommended procedure to ethanol precipitate genomic DNA to remove inhibitors Degraded sample DNA Confirm quality of genomic DNA sample Low PCR yield DNA lost during purification Gel images show PCR product but low OD Vacuum elution is not complete Ensure that filtering is complete for all wells matte dull look before stopping vacuum elution Insufficient purified PCR product for quantitation Volume in a particular well s on the elution catch plate is lt 2 uL after transferring 45 uL to the fragmentation plate Do the following in this order Add 2 uL Buffer EB to the corresponding wells on the fragmentation plate Mix by pipetting up and down
109. er Be sure to equilibrate the arrays to room temperature otherwise the rubber septa may crack and the array may leak An accurate hybridization temperature is critical for this assay Therefore we recommend that your hybridization ovens be serviced at least once per year to ensure that they are operating within specifications Gloves safety glasses and lab coats must be worn when preparing the Hybridization Master Mix Consult the appropriate MSDS for reagent storage and handling requirements chapter 4 Affymetrix Genome Wide Human SNP Nsp Sty 5 0 Assay 48 Sample Protocol 121 Prepare the Reagents Consumables and Other Components Prepare a 12X MES Stock Solution The 12X MES stock solution can be prepared in bulk and kept for at least one month if properly stored Proper storage Protect from light using aluminum foil e Keep at 4 C IMPORTANT Do not autoclave Store between 2 C and 8 C and shield from light using aluminum foil Discard solution if it turns yellow To prepare 1000 mL of 12X MES Stock Solution 1 25 M MES 0 89 M Na 1 Combine 70 4 g MES hydrate 193 3 g MES sodium salt 800 mL AccuGENE water 2 Mix and adjust volume to 950 mL Test the pH The pH should be between 6 5 and 6 7 Adjust the pH so it falls between 6 5 and 6 7 Adjust the volume to 1000 mL Filter the solution through a 0 2 um filter e Noo A Protect from light using aluminum foil and store at 4 C
110. er plate reader All multi channel pipettes chapter 4 Affymetrix Genome Wide Human SNP Nsp Sty 5 0 Assay 48 Sample Protocol 23 Workflow Recommendations Figure 4 1 shows the recommended workflow for one operator processing 48 samples Day 1 PCR Staging Room and Main Lab e Restriction Digest 1 Sty Plate Ligation 1 Sty Plate e PCR 3 Sty Plates Main Lab Day 2 PCR Staging Room and Main Lab e Restriction Digest 1 Nsp Plate Ligation 1 Nsp Plate e PCR 4 Nsp Plates Main Lab Day 3 Main Lab Nsp and Sty PCR Products Pooled PCR Product Purification Day 4 Main Lab Fragmentation and Labeling Hybridization onto Arrays Day 5 Main Lab 48 Arrays Washing Staining and Scanning Arrays 48 Arrays Figure 4 1 Workflow recommended for processing 48 samples Since WGSA involves a series of ordered stages the output of one stage directly impacts the performance of the subsequent stage For example the quantity and purity of the DNA after purification can affect the kinetics of the Fragmentation Reagent during the subsequent fragmentation stage 24 Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual To efficiently process samples in 96 well plates it is essential that you be proficient with the use of multi channel pipettes Attempting to use a single channel pipette for plate based samples requires too many pipetting steps thus creating too high of a chance
111. es 1X 4 C HOLD Can be held overnight appendix B Thermal Cycler Programs 209 GW5 0 Fragment GW5 0 Fragment Program 37 C 35 minutes 95 C 15 minutes 4 C Hold GW5 0 Label GW5 0 Label Program Temperature 37 C 4 hours 95 C 15 minutes 4 C Hold GW5 0 Hyb GW5 0 Hyb Program Temperature 95 C 10 minutes 49 C Hold 210 Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Appendix E GELS This appendix describes the use of E GelS for confirming Sty and Nsp PCR reactions Fragmentation reactions Before Using E Gels When Using the E Gel 48 2 Use the following reagents Loading solution Gel Loading Buffer from Sigma Aldrich Dilute this solution to 1 20 or 1 30 using H20 before use DNA Marker All Purpose Hi Lo DNA Marker from Bionexus Dilute this marker 1 3 with H20 before use For more information refer to Appendix A Reagents Equipment and Consumables When Using the E Gel 48 4 Use the following reagents Loading solution 5xSB Loading Medium from Faster Better Media Dilute this solution to 1 20 or 1 30 with H20 before use DNA Marker 25 bp DNA Ladder from Invitrogen 5xSB Loading Medium contains Orange G Because Orange G is known to affect DNA migration slightly and because E Gels are salt sensitive dilute the ladder and samples with the same loading solution For more information refer to
112. f Place the wash lines in a different bottle of deionized water than the one used for the shutdown protocol IMPORTANT To maintain the cleanliness of the fluidics station and obtain the highest quality image and data possible a weekly bleach protocol is highly recommended 146Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Chapter DATA ANALYSIS The purpose of this chapter is to Describe the necessary steps to analyze data from Genome Wide Human SNP Arrays 5 0 Present some guidelines for assessing data quality This information is intended as a supplement to the information sources listed below and does not replace them GeneChip Operating Software User s Guide GCOS BRLMM Analysis Workflow Document Genome Wide Human SNP Array 5 0 library files Software Requirements GeneChip Operating Software GCOS 1 4 client GCOS 1 3 server or higher Affymetrix Genome Wide Human SNP Array 5 0 library files BRLMM Analysis Tool 2 0 BAT 2 0 GCOS Service Pack 2 Custom Grid Patch GCOS SP2 148 Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Overview of the QC and Genotyping Analysis Workflow This section describes the QC and Genotyping Workflow for generating genotyping calls using BAT 2 0 This process is summarized in Figure 6 1 Note that acquisition of raw data using GCOS precedes analysis by BAT 2 0 Refer to the GeneChip Operating Software User s Gu
113. ffer 6X SSPE 0 01 Tween 20 For 1000 mL 300 mL of 20X SSPE 1 0 mL of 10 Tween 20 699 mL of water Filter through a 0 2 um filter Store at room temperature Wash B Stringent Wash Buffer 0 6X SSPE 0 01 Tween 20 For 1000 mL 30 mL of 20X SSPE 1 0 mL of 10 Tween 20 969 mL of water Filter through a 0 2 um filter Store at room temperature The pH should be 8 IMPORTANT Prepare Wash B in smaller quantities to avoid long term storage The container must be sealed tightly to avoid changes in salt concentration due to evaporation 0 5 mg mL Anti Streptavidin Antibody Resuspend 0 5 mg in 1 mL of water Store at 4 C 134 Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual 12X MES Stock Buffer 1 25 M MES 0 89 M Na For 1 000 mL 70 4g of MES hydrate 193 3g of MES Sodium Salt 800 mL of Molecular Biology Grade Water Mix and adjust volume to 1 000 mL The pH should be between 6 5 and 6 7 Filter through a 0 2 um filter IMPORTANT Do not autoclave Store at 2 C to 8 C and shield from light Discard solution if yellow 1X Array Holding Buffer Final 1X concentration is 100 mM MES 1M Nar 0 0196 Tween 20 For 100 mL 8 3 mL of 12X MES Stock Buffer 18 5 mL of 5 M NaCI 0 1 mL of 10 Tween 20 73 1 mL of water Store at 2 C to 8 C and shield from light chapter 5 Washing Staining and Scanning Arrays 135 Experiment and Fluidics Stati
114. fold Figure 4 12 Plate Stack Inside Vacuum Manifold with Deep Collar H When all of the wells appear dry 1 Turn off the vacuum and inspect each well using a flashlight The surface changes from sheeny reflective to matte non reflective when dry There should be no standing liquid in any of the wells being used 2 Ifall of the wells are not dry place the plate back on the manifold and resume filtering IMPORTANT Continue filtering and inspecting the wells with a flashlight until completely dry There should be no standing liquid in any of the wells being used 3 When all wells are completely dry remove the plate stack from the manifold 10 Seal the plate stack with an adhesive film and spin it down at room temperature for 5 minutes at 1400 rcf 94 Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Use the following formula to convert relative centrifugal force rcf to revolutions per minute rpm rpm 1000 X square root rcf 1 12r The radius r is equal to the distance in millimeters between the axis of rotation of the centrifuge and the bottom of the plate bucket For example on the Eppendorf 5804R spinning at 3100 rpm gives an rcf of 1400 assuming r 133 mm 11 Remove the elution catch plate from the filter plate 12 Using a 12 channel P200 pipette transfer 45 uL of eluate to a new PCR plate for fragmentation x NOTE If a particular well s contain less than 45
115. fymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Genotype Calls File The genotype calls file is a text file containing the genotype calls for the analysis run This file has the extension calls txt and consists of a comment section followed by the genotype calls The comment section is identified by the prefix and contains information about the analysis run including the program the algorithm library files and command line used to generate the file The genotype calls are tab delimited and are preceded by a header row The header row indicates which column contains the probeset_ids and the genotype calls for each CEL file The tab delimited section contains a header row indicating which column contains the probeset_ids and the genotype calls for each CEL file Calls are encoded as follows NoCall 0 1 AB e 2 brlmm calls txt Notepad Jog cmd probeset_id AFFX 2315060 AFFX 2315061 AFFX 2315062 AFFX 2315057 AFFX 2315058 AFFX 2315059 AFFX 2315063 AFFX 2315064 AFFX 2315065 AFFX 2315066 File Edit Format View Help ze 10000 chrX file C Documents and Settings jbumiMy DocumentsXData genotype l LibraryFiles Mapping250K_Nsp chnymax score 0 5 dm thresh 0 17 dm het mult 0 feat effect file target sketch file qmethod spec plier optmethod 1 prior fi le chrX force 0 force 0 analysis brimm cel count 6 cvs id BRLMM Analysis T ool 2 0 model file time str Tue Nov 14 20 30 45 2006 an
116. g Gels The instructions below are for running 2 TBE gels For information on running E Gel 48 2 agarose gels refer to Appendix C E gels on page 211 Before Running Gels To ensure consistent results take 3 uL aliquot from each PCR WARNING Wear the appropriate personal protective equipment when handling ethidium bromide Run the Gels When the GW5 0 PCR program is finished 1 a RON 7 8 9 Remove each plate from the thermal cycler Spin down plates at 2000 rpm for 30 sec Place plates in cooling chambers on ice or keep at 4 C Label three fresh 96 well reaction plates P Gel P2Gel and P3Gel Aliquot 3 uL of 2X Gel Loading Dye to each well in rows A through D of the fresh labeled P XGel plates Using a 12 channel P20 pipette transfer 3 uL of each PCR product from the 3 Sty PCR plates to the corresponding plate row and wells of the PXGel plates Example 3 uL of each PCR product from each well of row A on plate P1 is transferred to the corresponding wells of row A on plate P1Gel Seal the PXGel plates Vortex the center of each PXGel plate then spin them down at 2000 rpm for 30 sec Load the total volume from each well of each PXGel plate onto 2 TBE gels 10 Run the gels at 120V for 40 minutes to 1 hour 11 Verify that the PCR product distribution is between 250 bp to 1100 bp see Figure 4 5 chapter 4 Affymetrix Genome Wide Human SNP Nsp Sty 5 0 Assay 48 Sample Protocol 57
117. gment thermal cycler program time 1 hour Input Required from Previous Stage The input required from Stage 8 Quantitation is Quantity Item 1 Plate of quantitated PCR product in a cooling chamber on ice 104 Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Equipment and Consumables Required The following equipment and consumables are required for this stage Refer to Appendix A for vendor and part number information IMPORTANT Use only the PCR plate adhesive film and thermal cyclers listed in Table 4 1 on page 27 Table 4 43 Equipment and Consumables Required for Stage 9 Fragmentation Quantity Item 1 Cooler chilled to 20 C 1 Cooling chamber double chilled to 4 C do not freeze 1 Ice bucket filled with ice 1 Marker fine point permanent 1 Microcentrifuge 1 Pipette single channel P20 1 Pipette single channel P100 1 Pipette single channel P1000 1 Pipette 12 channel P20 accurate to within 5 As needed Pipette tips for pipettes listed above full racks 1 Plate centrifuge 1 Plate seal 1 Thermal cycler 2 Tube Eppendorf 1 5 mL 2 Tubes 12 strip 0 2 mL 1 Vortexer chapter 4 Affymetrix Genome Wide Human SNP Nsp Sty 5 0 Assay 48 Sample Protocol 105 Reagents Required The following reagents are required for this stage Refer to Appendix A for vendor and part number information The amounts listed are suffi
118. he DI water bottle Figure 8 5 At this step there is no need to be concerned about the bleach remaining in the lines Figure 8 5 Immerse the three wash and water lines in the DI water bottle 3 Press down on the needle levers to begin the rinse cycle The fluidics station will empty the lines and rinse the needles chapter 8 Vacuum Manifold and Fluidics Station Care and Maintenance 191 4 When the rinse is completed after approximately one hour the fluidics station will bring the temperature back to 25 C and drain the lines with air The LCD display will read CLEANING DONE 5 Discard the vials used for the bleach protocol 6 After completing the bleach protocol follow the suggestions for storage of the Fluidics Station 450 in Table 8 2 below Table 8 2 Storage Suggestions for the Fluidics Station 450 If Then do this Planning to use the system After running the bleach protocol remove the DI immediately water supply used in the rinse phase and install the appropriate reagents for use in the next staining and washing protocol including fresh DI water Perform a prime protocol without loading your probe arrays Failure to run a prime protocol will result in irreparable damage to the loaded hybridized probe arrays Not planning to use the system Since the system is already well purged with immediately water there is no need to run an additional shutdown protocol Remove the old DI water bottle and
119. he Fragmentation Reagent This reagent is extremely temperature sensitive and rapidly loses activity at higher temperatures To avoid loss of activity Handle the tube by the cap only Do not touch the sides of the tube as the heat from your fingers will raise the reagent temperature Dilute immediately prior to use Keep at 20 C until ready to use Transport and hold in a 20 C cooler Return to the cooler immediately after use Spin down so that the contents of the tube are uniform Perform these steps rapidly and without interruption This reagent is sticky and may adhere to the walls of some microfuge tubes and 96 well plates This reagent is viscous and requires extra care when pipetting Follow these guidelines Pipette slowly to allow enough time for the correct volume of solution to enter the pipette tip Avoid excess solution on the outside of the pipette tip chapter 4 Affymetrix Genome Wide Human SNP Nsp Sty 5 0 Assay 48 Sample Protocol 107 Prepare the Reagents Consumables and Other Components Thaw Reagents Thaw the Fragmentation Buffer 10X on ice Lu IMPORTANT Leave the Fragmentation Reagent at 20 C until ready to use Prepare Your Work Area To prepare the work area 1 Place a double cooling chamber and a cooler on ice 2 Place the AccuGENE water on ice 3 Prepare the Fragmentation Buffer as follows A Vortex 3 times 1 sec each time B Pulse spin for 3 sec C P
120. he strip tubes labeled Dig 2 Using a 12 channel P20 pipette add 14 75 uL of Nsp Digestion Master Mix to each DNA sample in the cooling chamber on ice The total volume in each well is now 19 75 uL Genomic DNA 50 ng uL 5 uL Nsp Digestion Master Mix 14 75 uL Total Volume 19 75 uL Seal the plate tightly with adhesive film Vortex the center of the plate at high speed for 3 sec Spin down the plate at 2000 rpm for 30 sec Ensure that the lid of thermal cycler is preheated M90 Load the plate onto the thermal cycler and run the GW5 0 Digest program Table 4 23 GW5 0 Digest Program GW5 0 Digest Program Temperature Time 37 C 120 minutes 65 C 20 minutes 4 C Hold 8 When the program is finished remove the plate and spin it down at 2000 rpm for 30 sec What To Do Next Do one of the following If following the recommended workflow Figure 4 1 on page 23 proceed immediately to Stage 5 Nsp Ligation on page 64 If not proceeding directly to the next step store the samples at 20 C 64 Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Stage 5 Nsp Ligation About this Stage During this stage the digested samples are ligated using the Nsp Adaptor You will 1 2 3 4 Prepare a Nsp Ligation Master Mix Add the master mix to the samples Place the samples onto a thermal cycler and the GW5 0 Ligate program is run Dilute the ligated sample
121. he writing of this manual it is BLEACHv2_450 for each of the respective modules in the Protocol drop down list Select all four modules 1 to 4 and click Run lt NOTE The fluidics station will not start until the needle lever is pressed down Figure 8 4 on page 189 The temperature will ramp up to 50 C 7 Follow the prompts on each LCD Load empty 1 5 mL vials onto each module if not already done so chapter 8 Vacuum Manifold and Fluidics Station Care and Maintenance 189 8 Press down on each of the needle levers to start the bleach protocol Figure 8 4 Figure 8 4 Press down on the needle levers to start the bleach protocol 9 The fluidics station will begin the protocol emptying the lines and performing the cleaning cycles using bleach solution 10 After approximately 30 minutes the LCD will prompt you when the bleach cycle is over and the rinse cycle is about to begin 190Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual The Rinse Cycle Once the bleach cycle has finished the second part of the protocol is a rinse step This step is essential to remove all traces of bleach from the system Failure to complete this step can result in damaged arrays 1 Follow the prompts on the LCD for each module Lift up on the needle levers and remove the bleach vials Load clean empty vials onto each module 2 Remove the three wash and water lines from the bleach bottle and transfer them to t
122. iDesktopXVnv setlNNAOBS33 Hw 301106 Vny C11 rl CEL C Documents and Settings jburri Desktop Vinv_set N406993_H W_301106_Vn _D04_11 CEL C Documents and Settings jburri Desktop Vinv_set N407019_HW_301106_VYn _BO5_11 CEL C Documents and Settings jburri Desktop Vinv_set N407022_HW_301106_Vn _A04_11 CEL C Documents and Settings jburri Desktop nv_set N407056_HWw_301106_Vn _402_11 CEL Figure 6 3 Import Options for QC and Genotyping Workflow 6 Verify that the correct algorithm is displayed in the Select the genotyping algorithm field The algorithm is determined automatically based on the library files selected on the Start Page For Genome Wide Human SNP Arrays 5 0 the algorithm is BRLMM P chapter 6 Data Analysis 151 7 Select the CEL files to be analyzed using one of the following methods Click Add to browse to the directory containing your CEL files then shift click or control click to select the CEL files the click Open Only one array type can be analyzed at a time Therefore only arrays that match the type selected in the Array Type field can be added If multiple array types are selected the dialog box shown below is displayed BRLMM Analysis Tool amp One or more files were not added The probe array type did not match Figure 6 4 Error Message Indicating Array Type Mismatch Click Import to browse to the directory containing a text file tha
123. icated equipment such as thermal cyclers microfuges pipets tips etc Once you enter the Main Lab do not return to the Pre PCR Room or the PCR Staging Room until you have showered and changed into freshly laundered clothing Maintain an ambient laboratory environment throughout the procedure Precautions that you can take to minimize contaminating pre PCR steps with amplified PCR product include the following Store reagents in the proper room according to the box label and reagent kit insert Restrict movements through labs containing amplified DNA Use proper gowning procedures Use dedicated equipment for pre PCR stages e g pipets tips thermal cyclers etc Print separate copies of the protocol for each room Pre PCR Clean Room The Pre PCR Clean Room or dedicated area such as a biosafety hood should be free of DNA template and PCR amplicons The master stocks of PCR primer and adaptor should be stored here with aliquots taken for use in the PCR Staging Room Reagent preparation tasks such as preparing master mixes should be done in this room The use of gowns booties and gloves is strongly recommended to prevent PCR carryover and to minimize the risk of trace levels of contaminants being brought into the Pre PCR Clean Room This room should contain dedicated pipets tips vortex etc Refer to Appendix A Reagents Equipment and Consumables for more information chapter 2 Laboratory Setup and Rec
124. ide for instructions on DAT and CEL file generation GCOS Figure 6 1 Analysis Workflow for the Genome Wide Human SNP Array 5 0 Using BAT 2 0 chapter 6 Data Analysis 149 QC and Genotyping Analysis Workflow The following instructions are for processing data from Genome Wide SNP Arrays using BAT 2 0 To process data from this array type you will use the BRLMM P algorithm x NOTE For BRLMM a minimum of 6 CEL files is required for the algorithm to run We recommend running a minimum of 80 samples at a time For BRLMM P there is no minimum You can run BRLMM P on a single file although performance may be poor We recommend running a minimum of 40 samples at a time although more samples is generally better For improved performance for BRLMM and BRLMM P the optimal cluster size is greater than or equal to 200 samples Performance on rare genotypes continues to improve as the number of samples increases Set up the OC Analysis To set up the OC analysis 1 Launch BRLMM Analysis Tool 2 0 from Start gt Programs gt Affymetrix gt BRLMM Analysis Tool BRLMM Analysis Tool tj Start Page gp Work Flow ac O Genotyping QC and Genotyping Array Type Select the array type GenomeWideSNP_5 v Library Files Select the library path C Documents and Settings jburri My Documents D ata genotype LibraryFiles Figure 6 2 OC and Genotyping Workflow Selected 150Affy
125. igure 4 15 For every 4 arrays A Load the arrays into an oven tray evenly spaced B Immediately place the tray into the hybridization oven Do not allow loaded arrays to sit at room temperature for more than approximately 1 5 minute Ensure that the oven is balanced as the trays are loaded and ensure that the trays are rotating at 60 rpm at all times Because you are loading 4 arrays per tray each hybridization oven will have a total of 32 arrays 126Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Operators 1 and 2 Load no more than 32 arrays in one hybridization oven at a time All 48 samples should be loaded within 1 hour Store the remaining samples and any samples not yet hybridized in a tightly sealed plate at 20 C Allow the arrays to rotate at 50 C 60 rpm for 16 to 18 hours IMPORTANT Allow the arrays to rotate in the hybridization ovens for 16 to 18 hours at 50 C and 60 rpm This temperature is optimized for this product and should be stringently followed Figure 4 15 Applying Tough Spots to the array cartridge chapter 4 Affymetrix Genome Wide Human SNP Nsp Sty 5 0 Assay 48 Sample Protocol 127 Method 2 Using an Applied Biosystems 2720 MJ Tetrad PTC 225 or MJ Tetrad 2 Thermal Cycler For this method you can use an Applied Biosystems 2720 Thermal Cycler MJ Tetrad PTC 225 Thermal Cycler MJ Tetrad 2 The thermal cycler must be located adjacent to th
126. in cooling chambers on ice or keep at 4 C Label four fresh 96 well reaction plates P Gel P2Gel P3Gel and P4Gel Aliquot 3 uL of 2X Gel Loading Dye to each well in rows A through D of the fresh labeled P XGel plates Using a 12 channel P20 pipette transfer 3 uL of each PCR product from the 4 Nsp PCR plates to the corresponding plate row and wells of the PXGel plates Example 3 uL of each PCR product from each well of row A on plate P1 is transferred to the corresponding wells of row A on plate P1Gel Seal the PXGel plates Vortex the center of each PXGel plate then spin them down at 2000 rpm for 30 sec Load the total volume from each well of each PXGel plate onto 2 TBE gels 10 Run the gels at 120V for 40 minutes to 1 hour 11 Verify that the PCR product distribution is between 250 bp to 1100 bp see Figure 4 6 on page 81 chapter 4 Affymetrix Genome Wide Human SNP Nsp Sty 5 0 Assay 48 Sample Protocol 81 ome ome t4 6 4 Figure 4 6 Example of PCR products run on 2 TBE agarose gel at 120V for 1 hour Average product distribution is between 250 to 1100 bp What To Do Next Do one of the following If following the recommended workflow Figure 4 1 on page 23 do one of the following Ifthe Nsp PCR plates are still on the thermal cyclers remove them now and run gels to confirm the PCR Running Gels on page 80 Then proceed to Stage 7 PCR Product Pooling and Purification on page 82 If the
127. ingle channel P200 As needed Pipette tips As needed 2 per sample Reaction plates 96 well As needed Plate seals 1 Spectrophotometer plate reader 1 Vortexer IMPORTANT Use only the PCR plate adhesive film and thermal cyclers listed in Table 4 1 on page 27 chapter 4 Affymetrix Genome Wide Human SNP Nsp Sty 5 0 Assay 48 Sample Protocol 31 Reagents Required The following reagents are required for this stage Refer to Appendix A for vendor and part number information Table 4 5 Reagents Required for Genomic DNA Plate Preparation Quantity As needed Reduced EDTA TE Buffer 10 mM Tris HCL 0 1 mM EDTA pH 8 0 Preparing the Genomic DNA Plate This protocol has been optimized using UV absorbance to determine genomic DNA concentrations Other quantitation methods such as PicoGreen may give different readings Therefore you should correlate readings from other methods to the equivalent UV absorbance reading To prepare the genomic DNA plate 1 Thoroughly mix the genomic DNA by vortexing at high speed for 3 sec 2 Determine the concentration of each genomic DNA sample 3 Based on OD measurements dilute each sample to 50 ng uL using reduced EDTA TE buffer Apply the convention that 1 absorbance unit at 260 nm equals 50 ug mL for double stranded DNA This convention assumes a path length of 1 cm Consult your spectrophotometer handbook for more informati
128. inin pipette tip referred to in Figure 4 11 below Figure 4 10 Ridge on Rainin Pipette Tips gett i isla If using Rainin pipette tips rest the ridge of the pipette tip on the lip of the plate when pipetting Buffer EB This technique will help ensure that Buffer EB is dispensed as close to the beads as possible without touching them Figure 4 11 Adding Buffer EB to Reactions on the Filter Plate 7 Inspect each well to verify that the beads are thoroughly resuspended The dry down after adding EtOH makes the beads very dry The beads must be thoroughly resuspended in Buffer EB so that the DNA can come off the beads 8 Remove the plate stack from the Jitterbug and remove the adhesive seal 9 Continue elution on the vacuum manifold as follows A Remove the manifold cover and insert the plate stack B Seal the empty wells with adhesive film C Place the deep well collar over the plate stack Figure 4 12 on page 93 D Turn the vacuum on to 20 to 24 in Hg and ensure that a seal has been formed between the collar and the base of the manifold E Ensure that the unused wells are completely sealed chapter 4 Affymetrix Genome Wide Human SNP Nsp Sty 5 0 Assay 48 Sample Protocol 93 F Cover the plate stack with the lid from a pipette tip box to protect it from environmental contaminants G Leave the plate stack on the manifold 15 to 30 minutes Deep well collar installed over filter plate on vacuum mani
129. ion select the correct experiment name from the drop down Experiment list The Probe Array Type appears automatically 2 In the Protocol drop down list select GenomeWideSNP5v1_450 3 Choose Run in the Fluidics Station dialog box to begin washing and staining Follow the instructions in the LCD on the fluidics station If you are unfamiliar with inserting and removing arrays from the fluidics station modules refer to the appropriate Fluidics Station User s Guide or Quick Reference Card P N 08 0093 for the Fluidics Station 450 chapter 5 Washing Staining and Scanning Arrays 141 10 11 12 Insert an array into the designated module of the fluidics station while the cartridge lever is in the Down or Eject position When finished verify that the cartridge lever is returned to the Up or Engaged position Remove any microcentrifuge vials remaining in the sample holders of the fluidics station module s being used When prompted to Load Vials 1 2 3 place the three vials into the sample holders 1 2 and 3 on the fluidics station Place one vial containing 600 uL Streptavidin Phycoerythrin SAPE stain solution mix in sample holder 1 Place one vial containing 600 uL anti streptavidin biotinylated antibody stain solution in sample holder 2 Place one vial containing 1 mL Array Holding Buffer in sample holder 3 Press down on the needle lever to snap needles into position and to start the run Once these step
130. ion Master Mix can be made ahead of time aliquoted and stored for 1 week at 20 C To prepare stored Hybridization Master Mix 1 Place the stored Hybridization Master Mix on the bench top and allow to warm to room temperature 2 Vortex at high speed until the mixture is homogeneous and without precipitates up to 5 minutes 3 Pulse spin for 3 sec 124 Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Method 1 Using a GeneAmp PCR System 9700 The thermal cycler used for this method must be a GeneAmp PCR System 9700 located adjacent to the hybridization ovens This particular thermal cycler is required because of the way the lid operates You can slide it back one row at a time as samples are loaded onto arrays Keeping the remaining rows covered prevents condensation in the wells Add Hybridization Master Mix and Denature the Samples To add Hybridization Master Mix and denature the samples 1 2 Pour 11 mL Hybridization Master Mix into a solution basin Using a 12 channel P200 pipette add 190 uL of Hybridization Master Mix to each sample on the Label Plate Total volume in each well is 263 uL Seal the plate tightly with adhesive film Hi IMPORTANT It is critical to seal the plate tightly Vortex the center of the plate for 3 minutes Spin down the plate at 2000 rpm for 30 sec Cut the adhesive film between each row of samples Do not remove the film Place the plate onto the ther
131. ior to scanning by turning the scanner on at least 10 minutes before use If the arrays were stored at 4 C allow them to warm to room temperature before scanning NOTE Refer to the GeneChip Operating Software User s Guide P N 701439 online help and tutorials for more information on scanning WARNING The scanner uses a laser and is equipped with a safety interlock system Defeating the interlock system may result in exposure to hazardous laser light Read and be familiar with the operation of the scanner before attempting to scan an array Refer to the GeneChip Scanner 3000 Quick Reference Card P N 08 0075 Prepare arrays for Scanning Follow the instructions in this section to prepare the arrays for scanning To prepare arrays for scanning 1 If necessary clean the glass surface of the array with a non abrasive towel or tissue before scanning Do not use alcohol to clean the glass On the back of the array cartridge clean excess fluid from around the septa Carefully cover both septa with Tough Spots See Figure 5 2 on page 143 Press to ensure the spots remain flat If the spots do not apply smoothly e g if you see bumps bubbles tears or curled edges do not attempt to smooth out the spot Remove the spot and apply a new spot Insert an array into the scanner and test the autofocus to ensure the spots do not interfere with the focus If a focus error message is observed remove the spot an
132. k kk kK KK KK KK es 84 Important Information About This Stage RR RR eee 84 Prepare the 75 EtOH kk ee kK eee KIR KK ee ee 85 Prepare the Reagents kk kk kk KK KK KK KK KK KK KIR KRI RR RR KK KK 85 Prepare the Vacuum Manifold Wa KK KIRI ee 85 Pool the PCR Products 0 0 ee eee 85 Purify th Pooled PCR products soccer W la a e eS 88 Elute the Purified Reactions AWA KK KK KK RR RR RR RR 91 What Ta Do Next er te Ih t ah ent ete de t KK Ker 94 Stage 8 Quantitation ee 95 Abo tthliS Stdge sieer tue AME ENNERELEREd MES EE 95 Location and Duration kk aaas kK KK KK KIR KI KI KI eee eee 95 Input Required from Previous Stage anaa aaa RR RR eee 95 Equipment and Consumables Required lll 95 Reagents Required vk kk kk KK KK KK KK es 96 Important Information About This Stage 0 0 00 KK 97 Prepare the Reagents Equipment and Consumables 97 contents v Prepare Diluted Aliquots of Purified Sample 98 Quantitate the Diluted PCR Product AWK KK RR RR RR KRI 99 Assess the OD Readings 0 0 00 RR RR RY RR RY 100 OD Troubleshooting Guidelines 000 ee 100 Vra TODONG Eo Ae 102 Stage 9 Fragmentation AA KK auaa ee 103 About thiS Stage s cinah E eV Ridge allay ak ese 103 Locationand Duration cere tetea y k A Wa wine Be TAA 103 Input Required from Previous Stage 0 000 eee 103 Equipment and Consumables Required
133. lace the buffer in the cooling chamber on ice 4 Label and place the following in the cooling chamber on ice Two strips of 12 tubes each one labeled Buffer and one labeled FR One 1 5 mL Eppendorf tube labeled Frag MM Plate of purified PCR product from the previous stage Preheat the Thermal Cycler Block The block must be heated to 37 C before samples are loaded To preheat the thermal cycler 1 Power on the thermal cycler and preheat the block to 37 C 2 Allow it to heat for 10 minutes before loading samples 108 Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Prepare the Samples for Fragmentation Add Fragmentation Buffer to Samples Hi IMPORTANT All additions in this procedure must be performed on ice To prepare the samples for Fragmentation 1 Aliquot 28 uL of 10X Fragmentation Buffer to each tube of the strip tubes labeled Buffer 2 Using a 12 channel P20 pipette add 5 uL of Fragmentation Buffer to each sample in the 96 well reaction plate Check your pipette tips each time to ensure that all of the buffer has been dispensed The total volume in each well is now 50 uL Dilute the Fragmentation Reagent IMPORTANT The concentration of stock Fragmentation Reagent U uL may vary from lot to lot Therefore read the label on the tube and record the stock concentration before diluting this reagent To dilute the Fragmentation Reagent 1 Read the Fragmentation Reagent tube label a
134. ldup before each use Clean the manifold regularly Refer to Chapter 8 for cleaning instructions If the flask trap is not placed below the level of the manifold some solution may splash back and adhere to the bottom of the filter plate Pool the PCR Products PA CAUTION Be very careful when pooling PCR products Avoid cross contaminating neighboring wells with small droplets To pool the PCR Plates on PF WN 2 a Thaw frozen PCR products to room temperature on the bench top in plate holders If any plates are on thermal cyclers remove them now Vortex the center of each plate at high speed for 3 sec Spin down each plate at 2000 rpm for 30 sec Place each PCR plate in a plate holder on the bench top Place a deep well pooling plate on the bench top On each PCR plate cut the seal between each row so that it can be removed one row at a time Using a 12 channel P200 pipette set to 110 uL 86 Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual A Remove the seal to expose row A only on each PCR plate B Transfer the reactions from row A of each PCR plate to the corresponding wells of row A on the pooling plate Table 4 36 below and Figure 4 7 on page 87 C Change your pipette tips Change pipette tips after the PCR product from the same row of each PCR plate has been pooled on the pooling plate D Remove the seal from each PCR plate to expose the next row E Transfer each reaction from the
135. lications and a list of references Chapter 2 Laboratory Setup Describes the appropriate laboratory configuration for running Genome Wide SNP 5 0 Assay experiments Chapter 3 Genomic DNA Preparation Describes the requirements for genomic DNA with recommended sources and methods for purification and quantitation Chapter 4 Genome Wide SNP 5 0 Assay for 48 Samples Includes a detailed step by step protocol for processing 48 samples of human genomic DNA Chapter 5 Washing Staining and Scanning Includes instructions and protocols for fluidics station and scanner operation 2 Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Chapter 6 Data Analysis Describes how to analyze data using the BRLMM Analysis Tool 2 0 Chapter 7 Troubleshooting Provides additional guidelines for obtaining optimal assay results including troubleshooting tips Chapter 8 Instrument Maintenance Includes maintenance recommendations and procedures for the vacuum manifold and fluidics station Appendix A Reagents Equipment and Supplies Required for the Genome Wide SNP 5 0 Assay Includes vendor and part number information for the equipment and reagents required Appendix B Thermal Cycler Programs Required for the Genome Wide SNP 5 0 Assay Lists the thermal cycler programs required Appendix C E Gels Describes the use of e gels for the protocol About Whole Genome Sampling Analysis Long before the completion of the h
136. mal cycler and close the lid Run the GW5 0 Hyb program Table 4 55 GW5 0 Hyb Thermal Cycler Program GW5 0 Hyb Program Temperature 95 C 10 minutes 49 C Hold chapter 4 Affymetrix Genome Wide Human SNP Nsp Sty 5 0 Assay 48 Sample Protocol 125 Load the Samples onto Arrays This procedure requires 2 operators working simultaneously Operator loads the samples onto the arrays Operator 2 covers the septa with Tough Spots and loads the arrays into the hybridization ovens To load the samples onto arrays Operator 1 Tasks 1 se g N 9 When the plate reaches 49 C slide back the lid on the thermal cycler enough to expose the first row of samples only Remove the film from the first row Using a single channel P200 pipette remove 200 uL of denatured sample from the first well Immediately inject the sample into an array Pass the array to Operator 2 NOTE The tasks for Operator 2 are listed below Remove 200 uL of sample from the next well and immediately inject it into an array Pass the array to Operator 2 Repeat this process one sample at a time until the entire row is loaded Place a fresh strip of adhesive film over the completed row 10 Slide the thermal cycler lid back to expose the next row of samples 11 Repeat steps 3 through 10 until all of the samples have been loaded onto arrays Operator 2 Tasks 1 2 Cover the septa on each array with a Tough Spot F
137. man SNP Nsp Sty 5 0 Assay 48 Sample Protocol 129 Operators 1 and 2 Load no more than 32 arrays in one hybridization oven at a time All 48 samples should be loaded within 1 hour Store the remaining samples and any samples not yet hybridized in a tightly sealed plate at 20 C Allow the arrays to rotate at 50 C 60 rpm for 16 to 18 hours IMPORTANT Allow the arrays to rotate in the hybridization ovens for 16 to 18 hours at 50 C and 60 rpm This temperature is optimized for this product and should be stringently followed 130Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual E a LL 1 Chapter WASHING STAINING AND SCANNING ARRAYS This chapter contains instructions for using the Fluidics Station 450 to wash and stain arrays GeneChip Scanner 3000 7G to scan arrays After completing the procedures in this chapter the scanned array image dat file is ready for analysis Equipment and Consumables Required The following equipment and consumables are required for washing staining and scanning arrays Table 5 1 Equipment and Consumables Required for Washing and Staining Arrays Item Vendor Part Number GeneChip Scanner 3000 7G Affymetrix _ GeneChip Fluidics Station 450 Affymetrix _ GeneChip Operating Software Affymetrix _ Sterile RNase free microcentrifuge vials USA Scientific 1415 2600 or 1 5 mL equivalent Micropipettors P 2 P 20 P 200 P
138. matically in the output folder All of the results for an individual analysis run are stored in this folder a NOTE The default batch name includes the date and time therefore it is unique for each run 10 Optional In the QC Output Option field manually change the name for the QC 11 report The default name is qc report txt This file contains the QC Call rates for the samples analyzed It is located in the batch subfolder Click Next The Algorithm Options for BRLMM P window is displayed Figure 6 6 on page 153 chapter 6 Data Analysis 153 Set Up the Genotyping Analysis To set up the genotyping analysis 1 Select the analysis parameters you want to use for this run BRLMM Analysis Tool tj Algorithm Options for BRLMM P Balaa Parameters Score Threshold 0 05 QC Call Rate Threshold 86 Select the QC report path C Documents and Settings jburri My Documents D ata genotype data BAT Output 20061208_140238 qce r Output Options Output CHP Files AGCC Format C Output text files C Output tables C Output summaries file Figure 6 6 Algorithm Options for the OC and Genotyping Workflow A Enter a value in the Score Threshold field Score Threshold is the maximum score at which the algorithm will make a genotype call Scores lower than the threshold are of lower confidence and will result in No Call The default for BRLMM P is 0 05 B Optional For advanced parameters click Ad
139. metrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual 2 Inthe Work Flow box select QC and Genotyping 3 Inthe Array Type box open the drop down menu and select Genome WideSNP_5 4 Inthe Library Files box click Browse and navigate to the folder containing the library files Library files are typically located in this folder C Program Files Affymetrix GeneChip Affy_Data Library The path you select is saved from session to session As long as the library files are not moved you only need to select the path the first time the software is used If the library files are moved you will need to change the library path lt NOTE The selected directory must contain the CDF Library file the chromosome X file and the SNP models file 5 Click Next The Next button is disabled until entries are made for each required field After clicking Next the Import Options window is displayed BRLMM Analysis Tool Import Options Balaa Select the genotyping algorithm BRLMM P x Array Type GenomewideSNP_5 CEL Files 9 files minimum of 4 ommended EN Xu C Documents and Settings Desktop Vnv setlNNAOBS85 Hw 301106 Vnv D10 rl CEL C Documents and SettingssjiburriDesktopXVnv setlNADBS931 Hw 301106 Vny DOT rl CEL C Documents and SettingssiburiDesktopXVnv setlNNADBS33 Hw 301106 Vny BOT rl CEL C Documents and SettingssiburriDesktopXVnv setlNNAOB333 Hw 301106 Vny B11 rl CEL C Documents and Settingssiburr
140. n deep well 1 Flashlight 1 Jitterbug As needed Kimwipes 1 Pipette 12 channel P20 1 Pipette 12 channel P200 1 Pipette 12 channel P1200 1 Pipette serological As needed Pipette tips for pipettes listed above full racks 1 Plate 96 well PCR 1 Plate centrifuge with deep well capacity 54mm H x 160g 1 Plate storage 2 4 mL deep well referred to as the pooling plate 1 Plate elution catch 96 well V bottom Plate 2ml 96 well format filter plate PES 0 45 um 1 requires a deep well collar on the vacuum manifold listed above 7 Plate holders 3 Plate seal 1 Solution basin 55 mL or larger 1 roll Tape lab 1 Vacuum Manifold Millipore 1 Vortexer IMPORTANT Use only the PCR plate adhesive film and thermal cyclers listed in Table 4 1 on page 27 84 Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Reagents Required The following reagents are required for this stage Refer to Appendix A for vendor and part number information The amounts listed are sufficient to process 48 reactions Table 4 35 Reagents Required for Stage 7 PCR Product Pooling and Purification Quantity Required for Reagent 48 Samples 3 mL Elution Buffer Buffer EB 100 mL 75 EtOH ACS grade ethanol diluted to 75 using AccuGENE water 50 mL Magnetic Beads Important Information About This Stage To help ensure the best results carefully read the information below before you begin this stage of the proto
141. n the DNA binds to the magnetic beads To add magnetic beads and incubate 1 Mix the magnetic bead stock very well by vigorously shaking the bottle Beads will settle overnight Examine the bottom of the bottle and ensure that the solution appears homogenous 2 Pour or pipette 50 mL of magnetic beads to a solution basin 1 mL of magnetic beads is required for each reaction You can add in multiple batches if the solution basin is not large enough 3 Using a 12 channel P1200 pipette A Add 1 0 mL of magnetic beads to each well of pooled PCR product B Mix well by pipetting up and down 5 times using the following technique Mixing Technique 1 Depress the plunger and place the pipette tips into the top of the solution 2 Move the pipette tips down aspirating at the same time until the tips are near the bottom of each well 3 Raise the tips out of the solution 4 Place the pipette tips against the wall of each well just above each reaction and carefully dispense the solution IMPORTANT The solution is viscous and sticky Pipette carefully to ensure that you aspirate and dispense 1 mL Thorough mixing is critical to ensure that the PCR products bind to the beads 5 Change pipette tips for each row 4 Cover the plate to protect the samples from environmental contaminants and allow to incubate at room temperature for 10 minutes You can use the lid from a pipette tip box to cover the wells Transfer
142. n page 27 Table 4 20 Equipment and Consumables Required for Stage 4 Nsp Restriction Enzyme Digestion Quantity Item 1 Cooler chilled to 20 C 1 Cooling chamber double chilled to 4 C do not freeze 1 Ice bucket filled with ice 1 Marker fine point permanent 1 Microcentrifuge 1 Pipette single channel P100 1 Pipette single channel P200 1 Pipette single channel P1000 1 Pipette 12 channel P20 As needed Pipette tips for pipettes listed above full racks 1 Plate centrifuge 1 Plate seal 1 Thermal cycler 1 strip Tubes 12 strip 0 2 mL 1 Tube Eppendorf 2 0 mL 1 Vortexer 60 Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Reagents Required The following reagents are required for this stage Refer to Appendix A for vendor and part number information The amounts listed are sufficient for processing 48 samples Table 4 21 Reagents Required for Stage 4 Nsp Restriction Enzyme Digestion Quantity Reagent 1 vial BSA 100X 10 mg mL 1 vial NE Buffer 2 10X 1 vial Nsp I 10 U uL NEB 2 5 mL AccuGENE Water molecular biology grade Important Information About This Stage To help ensure the best results carefully read the information below before you begin this stage of the protocol IMPORTANT The same team or individual operator should not perform Nsp 1 and Sty 1 digestion reactions on the same day About Using Control
143. nd record the concentration 2 Dilute the Fragmentation Reagent to 0 1 U uL as described below using the appropriate recipe from Table 4 46 Table 4 46 Dilution Recipes for the Fragmentation Reagent Reagent Fragmentation Reagent Concentration 2 25U pL 2 5 U jiL 2 75 U L AccuGENE water 306 uL 308 uL 309 6 uL 310 9 uL 312 uL 10X Fragmentation Buffer 36 uL 36 uL 36 uL 36 uL 36 uL Fragmentation Reagent 18 uL 16 uL 14 4 uL 13 1 uL 12 uL Total 360 pL 360 uL 360 uL 360 uL 360 uL enough for 48 samples chapter 4 Affymetrix Genome Wide Human SNP Nsp Sty 5 0 Assay 48 Sample Protocol 109 C D E To the 1 5 mL Eppendorf tube on ice 1 Add the AccuGENE water and Fragmentation Buffer 2 Allow to cool on ice for 5 minutes Remove the Fragmentation Reagent from the freezer and 1 Immediately pulse spin for 3 sec Spinning is required because the Fragmentation Reagent tends to cling to the top of the tube making it warm quicker 2 Immediately place in a cooler Add the Fragmentation Reagent to the 1 5 mL Eppendorf tube Vortex the diluted Fragmentation Reagent at high speed 3 times 1 sec each time Pulse spin for 3 sec and immediately place on ice 3 Proceed immediately to the next set of steps Add Diluted Fragmentation Reagent to the Samples Add Diluted Fragmentation Reagent to the Samples To add diluted Fragmentation Reagent to the samples 1 Quickly and on ice aliquot 28 uL of diluted F
144. nes novel stage and location dependent allelic imbalances in human bladder tumors Cancer Res 2005 65 34 45 Raghavan M Lillington DM Skoulakis S Debernardi S Chaplin T Foot NJ Lister TA Young BD Genome wide single nucleotide polymorphism analysis reveals frequent partial uniparental disomy due to somatic recombination in acute myeloid leukemias Cancer Res 2005 65 375 8 Irving JA Bloodworth L Bown NP Case MC Hogarth LA Hall AG Loss of heterozygosity in childhood acute lymphoblastic leukemia detected by genome wide microarray single nucleotide polymorphism analysis Cancer Res 2005 65 3053 8 Slater HR Bailey DK Ren H Cao M Bell K Nasioulas S Henke R Choo KH Kennedy GC High Resolution Identification of Chromosomal Abnormalities Using Oligonucleotide Arrays Containing 116 204 SNPs 4m J Hum Genet 2005 77 709 26 chapter 1 Overview 11 74 75 76 77 78 79 80 81 82 83 84 Liang D Wu L Pan Q Harada N Long Z Xia K Yoshiura K Dai H Niikawa N Cai F et al A father and son with mental retardation a characteristic face inv 12 and insertion trisomy 12p12 3 p11 2 4m J Med Genet A 2006 140 238 44 Bruce S Leinonen R Lindgren CM Kivinen K Dahlman Wright K Lipsanen Nyman M Hannula Jouppi K Kere J Global analysis of uniparental disomy using high density genotyping arrays J Med Genet 2005 42 847 51 Teh MT Blay
145. ng for troubleshooting tips chapter 6 Data Analysis 167 Date 12 18 2006 10 53 33 AM Report File Name C Documents and Settings iburri My Documents Data genotype data BAT Output 20061218_102823 qc report txt Batch 20061218_102823 Probe Array Type GenomeWideSNP_5 Analysis method1 analysis_name QC call rate all analysis dm group_name all_qc as percentage 1 dm cutoff 0 33 dm hetMult 1 25 precision 4 QC call rate QC call rate QC call rate QC call rate Chip Gender Nsp e Sty fall NAO6985_HW_301106_Vn _D10_r1 CEL female 93 83 96 49 89 21 94 31 NA06991_H W_301106_Vni _DO1_11 CEL female 93 57 95 5 88 89 93 65 N 06333 Hw 301106 Vnv BOT rl CEL male 91 52 95 07 89 05 92 92 NA 6333 Hw 301106 Vnv B11 rl CEL male 97 94 98 34 95 01 97 55 N 06333 Hw 301106 Vnv C11 rt CEL 35 37 36 61 30 38 35 14 Figure 6 19 Example of a OC Call Rate Report Genomic DNA Quality Genomic DNA should be prepared following the guidelines in Chapter 3 of this manual DNA prepared outside of these guidelines e g degraded DNA nicked DNA or DNA with inhibitors may produce lower Call Rates without necessarily reducing accuracy A gel image of the DNA before restriction digestion should be used to evaluate DNA quality Direct comparison to the Reference Genomic DNA 103 control is one way to accomplish this If an alternate genomic DNA preparation method is used we highly recommended that a small pilot experiment be conducted to evalua
146. nome Wide Human SNP 5 0 Nsp Sty Assay Manual 42 45 46 47 48 49 50 5 __E 52 Mitra N Ye TZ Smith A Chuai S Kirchhoff T Peterlongo P Nafa K Phillips MS Offit K Ellis NA Localization of cancer susceptibility genes by genome wide single nucleotide polymorphism linkage disequilibrium mapping Cancer Res 2004 64 8116 25 Butcher LM Meaburn E Knight J Sham PC Schalkwyk LC Craig IW Plomin R SNPs microarrays and pooled DNA identification of four loci associated with mild mental impairment in a sample of 6000 children um Mol Genet 2005 14 1315 25 Kulle B Schirmer M Toliat MR Suk A Becker C Tzvetkov MV Brockmoller J Bickeboller H Hasenfuss G Nurnberg P et al Application of genomewide SNP arrays for detection of simulated susceptibility loci Hum Mutat 2005 25 557 65 Godde R Rohde K Becker C Toliat MR Entz P Suk A Muller N Sindern E Haupts M Schimrigk S et al Association of the HLA region with multiple sclerosis as confirmed by a genome screen using 710 000 SNPs on DNA arrays J Mol Med 2005 83 486 94 Horvath A Boikos S Giatzakis C Robinson White A Groussin L Griffin KJ Stein E Levine E Delimpasi G Hsiao HP et al A genome wide scan identifies mutations in the gene encoding phosphodiesterase 11A4 PDE11A in individuals with adrenocortical hyperplasia Nat Genet 2006 38 794 800 Matsuzaki H Dong S Loi
147. ome Wide Human SNP 5 0 Nsp Sty Assay Manual Chapter g GENOMIC DNA GENERAL REQUIREMENTS This chapter describes the general requirements for genomic DNA sources and extraction methods The success of this assay requires the amplification of PCR fragments between 200 and 1100 bp in size throughout the genome To achieve this the genomic DNA must be of high quality and must be free of contaminants that would affect the enzymatic reactions carried out A genomic DNA control Reference Genomic DNA 103 is provided in the Genome Wide Human SNP Nsp Sty Assay Kit 5 0 This control DNA meets the requirements outlined below The size of the starting genomic DNA can be compared with Ref103 DNA to assess the quality The control DNA should also be used as a routine experimental positive control and for troubleshooting Assay performance may vary for genomic DNA samples that do not meet the general requirements described below However the reliability of any given result should be assessed in the context of overall experimental design and goals General Requirements DNA must be double stranded not single stranded This requirement relates to the restriction enzyme digestion step in the protocol DNA must be free of PCR inhibitors Examples of inhibitors include high concentrations of heme from blood and high concentrations of chelating agents 1 e EDTA The genomic DNA extraction purification method should render DNA that i
148. ome Wide Human SNP 5 0 Nsp Sty Assay Manual Stain Buffer Mix well Table 5 3 Stain Buffer Components 1X Final Concentration H O 800 04 uL SSPE 20X 360 uL 6X Tween 20 3 3 96 uL 0 01 Denhardt s 50X 24 uL 1X Subtotal 1188 uL Subtotal 2 594 uL SAPE Stain Solution Streptavidin Phycoerythrin SAPE should be stored in the dark at 4 C either foil wrapped or in an amber tube Remove SAPE from refrigerator and tap the tube to mix well before preparing stain solution Always prepare the SAPE stain solution immediately before use Mix well Do not freeze either concentrated SAPE or diluted SAPE stain solution Table 5 4 SAPE Solution Mix Components Volume Final Concentration Stain Buffer 594 uL 1X 1 mg mL Streptavidin Phycoerythrin SAPE 6 0 uL 10 ug mL Total 600 uL x NOTE A vial containing SAPE Stain Solution must be placed in sample holder 1 for each module used chapter 5 Washing Staining and Scanning Arrays 139 Antibody Stain Solution Mix well Table 5 5 Antibody Solution Mix Components Volume Final Concentration Stain Buffer 594 uL 1X 0 5 mg mL biotinylated antibody 6 uL 5 ug mL Total 600 uL x NOTE A vial containing Antibody Stain Solution must be placed in sample holder 2 for each module used Array Holding Buffer Mix well Table 5 6 Array Holding Buffer Components Volume MES Stock Buffer 12X 8 3 mL 5
149. ommendations 15 PCR Staging Room The PCR Staging Room is a low copy template lab which should be free from any PCR product amplicons It is the area where non amplified template genomic DNA should be handled The digestion and ligation reactions should be conducted in this area The PCR reactions should be prepared in this area The use of gowns booties and gloves is recommended to prevent PCR carryover Main Lab The Main Lab has airborne contamination with PCR product and template After entering the main lab it is inadvisable to re enter the Pre PCR Clean Area or the PCR Staging Room without first showering and changing into freshly laundered clothes Safety Precautions The Genome Wide Human SNP Nsp Sty Assay Kit 5 0 as well as the Genome Wide Human SNP Array 5 0 are for research use only All blood and other potentially infectious materials should be handled as if capable of transmitting infection and disposed of with proper precautions in accordance with federal state and local regulations lt NOTE Some components required for this assay may pose significant health risks Follow prudent laboratory practices when handling and disposing of carcinogens and toxins Refer to the manufacturer s Material Safety Data Sheet for additional information Wear appropriate personal protective equipment when performing this assay At a minimum safety glasses and chemical resistant gloves should be worn 16 Affymetrix Gen
150. on If using a quantitation method other than UV absorbance correlate the reading to the equivalent UV absorbance reading 4 Thoroughly mix the diluted DNA by vortexing at high speed for 3 sec IMPORTANT An elevated EDTA level may interfere with subsequent reactions 32 Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Aliquoting Prepared Genomic DNA To aliquot the prepared genomic DNA 1 Vortex the plate of genomic DNA at high speed for 10 sec then spin down at 2000 rpm for 30 sec 2 Aliquot 5 uL of each DNA to the corresponding wells of two 96 well reaction plates 5 uL of the 50 ng uL working stock is equivalent to 250 ng genomic DNA per well Two replicates of each sample are required for this protocol one for Nsp and one for processing Sty 3 Sealeach plate with adhesive film What To Do Next Do one of the following Proceed to the next stage processing one plate of samples one enzyme at a time Store the sealed plates of diluted genomic DNA at 20 C chapter 4 Affymetrix Genome Wide Human SNP Nsp Sty 5 0 Assay 48 Sample Protocol 33 Stage 1 Sty Restriction Enzyme Digestion About this Stage During this stage the genomic DNA is digested by the Sty I restriction enzyme You will 1 Prepare a Sty Digestion Master Mix 2 Add the master mix to one set of 48 samples 3 Place the samples onto a thermal cycler and run the GW5 0 Digest program Location and Duration
151. on Setup The following instructions are for GeneChip Operating Software GCOS 1 4 client 1 3 server Register a New Experiment in GCOS To register a new experiment 1 From the File menu click New Experiment The New Experiment window appears in the display pane The top half of the display pane refers to the sample and the bottom half refers to the experiment 19 Software Exp 8 ris tA Wm R n Tooke Wedew Heb eusimmmm Bee esi E1 ZC gal No tergiste 2 wr E esti ctt Hai 9 en Un pu ti SR tg i C deeper perat Ban 4 lic 12 02 03 02 04 16 Initiating the scanner wy 12 02 02 02 04 20 GeneChe Operating Software inisakzation complete Figure 5 1 GCOS Sample Entry Pane 2 Enter information into the appropriate boxes Fields that are highlighted in bold require an entry 136 Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Drop down menus are available for Sample Project information default information can be used or new information can be entered The Experiment Name must be unique Appropriate library files must be installed for a array to appear in the drop down menu 3 From the File menu click Save As or click the Save icon on the tool bar to register the experiment into the database TIP The Sample Information fields can be customized See the GeneChip Operating Software User s Guide for further information Prepare the
152. ooling chamber One strip of 12 tubes labeled Lig A 2 0 mL Eppendorf tube labeled Lig MM Solution basin 3 Prepare the digested samples as follows A Vortex the center of the plate at high speed for 3 sec B Spin down the plate at 2000 rpm for 30 sec C Place back in the cooling chamber on ice 4 To prepare the reagents A Vortex at high speed 3 times 1 sec each time except for the enzyme B Pulse spin for 3 sec C Place in the cooling chamber IMPORTANT T4 DNA Ligase Buffer 10X contains ATP and should be thawed on ice Vortex the buffer as long as necessary before use to ensure precipitate is re suspended and that the buffer is clear Avoid multiple freeze thaw cycles per vendor instructions Preheat the Thermal Cycler Lid Power on the thermal cycler to preheat the lid Leave the block at room temperature The lid must be preheated before samples are loaded 68 Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Prepare the Nsp Ligation Master Mix Keeping all reagents and tubes on ice prepare the Nsp Ligation Master Mix as follows 1 g NOT Ww To the 2 0 mL Eppendorf tube add the following reagents based on the volumes shown in Table 4 26 Adaptor Nsp T4 DNA Ligase Buffer 10X Remove the T4 DNA Ligase from the freezer and immediately place in the cooler on ice Pulse spin the T4 DNA Ligase for 3 sec Immediately add the T4 DNA Ligase to the master mix then place back in the
153. otocol 49 Reagents Required The following reagents are required for this stage Refer to Appendix A for vendor and part number information The amounts listed are sufficient to process 48 samples Table 4 15 Reagents Required for Stage 3 Sty PCR Quantity Reagent 15 mL AccuGENE water molecular biology grade 1 vial PCR Primer 002 100 uM The following reagents from the Clontech TITANIUM DNA Amplification Kit dNTPs 2 5 mM each e GC Melt 5M TITANIUM Taq DNA Polymerase 50X e TITANIUM Taq PCR Buffer 10X Gels and Related Materials Required Verifying the PCR reaction is required for this stage You can use the following gels and related materials or E Gels as described in Appendix C E gels on page 211 The amounts listed are sufficient to process 48 Sty samples Refer to Appendix A for vendor and part number information Table 4 16 Gels and Related Materials Required for Stage 3 Sty PCR Quantity Reagent 190 uL DNA Marker 19 Gels 2 TBE As needed Gel loading solution 3 Plates 96 well reaction 50 Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Important Information About This Stage To help ensure the best results carefully read the information below before you begin this stage of the protocol IMPORTANT e Make sure the Sty ligated DNA was diluted to 100 uL with AccuGENE water Set up the PCRs in PCR Staging Area
154. otyping accuracy QC call rate is determined by the DM algorithm on a carefully selected set of 3022 SNPs tiled with both perfect matches and mismatches This set of SNPs is also enriched for lower performing SNPs to serve as a more effective predictor of performance in a clustering analysis We have chosen a QC call rate of 86 as the default This default is expected to result in reasonable call rates and accuracy Samples right at the 86 QC call rate threshold are expected to have a call rate around 97 with BRLMM P analysis with an average accuracy of 99 A strong correlation exists between the QC call rate and BRLMM P performance The more a sample exceed the 86 threshold the better the performance For BRLMM P analysis we have chosen a confidence score value of 0 05 as the default This default provides a good compromise between accuracy and call rate This value is adjustable in Affymetrix genotyping software to give flexibility to increase call rates with lower genotyping accuracy or to decrease call rates with greater genotyping accuracy 172Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Chapter p TROUBLESHOOTING Assay Recommendations Genotyping applications require very high accuracy to achieve maximum power Therefore great care should be taken to avoid possible sources of cross contamination that would lead to genotyping errors As with any assay using PCR the GeneChip Mapping Assay ha
155. p 10 to 15 minutes Insert a 200 uL pipette tip into the upper right septum of each array IMPORTANT To ensure that the data collected during scanning is associated with the correct sample number the arrays in a meaningful way lt is critical that you know which sample is loaded onto each array Prepare the Hybridization Master Mix As an option you can prepare a larger volume of Hybridization Master Mix than required The extra mix can be aliquoted and stored at 20 C for up to one week Preparing Fresh Hybridization Master Mix To prepare the Hybridization Master Mix 1 To the 50 mL centrifuge tube add the reagents in the order shown in Table 4 54 DMSO addition pipette directly into the solution of other reagents Avoid pipetting along the side of the tube chapter 4 Affymetrix Genome Wide Human SNP Nsp Sty 5 0 Assay 48 Sample Protocol 123 2 Mix well 3 If making a larger volume aliquot out 11 mL and store the remainder at 20 C for up to one week Table 4 54 Hybridization Master Mix Reagent 1 Array 48 Arrays 15 extra MES 12X 1 25 M 12 uL 660 uL Denhardt s Solution 50X 13 uL 715 uL EDTA 0 5 M 3 uL 165 uL HSDNA 10 mg mL 3 uL 165 uL OCR 0100 2 uL 110 uL Human Cot 1 DNA 1 mg mL 3 uL 165 uL Tween 20 3 1 uL 55 uL DMSO 100 13 uL 715 uL TMACL 5 M 140 uL 7 7 mL Total 190 uL 10 45 mL Using Premixed Hybridization Master Mix Hybridizat
156. p Sty Assay Manual Other Equipment Required Table A 5 Other Equipment Required to Run the Genome Wide Human SNP 5 0 Nsp Sty Assay Equipment Quantity Manufacturer Part Number Laboratory Distributor Location Collar Multiscreen Deep well for 1 Millipore MSVMHTSOD Main Lab vacuum manifold StrataCooler Lite 400012 Pre PCR and Benchtop Main Lab Cooler 2 Stratagene 20 C StrataCooler II 400002 blue Benchtop 400008 red Cooling chamber single gold block 3 double Diversified CHAM 1000 2 double and 1 1 single Biotech single single in PCR Staging Area double gold block CHAM 1020 1 double in double Main Lab Ice bucket 2 Pre PCR and 4 to 9 liters Main Lab In the U S A 11 701 13 Fisher Scientific Jitterbug 1 Main Lab Microplate Incubator In the U S A 35821 065 Shaker VWR Inthe U S A and 130000 115V all other 130000 2 230V countries Boekel Scientific Vacuum Manifold MultiScreenurs 1 Millipore MSVMHTS0O Main Lab Microcentrifuge PicoFuge 2 Stratagene 400550 Pre PCR and maximum rotation 6000 rpm Main Lab Pipet Lite Magnetic Assist single channel 2 Rainin L 20 Pre PCR and P20 Main Lab Pipet Lite Magnetic Assist single channel 2 Rainin L 200 Pre PCR and P200 Main Lab Pipet Lite Magnetic Assist single channel 2 Rainin L 1000 Main Lab P1000 Pipette 12 channel P20 2 Rainin P N L12 20 Pre PCR and accurate to within 5
157. p Sty Assay Manual Output File Formats qc report txt The qc report txt file contains the QC call rate for all CEL files analyzed P qc report txt Notepad File Edit Format View Help format_version 1 content_version 1 default_analysis_name QC call rate all library file C Documents and Settings jburri My Documents Data genotype LibraryFiles GenomewideSNP_5 cdf gender information male 0 female 1 unknown 1 method1 analysis_name QC call rate all analysis dm group_name al1_qc as percentage 1 dm cutoff 0 33 dm hetMult 1 method2 analysis_name QC call rate Nsp analysis dm group name nsp qc as percentage 1 dm cutoff 0 33 dm hetMult 1 method3 analysis_name QC call rate Sty analysis dm group name sty qc as percentage 1 dm cutoff 0 33 dm hetMult 1 method4 analysis_name QC call rate Nsp Sty overlap analysis dm group name nsp sty qc as percentage 1 dm cutoff 0 analysis name Gender analysis gender group name chrX as text 1 em cutoff 0 5 em thresh 0 05 gender cutoff 0 Gender QC call rate Nsp QC call rate Nsp Sty overlap QC call rate Sty QC call rate all female 93 83 96 49 89 21 94 31 female 93 57 88 89 93 65 male 91 52 3 92 male 97 94 a a 59 male 95 37 i d 14 Figure 6 11 OC Call Rate Report Clustering Report File This file is a summary report created in the output directory with the extension report txt It provides a quick overview of the CEL file
158. pL of purified PCR product see page 177 of Chapter 7 Troubleshooting for instructions What To Do Next Take an OD measurement using the remaining eluate as described below Do one of the following If following the recommended workflow Figure 4 1 on page 23 seal the plate containing the eluate and store it overnight at 20 C Proceed directly to Stage 9 Fragmentation on page 103 chapter 4 Affymetrix Genome Wide Human SNP Nsp Sty 5 0 Assay 48 Sample Protocol 95 Stage 8 Quantitation About this Stage During this stage you will prepare one dilution of each PCR product in optical plates You will then quantitate the diluted PCR products Location and Duration Main Lab Hands on time 20 minutes Input Required from Previous Stage Input required from Stage 7 PCR Product Pooling and Purification is Quantity Item 1 Plate of purified PCR product Equipment and Consumables Required The following equipment and consumables are required for this stage Refer to Appendix A for vendor and part number information IMPORTANT Use only the PCR plate adhesive film and thermal cyclers listed in Table 4 1 on page 27 96 Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Table 4 37 Equipment and Consumables Required for Stage 8 Quantitation Quantity Item 1 Marker fine point permanent 1 Pipette single channel P20 1 Pipette single channel P200 1 Pipe
159. pare the Nsp PCR Master Mix To prepare the Nsp PCR Master Mix IMPORTANT The PCR reaction is sensitive to the concentration of primer used It is critical that the correct amount of primer be added to the Nsp PCR Master Mix to achieve the correct distribution of fragments 200 to 1100 bp in the products Check the PCR reactions on a gel to ensure that the distribution is correct 1 Keeping the 50 mL Falcon tube in the cooling chamber add the reagents as shown in Table 4 31 on page 77 except for the Tag DNA polymerase 2 Remove the TITANIUM 7aq DNA Polymerase from the freezer and immediately place in a cooler 3 Pulse spin the Taq DNA polymerase for 3 sec 4 Immediately add the Taq DNA polymerase to the master mix then return the tube to the cooler on ice 5 Vortex the master mix at high speed 3 times sec each time 6 Pour the mix into the solution basin keeping the basin on ice chapter 4 Affymetrix Genome Wide Human SNP Nsp Sty 5 0 Assay 48 Sample Protocol 77 Table 4 31 Nsp PCR Master Mix for 48 Samples Reagent For 1 Reaction 4 PCR Plates 15 extra AccuGENE water 39 5 uL 8 722 mL TITANIUM Taq PCR Buffer 10X 10 uL 2 208 mL GC Melt 5M 20 uL 4 416 mL dNTP 2 5 mM each 14 uL 3 091 mL PCR Primer 002 100 uM 4 5 uL 0 994 mL TITANIUM Taq DNA Polymerase 50X 2 uL 0 442 mL do not add until ready to aliquot master mix to ligated samples Total 90 uL 19 873 mL Add Nsp
160. pha 12 connexin 46 6 cause Pelizaeus Merzbacher like disease 4m J Hum Genet 2004 75 251 60 chapter 1 Overview 7 31 32 33 34 35 36 37 38 39 40 41 Janecke AR Thompson DA Utermann G Becker C Hubner CA Schmid E McHenry CL Nair AR Ruschendorf F Heckenlively J et al Mutations in RDH12 encoding a photoreceptor cell retinol dehydrogenase cause childhood onset severe retinal dystrophy Nat Genet 2004 36 850 4 Hao K Li C Rosenow C Hung Wong W Estimation of genotype error rate using samples with pedigree information an application on the GeneChip Mapping 10K array Genomics 2004 84 623 30 Weber S Mir S Schlingmann KP Nurnberg G Becker C Kara PE Ozkayin N Konrad M Nurnberg P Schaefer F Gene locus ambiguity in posterior urethral valves prune belly syndrome Pediatr Nephrol 2005 20 1036 1042 Metherell LA Chapple JP Cooray S David A Becker C Ruschendorf F Naville D Begeot M Khoo B Nurnberg P et al Mutations in MRAP encoding a new interacting partner of the ACTH receptor cause familial glucocorticoid deficiency type 2 Nat Genet 2005 37 166 70 Morgan NV Pasha S Johnson CA Ainsworth JR Eady RA Dawood B McKeown C Trembath RC Wilde J Watson SP et al A germline mutation in BLOCIS3 reduced pigmentation causes a novel variant of Hermansky Pudlak syndrome HPS8 4m J Hum Genet 2006 78 160 6 Sayer JA Otto
161. proved CNV detection In summary the Genome Wide Human SNP Array 5 0 leverages a DNA target prep that is successfully used in the GeneChip Mapping 500K array set such that nearly 500 000 SNPs are genotyped on a single array The array also contains non polymorphic probes for improved detection of copy number variants present in the genome The Genome Wide Human SNP Array 5 0 thus provides a robust flexible cost effective approach for scoring SNP genotypes in large numbers of samples and will provide a new technological paradigm for the design of whole genome SNP based association studies References 1 Botstein D White RL Skolnick M Davis RW Construction of a genetic linkage map in man using restriction fragment length polymorphisms 4m J Hum Genet 1980 32 314 31 2 Lander ES Linton LM Birren B Nusbaum C Zody MC Baldwin J Devon K Dewar K Doyle M FitzHugh W et al Initial sequencing and analysis of the human genome Nature 2001 409 860 921 3 Venter JC Adams MD Myers EW Li PW Mural RJ Sutton GG Smith HO Yandell M Evans CA Holt RA et al The sequence of the human genome Science 2001 291 1304 51 4 Botstein D Risch N Discovering genotypes underlying human phenotypes past successes for mendelian disease future approaches for complex disease Nat Genet 2003 33 Suppl 228 37 5 Carlson CS Eberle MA Kruglyak L Nickerson DA Mapping complex disease loci in whole genome asso
162. r and immediately place in a cooler e Pulse spin the enzyme for 3 sec 2 Immediately add the enzyme to the master mix then place remaining enzyme back in the cooler Vortex the master mix at high speed 3 times 1 sec each time Pulse spin for 3 sec Place in the cooling chamber Return any remaining enzyme to the freezer e g SN 9 g Proceed immediately to Add Sty Digestion Master Mix to Samples on page 38 38 Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Table 4 8 Sty Digestion Master Mix Reagent 1 Sample 48 Samples 15 extra AccuGENE Water 11 55 uL 637 6 uL NE Buffer 3 10X 2 uL 110 4 uL BSA 100X 10 mg mL 0 2 uL 11 uL Sty I 10 U pL 1 uL 55 2 uL Total 14 75 uL 814 2 pL Add Sty Digestion Master Mix to Samples To add the Sty Digestion Master Mix to samples 1 Noo PR Ww Using a single channel P200 pipette aliquot 67 uL of Sty Digestion Master Mix to each tube of the strip tubes labeled Dig Using a 12 channel P20 pipette add 14 75 uL of Sty Digestion Master Mix to each DNA sample in the cooling chamber on ice The total volume in each well is now 19 75 uL Genomic DNA 50 ng L 5 uL Digestion Master Mix 14 75 uL Total Volume 19 75 uL Seal the plate tightly with adhesive film Vortex the center of the plate at high speed for 3 sec Spin down the plate at 2000 rpm for 30 sec Ensure that the lid of thermal cycler is p
163. ragmentation Reagent to each tube of the strip tubes labeled FR Avoid introducing air bubbles at the bottom of the strip tubes to ensure the accurate transfer of 5 uL diluted DNA to each sample 2 Using a 12 channel P20 pipette add 5 uL of diluted Fragmentation Reagent to each sample Do not pipette up and down Sample with Fragmentation Buffer 50 uL Diluted Fragmentation Reagent 0 1 U uL 5 uL Total 55 uL 3 Seal the plate and inspect the edges to ensure that it is tightly sealed IMPORTANT To minimize solution loss due to evaporation make sure that the plate is tightly sealed prior to loading onto the thermal cycler The MJ thermal cyclers are more prone to evaporation 4 Vortex the center of the plate at high speed for 3 sec 110Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual 5 Place the plate in a chilled plastic plate holder and spin it down at 4 C at 2000 rpm for 30 sec 6 Immediately load the plate onto the pre heated block of the thermal cycler 37 C and run the GW5 0 Fragment program Table 4 47 GW5 0 Fragment Thermal Cycler Program GW5 0 Fragment Program Temperature Time 37 C 35 minutes 95 C 15 minutes 4 C Hold 7 Discard any remaining diluted Fragmentation Reagent Diluted Fragmentation Reagent should never be reused What To Do Next Proceed directly to the next stage Concurrently check the fragmentation reaction by running gel
164. reheated Load the plate onto the thermal cycler and run the GW5 0 Digest program chapter 4 Affymetrix Genome Wide Human SNP Nsp Sty 5 0 Assay 48 Sample Protocol 39 Table 4 9 GW5 0 Digest Program GW5 0 Digest Program Temperature Time 37 C 120 minutes 65 C 20 minutes 4 C Hold 8 When the program is finished remove the plate and spin it down at 2000 rpm for 30 sec What To Do Next Do one of the following If following the recommended workflow Figure 4 1 on page 23 place the plate in a cooling chamber on ice and proceed immediately to Stage 2 Sty Ligation on page 40 If not proceeding directly to the next step store the samples at 20 C 40 Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Stage 2 Sty Ligation About this Stage During this stage the digested samples are ligated using the Sty Adaptor You will 1 2 3 4 Prepare a Sty Ligation Master Mix Add the master mix to the samples Place the samples onto a thermal cycler and the GW5 0 Ligate program is run Dilute the ligated samples with AccuGENE water Location and Duration Pre PCR Clean Area Hands on time 30 minutes e GWS5 0 Ligate thermal cycler program time 3 3 hours Input Required From Previous Stage The input required from Stage 1 Sty Restriction Enzyme Digestion is Quantity Item 48 samples Sty digested samples in a cooling chamber on ice chapter 4 Affymetrix
165. rium LD in which a disease predisposing allele co segregates with a particular allele of a SNP have been hampered by the lack of whole genome genotyping methodologies 10 As new genotyping technologies develop coupled with ongoing studies into LD patterns and haplotype block structure across the genome improvements in the design and power of association studies will be feasible 11 18 We have developed an assay termed whole genome sampling analysis WGSA for highly multiplexed SNP genotyping of complex DNA 19 20 This method reproducibly amplifies a subset of the human genome through a single primer amplification reaction using restriction enzyme digested adapter ligated human genomic DNA This assay was first developed for simultaneous genotyping of over 10 000 SNPs on a single array GeneChip Human Mapping 10K Array Xba 142 2 0 and has been used to date for both linkage studies 21 40 and association studies 41 46 The WGSA assay was extended to allow highly accurate SNP genotyping of over 100 000 SNPs using the two array GeneChip Mapping 100K Set 47 These arrays have also been used for genome wide LD studies 48 as well as landmark whole genome association studies in age related macular degeneration multiple sclerosis and cardiac repolarization 49 51 The WGSA assay was again extended in 2005 with the fourth generation product known as the GeneChip Mapping 500K Assay in which 500 000 SNPs are queried using a two array
166. rmation IMPORTANT Increased variability in Genome Wide SNP 5 0 Assay performance has been observed in GeneChip Hybridization Oven 640 models P N 800138 or 800189 manufactured prior to 2001 Check the serial number of your hybridization oven s If the serial numbers are 11214 or lower contact Affymetrix for an upgrade The following table lists the equipment and consumables required Table 4 52 Equipment and Consumables Required for Stage 11 Target Hybridization Quantity Item 1 Cooling chamber chilled to 4 C do not freeze 48 Genome Wide Human SNP Array 5 0 one array per sample 1 GeneChip Hybridization Oven 640 1 Ice bucket filled with ice 1 Pipette single channel P200 1 Pipette single channel P1000 As needed Pipette tips for pipettes listed above full racks 1 Plate Bio Rad 96 well P N MLP 9601 1 Plate centrifuge 2 Plate holders centrifuge 1 Plate seal 1 Solution basin 55 mL 1 Thermal cycler See About this Stage on page 117 2 per array Tough Spots 1 Tube centrifuge 50 mL 1 Vortexer chapter 4 Affymetrix Genome Wide Human SNP Nsp Sty 5 0 Assay 48 Sample Protocol 119 IMPORTANT Use only the PCR plate adhesive film and thermal cyclers listed in Table 4 1 on page 27 Reagents Required The following reagents are required for this stage Refer to Appendix A for vendor and part number information The amounts listed are sufficient
167. rminal Deoxynucleotidyl Transferase Buffer TdT Buffer 5X 114Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Important Information About This Stage To help ensure the best results carefully read the information below before you begin this stage of the protocol IMPORTANT To minimize sample loss due to evaporation be sure that the plate is tightly sealed before running the GW5 0 Label thermal cycler program Prepare the Reagents Consumables and Other Components Thaw Reagents Thaw the following reagents on ice 5X TdT Buffer DNA Labeling Reagent Lu IMPORTANT Leave the TdT enzyme at 20 C until ready to use Prepare Your Work Area To prepare the work area 1 Place a double cooling chamber and a cooler on ice 2 Prepare the reagents as follows A Vortex each reagent at high speed 3 times 1 sec each time B Pulse spin for 3 sec C Place in the cooling chamber 3 Label one 15 mL centrifuge tube MM and place on ice 4 Label and place the following in the cooling chamber One strip of 12 tubes labeled MM Plate of fragmented reactions from the previous stage Preheat the Thermal Cycler Block The block must be heated to 37 C before samples are loaded To preheat the thermal cycler block 1 Turn on the thermal cycler and preheat the block to 37 C 2 Allow it to heat for 10 minutes before loading samples chapter 4 Affymetrix Genome Wide Human SNP Nsp Sty
168. rmine the OD260 measurement for the water blank and average 3 Determine the concentration of each PCR product as follows A Take 1 OD reading for every sample OD sample OD average water blank OD B Calculate the undiluted sample concentration for each sample using the Sample OD Sample concentration in ug uL OD X 0 05 ug uL X 100 Apply the convention that 1 absorbance unit at 260 nm equals 50 ug mL equivalent to 0 05 ug uL for double stranded PCR products This convention assumes a path length of 1 cm Consult your spectrophotometer handbook for further information 100Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Assess the OD Readings Follow the guidelines below for assessing and troubleshooting OD readings Sample OD A typical sample OD is 1 0 to 1 2 This OD range is equivalent to a final PCR product concentration of 5 0 to 6 0 ug uL It is based on the use of a conventional UV spectrophotometer plate reader and assumes a path length of 1 cm Process Control Metrics Evaluate the process control metrics as follows The OD260 0D280 ratio should be between 1 8 and 2 0 Do not proceed if this metric falls outside of this range The OD320 measurement should be very close to zero 0 0 005 OD Troubleshooting Guidelines Refer to the tables below when troubleshooting OD readings Table 4 39 PROBLEM Sample OD is greater than 1 2 6 ug uL If the sample OD is greater than 1 2 calculated con
169. rnquist M D Bigler J Patterson R E Kristal A R Shattuck A L Potter J D White E Abouta J S Buccal cell DNA yield quality and collection costs comparison of methods for large scale studies Cancer Epidemiol Biomarkers Prev 11 10 Pt 1 1130 3 2002 Lench N Stanier P Williamson R Simple non invasive method to obtain DNA for gene analysis Lancet Jun 18 1 8599 1356 1358 1988 Paez J G Lin M Beroukhim R Lee J C Zhao X Richter D J Gabriel S Herman P Sasaki H Altshuler D Li C Meyerson M Sellers W R Genome coverage and sequence fidelity of phi29 polymerase based multiple strand displacement whole genome amplification Nucleic Acids Research 32 9 2004 Tzvetkov M V Becker C Kulle B Nurnberg P Brockmoller J Wojnowski L Genome wide single nucleotide polymorphism arrays demonstrate high fidelity of multiple displacement based whole genome amplification Electrophoresis Feb 26 3 710 5 2005 Wong K K Tsang Y T M Shen J Cheng R S Chang Y Man T Lau C C Allelic imbalance analysis by high density single nucleotide polymorphic allele SNP array with whole genome amplified DNA Nucleic Acids Res May 17 32 9 e69 2004 20 Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Chapter i f HS aii Pal AFFYMETRIX GENOME WIDE HUMAN SNP Nsp Sty 5 0 ASSAY 48 SAMPLE PROTOCOL About This Protocol The Genome Wi
170. rogress Page Status 11 17 2006 9 43 18 AM 11 17 2006 9 43 19 AM 11 17 2006 9 43 22 AM 11 17 2006 9 43 22 AM 11 17 2006 9 43 22 AM 11 17 2006 9 43 23 AM 11 17 2006 9 43 25 AM 11 17 2006 3 43 25 AM 11 17 2006 3 43 25 AM 11 17 2006 3 44 03 AM 11 17 2006 8 44 08 AM 11 17 2006 8 44 08 AM 11 17 2006 9 44 46 AM 11 17 2006 9 44 46 AM 11 17 2006 9 44 46 AM ss Initiating analysis Loading parameters Initializing QC Process Checking chip type on all cel files Checking methods Checking SNP lists Processing CEL files for genotype QC analysis progress in 1 per dot Processing 1 of 6 C Documents and Settings jburri My Documents D ata genotype data NA108 Processing probesets Donel Processing 2 of 6 C Documents and Setlings jburri My Documents Data genotype data NA108 Processing probesets Donel Processing 3 of 6 C Documents and Settings jburri My Documents D ata genotype data NA1 08 Processing probesets Overall Progress QC Processing probesets Figure 6 9 Progress Page for the OC and Genotyping Workflow 5 Optional To cancel the analysis click Cancel The process that is currently running will finish then the analysis will be cancelled Depending on the process that is running cancellation may take a few minutes 6 Once the analysis is complete do one of the following Click Finish to close the application Click Back to re
171. ructions on page 80 Before Running Gels To ensure consistent results take 3 uL aliquot from each PCR WARNING Wear the appropriate personal protective equipment when handling ethidium bromide Run the Gels When the GW5 0 PCR program is finished 1 Remove each plate from the thermal cycler Spin down plates at 2000 rpm for 30 sec Place plates in cooling chambers on ice or keep at 4 C Label four fresh 96 well reaction plates P Gel P2Gel P3Gel and P4Gel Aliquot 12 uL of diluted gel loading buffer to each well in rows A through D of the fresh labeled PXGel plates a PWN appendix C E gels 215 6 Using a 12 channel P20 pipette transfer 3 uL of each PCR product from the 4 Nsp PCR plates to the corresponding plate row and wells of the PXGel plates Example 3 uL of each PCR product from each well of row A on plate P1 is transferred to the corresponding wells of row A on plate P1Gel 7 Seal the PXGel plates Vortex the center of each PXGel plate then spin them down at 2000 rpm for 30 sec 9 Load the total volume of 15 uL from each well of each PXGel plate onto E Gel 48 2 agarose gels 10 Run the gels for 22 min 11 Verify that the PCR product distribution is between 250 bp to 1100 bp see Figure C 1 on page 213 216 Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Modifications for Stage 9 Fragmentation Follow the Stage 9 instructions in Stage 9 Fragmentation on page 10
172. rvoir A to Non Stringent Wash Buffer and intake buffer reservoir B to Stringent Wash Buffer 4 Click Run for each module to begin priming 5 Follow LCD instructions NOTE All modules can be selected by selecting the All Modules button in the fluidics dialog box Array Wash and Stain The Affymetrix staining protocol for mapping arrays is a three stage process The process consists of 1 a Streptavidin Phycoerythin SAPE stain 2 an antibody amplification step and 3 a final stain with Streptavidin Phycoerythin SAPE Following staining the array is filled with Array Holding Buffer prior to scanning To wash and stain arrays 1 After 16 to 18 hours of hybridization remove the hybridization cocktail from the array and transfer it to the corresponding well of a 96 well plate Store on ice during the procedure or at 80 C for long term storage 2 Fill the array completely with 270 uL of Array Holding Buffer See Array Holding Buffer on page 139 for buffer recipe f NOTE Arrays can be stored in the Array Holding Buffer at 4 C for up to 3 hours before proceeding with washing and staining Equilibrate arrays to room temperature before washing and staining Prepare Buffers and Solutions Prepare the following buffers and solutions recipes follow Volumes given are sufficient for one array Mix well Stain Buffer SAPE Stain Solution Antibody Stain Solution Array Holding Buffer 138 Affymetrix Gen
173. s Positive Controls We recommend including one positive and one negative control with every set of samples run Reference Genomic DNA 103 can be used as a positive control It is supplied in the Genome Wide Human SNP Nsp Sty Assay Kit 5 0 A process negative control can be included at the beginning of the assay to assess the presence of contamination Refer to Chapter 3 and Chapter 7 for more information chapter 4 Affymetrix Genome Wide Human SNP Nsp Sty 5 0 Assay 48 Sample Protocol 61 Prepare the Reagents Equipment and Consumables Thaw Reagents and Genomic DNA 1 Allow the following reagents to thaw on ice NE Buffer 2 BSA If the genomic DNA is frozen allow it to thaw in a cooling chamber on ice F IMPORTANT Leave the NSP enzyme at 20 C until ready to use Prepare Your Work Area To prepare the work area 1 2 Place a double cooling chamber and a cooler on ice Label the following tubes then place in the cooling chamber One strip of 12 tubes labeled Dig A 2 0 mL Eppendorf tube labeled Dig MM Place the AccuGENE water on ice Prepare the plate with genomic DNA as follows A Vortex the center of the plate at high speed for 3 sec B Spin down the plate at 2000 rpm for 30 sec C Place back in the cooling chamber on ice Prepare the reagents except for the enzyme as follows A Vortex 3 times 1 sec each time B Pulse spin for 3 sec C Place in the cooling chamber Pr
174. s amp positive control affected Problem with master mixes or individual reagents Ensure all reagents added to master mixes and enzymes are stored at 20 C Work quickly with enzymes and return to 20 C directly after use to prevent loss of activity Failed restriction digest Use restriction enzyme to digest a known good DNA sample Run gel to confirm restriction enzyme activity Use the correct concentration of BSA Failed adaptor ligation reaction Confirm enzyme activity Ligase buffer contains ATP and should be defrosted held at 4 C Vortex ligase buffer thoroughly before use to ensure precipitate is re suspended Avoid multiple freeze thaw cycles Try a fresh tube of buffer Reduced adaptor ligation efficiency due to adaptor self ligation DNA re ligation To prevent self ligation of adaptor work rapidly and add DNA ligase last Failed PCR reaction Check PCR reagents Take care with preparation of master mixes and ensure accurate pipetting and thorough mixing Reduced PCR reaction yield non optimal PCR conditions Use a validated thermal cycler check PCR programs Use recommended thin walled reaction tubes Thoroughly mix PCR reaction Ligation mix not diluted prior to PCR reaction Ligation mixture diluted 1 4 with molecular biology grade water to remove potential inhibitors and maintain optimal pH and salt concentration Incorrect concentration of nucl
175. s Stage The input required from Stage 9 Fragmentation is Quantity Item 1 Plate of fragmented DNA Equipment and Consumables Required The following equipment and consumables are required for this stage Refer to Appendix A for vendor and part number information IMPORTANT Use only the PCR plate adhesive film and thermal cyclers listed in Table 4 1 on page 27 chapter 4 Affymetrix Genome Wide Human SNP Nsp Sty 5 0 Assay 48 Sample Protocol 113 Table 4 48 Equipment and Consumables Required for Stage 10 Labeling Quantity Item 1 Cooler chilled to 20 C 1 Cooling chamber double chilled to 4 C do not freeze 1 Ice bucket filled with ice 1 Marker fine point permanent 1 Microcentrifuge 1 Pipette single channel P200 1 Pipette single channel P1000 1 Pipette 12 channel P20 accurate to within 5 As needed Pipette tips for pipettes listed above full racks 1 Plate centrifuge 1 Plate seal 1 Thermal cycler 1 Tube centrifuge 15 mL 1 Tubes 12 strip 0 2 mL 1 Vortexer Reagents Required The following reagents are required for this stage Refer to Appendix A for vendor and part number information The amounts listed are sufficient to process 48 samples Table 4 49 Reagents Required for Stage 10 Labeling Quantity Reagent 1 vial DNA Labeling Reagent 30 mM 1 vial Terminal Deoxynucleotidyl Transferase TdT 30 U uL 1 vial Te
176. s an inherent risk of contamination with PCR product from previous reactions In Chapter 2 Laboratory Setup and Recommendations we recommend a workflow to minimize the risk of cross contamination during the assay procedure It is essential to adhere to workflow recommendations PCR reactions should only be carried out in the main laboratory Personnel should not re enter the Pre PCR Clean and PCR Staging areas following potential exposure to PCR product without first showering and changing into clean clothes It is essential to carefully read and follow the protocol as written The assay in this manual has been validated using the reagents and suppliers listed Substitution of reagents and shortcuts are not recommended as they could result in suboptimal results For example always use AccuGENE water from Cambrex and ligase and restriction enzymes from New England Biolabs Additional recommendations are as follows Think ahead to ensure that the reagents and equipment you require including pipettes are in the correct work area Ensuring the proper equipment is available in the proper laboratory areas will make the workflow easier and will help reduce the risk of sample contamination Pay particular attention to the storage and handling of reagents Proper storage and handling is particularly important for enzymes such as DNA Ligase and the Fragmentation Reagent DNase I Both ofthese enzymes are sensitive to temperatures exceeding
177. s analyzed The format of the file is tab delimited text with a header line followed by a line for each CEL file analyzed The content of each column is described in Table 6 1 on page 159 Some of the metrics provided can potentially be used to track and identify outlier arrays brimm p report txt Notepad Joe File Edit Format View Help gender brlmm p_call_rate AB percent AA percent BB_percent raw intensity mean raw_intensity_stdev allele_summarization_mean allele summarization stdev allele deviation mean allele deviation stdev allele mad residuals mean allele mad residuals stdev cluster distance mean cluster distance stdev cel filel cel female 9926 2761 36 58 35 08 8 69133 1 15201 8 96336 1 12884 0 29147 0 43243 00445 0 04234 0 0 cel_file2 cel female 99 09 27 73 36 51 3484 9 49777 1 14045 8 95527 11256 0 22596 0 35553 0 04352 004186 0 0 cel_file3 cel male 99 34 2727 36 75 3531 937524 120563 897358 1 12414 0 32752 0 42996 0 08237 0 06055 0 cel file4 cel male 993 2704 3684 35 42 08518 1 1561 895773 1 13904 0 36313 0 49814 0 04982 0 04867 0 0 cel file5 cel female 9674 282 3515 33 39 7 30338 094595 894939 1 11732 038371 0 48072 0 07671 0 0673 0 0 Figure 6 12 Example of a Clustering Report File chapter 6 Data Analysis 159 Table 6 1 Description of Content of brimm report txt Files Column Header Content The CEL file name Gender The estimated gender based upon chrX SNP calls Brlmm_call_ra
178. s are complete the fluidics protocols begin The Fluidics Station dialog box at the workstation terminal and the LCD window displays the status of the washing and staining steps When staining is finished remove the microcentrifuge vials containing stain and replace with three empty microcentrifuge vials as prompted Remove the arrays from the fluidics station by first pressing down the cartridge lever to the eject position Check the array window for large bubbles or air pockets If bubbles are present 1 use a pipette to manually fill the array with Array Holding Buffer 2 remove one half of the solution then 3 manually fill the array with Array Holding Buffer IMPORTANT If a bubble is present do not return the array to the array holder The array must be filled manually with Array Holding Buffer If the array has no large bubbles it is ready for scanning Pull up on the cartridge lever to engage wash block and proceed to Scanning Arrays on page 142 If the arrays cannot be scanned promptly store them at 4 C in the dark until ready for scanning Scan must be performed within 24 hours When finished washing and staining shut down the fluidics station following the procedure listed under Shutting Down the Fluidics Station on page 145 142 Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Scanning Arrays The GeneChip Scanner 3000 7G is also controlled by GCOS Software 1 4 Make sure the laser is warmed up pr
179. s as described under Check the Fragmentation Reaction on page 111 chapter 4 Affymetrix Genome Wide Human SNP Nsp Sty 5 0 Assay 48 Sample Protocol 111 Check the Fragmentation Reaction The instructions below are for running 4 TBE gels For information on running E Gel 48 4 agarose gels refer to Appendix C E gels on page 211 To ensure that fragmentation was successful 1 When the GW5 0 Fragment program is finished A Remove the plate from the thermal cycler B Spin down the plate at 2000 rpm for 30 sec and place in a cooling chamber on ice Dilute 1 5 uL of each fragmented PCR product with 4 uL gel loading dye 3 Run on 4 TBE gel with the BioNexus All Purpose Hi Lo ladder at 120V for 30 minutes to 1 hour 4 Inspect the gel and compare it against the example shown in Figure 4 14 below Figure 4 14 Typical example of fragmented PCR products run on 4 TBE agarose gel at 120V for 30 minutes to 1 hour Average fragment size is 180 bp 112Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Stage 10 Labeling About this Stage During this stage you will Label the fragmented samples using the DNA Labeling Reagent Prepare the Labeling Master Mix Add the mix to each sample Place the samples onto a thermal cycler and run the GW5 0 Label program Location and Duration Main Lab Hands on time 30 minutes e GWS5 0 Label thermal cycler program time 4 25 hours Input Required from Previou
180. s generally salt free because high concentrations of certain salts can also inhibit PCR and other enzyme reactions DNA should be prepared as described in Chapter 4 Affymetrix Genome Wide Human SNP Nsp Sty 5 0 Assay 48 Sample Protocol DNA must not be contaminated with other human genomic DNA sources or with genomic DNA from other organisms PCR amplification of the ligated genomic DNA is not human specific so sufficient quantities of non human DNA may also be amplified and could potentially result in compromised genotype calls Contaminated or mixed DNA may manifest as high detection rates and low call rates 18 Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual DNA must not be highly degraded For any particular SNP the genomic DNA fragment containing the SNP must have Nsp I or Sty I restriction sites intact so that ligation can occur on both ends of the fragment and PCR can be successful The approximate average size of genomic DNA may be assessed on a 1 or 2 agarose gel using an appropriate size standard control Reference Genomic DNA 103 can be run on the same gel for side by side comparison High quality genomic DNA will run as a major band at approximately 10 20 kb on the gel Genomic DNA amplified with the Repli G Kit a 29 whole genome amplification kit QIAGEN has been tested successfully with the Genome Wide Human SNP 5 0 Nsp Sty Assay The Repli G Kit was used to amplify 30 ng genomic DNA The amplified
181. s with AccuGENE water Location and Duration Pre PCR Clean Area Hands on time 30 minutes e GWS5 0 Ligate thermal cycler program time 3 3 hours Input Required From Previous Stage The input required from Stage 4 Nsp Restriction Enzyme Digestion is OTELA Item 48 samples Nsp digested samples in a cooling chamber on ice chapter 4 Affymetrix Genome Wide Human SNP Nsp Sty 5 0 Assay 48 Sample Protocol 65 Equipment and Consumables Required The following equipment and consumables are required for this stage Refer to Appendix A for vendor and part number information Table 4 24 Equipment and Consumables Required for Stage 5 Nsp Ligation Quantity Item 1 Cooler chilled to 20 C 1 Cooling chamber double chilled to 4 C do not freeze 1 Ice bucket filled with ice 1 Marker fine point permanent 1 Microcentrifuge 1 Pipette single channel P100 1 Pipette single channel P1000 1 Pipette 12 channel P20 1 Pipette 12 channel P200 As needed Pipette tips for pipettes listed above full racks 1 Plate centrifuge 2 Plate seal 1 Solution basin 55 mL 1 Thermal cycler 1 strip Tubes 12 strip 0 2 mL 1 Tube Eppendorf 2 0 mL 1 Vortexer IMPORTANT Use only the PCR plate adhesive film and thermal cyclers listed in Table 4 1 on page 27 66 Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Reagents Required The following r
182. say a comparison with microsatellite marker assays and finding of significant linkage to chromosome 6q22 Am J Hum Genet 2004 74 886 97 Shrimpton AE Levinsohn EM Yozawitz JM Packard DS Jr Cady RB Middleton FA Persico AM Hootnick DR A HOX gene mutation in a family with isolated congenital vertical talus and Charcot Marie Tooth disease Am J Hum Genet 2004 75 92 6 Puffenberger EG Hu Lince D Parod JM Craig DW Dobrin SE Conway AR Donarum EA Strauss KA Dunckley T Cardenas JF et al Mapping of sudden infant death with dysgenesis of the testes syndrome SIDDT by a SNP genome scan and identification of TSPYL loss of function Proc Natl Acad Sci U S A 2004 101 11689 94 Kaindl AM Ruschendorf F Krause S Goebel HH Koehler K Becker C Pongratz D Muller Hocker J Nurnberg P Stoltenburg Didinger G et al Missense mutations of ACTA1 cause dominant congenital myopathy with cores J Med Genet 2004 41 842 8 Gissen P Johnson CA Morgan NV Stapelbroek JM Forshew T Cooper WN McKiernan PJ Klomp LW Morris AA Wraith JE et al Mutations in VPS33B encoding a regulator of SNARE dependent membrane fusion cause arthrogryposis renal dysfunction cholestasis ARC syndrome Nat Genet 2004 36 400 4 Uhlenberg B Schuelke M Ruschendorf F Ruf N Kaindl AM Henneke M Thiele H Stoltenburg Didinger G Aksu F Topaloglu H et al Mutations in the gene encoding gap junction protein al
183. sed for each digestion and ligation stage chapter 7 Troubleshooting 179 OD Troubleshooting Guidelines Refer to the tables below when troubleshooting OD readings Table 7 1 PROBLEM Sample OD is greater than 1 2 6 pg pL If the sample OD is greater than 1 2 calculated concentration greater than 6 ug uL a problem exists with either the elution of PCR products or the OD reading The limit on PCR yield is approximately 6 ug uL as observed in practice and as predicted by the mass of dNTPs in the reaction Possible causes include The purified PCR product was eluted in a volume less than 55 uL The purified PCR product was not mixed adequately before making the 1 100 dilution The diluted PCR product was not mixed adequately before taking the OD reading The water blank reading was not subtracted from each sample OD reading The spectrophotometer plate reader may require calibration Pipettes may require calibration There may be air bubbles or dust in the OD plate There may be defects in the plastic of the plate The settings on the spectrophotometer plate reader or the software may be incorrect OD calculations may be incorrect and should be checked Table 7 2 PROBLEM Sample OD is Less Than 1 0 5 ug L If the sample OD is less than 1 0 calculated concentration less than 5 ug uL a problem may exist with either the genomic DNA the PCR reaction the elution of purified PCR products or the
184. shown in Figure 8 2 on page 187 A Place on the fluidics station an empty one liter waste bottle a 500 mL bottle of bleach and a one liter bottle of DI water The Bleach protocol requires approximately one liter of DI water B Insert the waste line into the waste bottle C Immerse all three wash and water lines into the bleach solution Hi IMPORTANT Do NOT immerse the waste line into the bleach chapter 8 Vacuum Manifold and Fluidics Station Care and Maintenance 187 Figure 8 2 The bleach cycle Immerse the tubes into the 0 525 sodium hypochlorite solution The waste line remains in the waste bottle 4 Open GeneChip Operating Software GCOS Microarray Suite or the current version of the Affymetrix control software 188 Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual 5 Click Run gt Fluidics from the menu Alternatively click the down arrow on the Protocol list on the tool bar The protocol window appears Figure 8 3 Fluidics Station 3 ma x Module 1 Module 2 Module 3 Module 4 Experiment Probe Array Type View Protocol Fluidics Protocol BLEACHv2_450 Version 2 0 Thoroughly clean FS450 by running bleach then DI Water thru all lines and all needles ET 1hr 35min Current Stage Time Cycle Temp Time Remaining Close Figure 8 3 The Fluidics Station protocol window select all modules 6 Choose the current bleach protocol as of t
185. sually distinguish between silver and aluminum blocks chapter 4 Affymetrix Genome Wide Human SNP Nsp Sty 5 0 Assay 48 Sample Protocol 79 Table 4 32 GW5 0 PCR Thermal Cycler Program for the GeneAmp PCR System 9700 silver or gold plated silver blocks GW5 0 PCR Program for GeneAmp PCR System 9700 Temperature Time Cycles 94 C 3 minutes 1X 94 C 30 sec 60 C 45 sec 30X 68 C 15 sec 68 C 7 minutes 1X 4 C HOLD Can be held overnight Volume 100 uL Specify Maximum mode Table 4 33 GW5 0 PCR Thermal Cycler Program for the MJ Tetrad PTC 225 GW5 0 PCR Program for MJ Tetrad PTC 225 Temperature Time Cycles 94 C 3 minutes 1X 94 C 30 sec 60 C 30 sec 30X 68 C 15 sec 68 C 7 minutes 1X 4 C HOLD Can be held overnight Volume 100 uL Use Heated Lid and Calculated Temperature 80 Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Running Gels The instructions below are for running 2 TBE gels For information on running E Gel 48 2 agarose gels refer to Appendix C E gels on page 211 Before Running Gels To ensure consistent results take 3 uL aliquot from each PCR WARNING Wear the appropriate personal protective equipment when handling ethidium bromide Run the Gels When the GW5 0 PCR program is finished 1 a RON 7 8 9 Remove each plate from the thermal cycler Spin down plates at 2000 rpm for 30 sec Place plates
186. t This Stage 0000 eee 120 vi Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Prepare the Reagents Consumables and Other Components 121 Prepare the Arrays 2 0 eee 122 Prepare the Hybridization Master Mix AW R KIRI III IS 122 Method 1 Using a GeneAmp PCR System 9700 124 Method 2 Using an Applied Biosystems 2720 MJ Tetrad PTC 225 or MJ Tetrad 2 Thermal Cycler 127 Chapter 5 Washing Staining and Scanning Arrays 131 Equipment and Consumables Required 131 Reagents Required 0 k kK KK KK KK KK KK KK RR KK KIRI KK KK 132 Reagent Preparation 2 25 09 3 osos vet KIIR III EUR KI KIRI KI KII K eee es 133 Experiment and Fluidics Station Setup kk RR RR RR RR 135 Register a New Experiment in GCOS ZEKI 135 Prepare the Fluidics Station M K KK KK eee 136 Array Wash and Staln lt xer Ziyan ein ied SIN edie ee RIO ee 137 Prepare Buffers and Solutions lille 137 Washing and Staining Arrays WW kk kk KK KK eee 140 SCANNING AMAVS ee tay d alay e E AL da ana qe ER RU s 142 Prepare arrays for Scanning snas ee eee eas ttis mu aes 142 Scanning the AL y v ccs kd xan aka eres te ere Te vot dt es 143 Shutting Down the Fluidics Station KK KIRR eee 145 Chapter 6 Data Analysis ons lena bank d ES od EET S ee eee oie do en aie 147 Software Requirements cles 147 Overview of the OC and Genotyping Analysis Workflow
187. t lists the CEL files to process The first line of the file must read cel_files Subsequent lines contain the full path name of the CEL files to process List one file only per line To remove CEL files from the list select the CEL file name and click Remove 8 Click Next The Next button remains disabled until at least one CEL file is displayed After clicking Next the Output Options window is displayed Figure 6 5 on page 152 9 In the General Output Options box A Select a location for the analysis output using one of the following methods Click Browse and select a folder Manually enter the full path to a folder The output location selected becomes the default from session to session You can change the location at any time by clicking Browse and selecting a new folder or by entering the path to a new folder The Next button remains inactive until a valid path is entered correctly 152Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual BRLMM Analysis Tool Output Options General Output Options Select the output location C Documents and SettingsV felton P GENE Desktop Output Folder Batch Name 20061211 154047 QC Output Option Enter the QC report filename qc reporttxt Figure 6 5 Output Options for the OC and Genotyping Workflow B Optional Manually change the batch name The batch name is generated automatically This name is assigned to a subfolder created auto
188. tage Refer to Appendix A for vendor and part number information The amounts listed are sufficient for processing 48 samples Table 4 7 Reagents Required for Stage 1 Sty Restriction Enzyme Digestion Quantity Reagent 1 vial BSA 100X 10 mg mL 1 vial NE Buffer 3 10X 1 vial Sty 10 U uL NEB 2 5 mL AccuGENE Water molecular biology grade Important Information About This Stage To help ensure the best results carefully read the information below before you begin this stage of the protocol IMPORTANT The same team or individual operator should not perform Nsp I and Sty digestion reactions on the same day About Using Controls Positive Controls We recommend including one positive and one negative control with every set of samples run Reference Genomic DNA 103 can be used as a positive control It is supplied in the Genome Wide Human SNP Nsp Sty Assay Kit 5 0 A process negative control can be included at the beginning of the assay to assess the presence of contamination Refer to Chapter 3 and Chapter 7 for more information 36 Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Prepare the Reagents Equipment and Consumables Thaw Reagents and Genomic DNA 1 Allow the following reagents to thaw on ice NE Buffer 3 BSA If the genomic DNA is frozen allow it to thaw in a cooling chamber on ice Lu IMPORTANT Leave the STY I enzyme at 20 C until ready to use
189. tative linear range of the instrument 0 2 to 2 0 OD Hybridization ovens should be serviced at least once per year to ensure that they are operating within the manufacturer s specifications chapter 7 Troubleshooting 175 Important Differences Between Genome Wide Human SNP Arrays 5 0 and GeneChip Expression Arrays For laboratories that also run GeneChip Expression arrays always check the temperature setting on the Hybridization Oven 640 For the Genome Wide Human SNP Array 5 0 ovens should be set to 50 C The temperature for hybridization on expression arrays is 45 C Buffer B is different for the expression and DNA arrays Using the MES based buffer B from the Expression protocol will result in substantially reduced call rates for the Genome Wide Human SNP Array 5 0 Also care should be taken to ensure the fluidics station is properly maintained and primed with the correct buffers prior to use Both the Genome Wide Human SNP 5 0 Nsp Sty Assay and Expression protocols use the same stain reagents for each staining step However after the last wash the Genome Wide Human SNP Array 5 0 is filled with Array Holding Buffer Genome Wide Human SNP Arrays 5 0 are scanned once at 570 nm on the GeneChip Scanner 3000 7G 176Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Troubleshooting the Genome Wide Human SNP 5 0 Nsp Sty Assay Problem Likely Cause Faint absent bands on PCR gel Solution Both sample
190. te The call rate at the default or user specified threshold AB_percent The percentage of SNPs called AB i e the heterozygosity AA_percent The percentage of SNPs called AA BB_percent The percentage of SNPs called BB Raw_intensity_mean The average of the raw PM probe intensities Raw_intensity_stdev The standard deviation of the raw PM probe intensities Allele_summarization_mean The average of the allele signal estimates log2 scale Allele_summarization_stdev The standard deviation of the allele signal estimates log2 scale Allele_deviation_mean The average of the absolute difference between the log2 allele signal estimate and its median across all arrays Allele_deviation_stdev The standard deviation of the absolute difference between the log2 allele signal estimate and its median across all arrays Allele_mad_residuals_mean The average of the median absolute deviation MAD between observed probe intensities and probe intensities fitted by the model Allele_mad_residuals_stdev The standard deviation of the median absolute deviation MAD between observed probe intensities and probe intensities fitted by the model Cluster_distance_mean The average distance to the cluster center for the called genotype BRLMM only Cluster_distance_stdev The standard deviation of the distance to the cluster center for the called genotype BRLMM only 160Af
191. te All OC call rate for all SNPs QC Call Rate QC Call Rate is displayed in the QC Results window Figure 6 19 upon completion of analysis Once this window is closed the file qc results txt can be opened in an application such as Notepad It is an indicator of the overall performance of the assay for Genome Wide Human SNP Array 5 0 A QC Call Rate in excess of 86 indicates that all steps from restriction digestion through scanning worked as expected A reduced Call Rate may result if an error in any of the assay steps occurs or if lower quality DNA samples are processed It is also common to observe lower Call Rates in circumstances where a new operator is learning the assay or the number of samples processed at one time increases In these later examples it may be prudent to budget time for additional practice for the operator in order to increase proficiency with the assay and achieve higher performance Some other factors that can lead to a reduced Call Rate include Deviation from the assay protocol Contaminated DNA Expired reagents For a sample with a lower Call Rate it is important to take into consideration the reasons for the lower Call Rate as well as the degree to which accuracy is compromised It may be necessary to repeat target preparation for that sample depending on the degree to which the lower Call Rate and decrease in accuracy affects the overall experimental goals Refer to Chapter 7 Troubleshooti
192. te P1 Plate P2 Plate P3 An equal aliquot of each sample from the Ligation Plate is transferred to the corresponding well of each PCR Plate For example an equal aliquot of each sample from row A on the Sty Ligation Plate is transferred to the corresponding wells of row A on PCR Plates P1 P2 and P3 Figure 4 4 Transferring Equal Aliquots of Diluted Ligated Sty Samples to Three Reaction Plates Prepare the Sty PCR Master Mix Location Pre PCR Clean Room Prepare the Sty PCR Master Mix To prepare the Sty PCR Master Mix IMPORTANT The PCR reaction is sensitive to the concentration of primer used It is critical that the correct amount of primer be added to the PCR Master Mix to achieve the correct distribution of fragments 200 to 1100 bp in the products Check the PCR reactions on a gel to ensure that the distribution is correct 1 Keeping the 50 mL Falcon tube in the cooling chamber add the reagents as shown in Table 4 17 on page 53 except for the Tag DNA polymerase chapter 4 Affymetrix Genome Wide Human SNP Nsp Sty 5 0 Assay 48 Sample Protocol 53 2 Remove the TITANIUM Taq DNA Polymerase from the freezer and immediately place in a cooler 3 Pulse spin the Taq DNA polymerase for 3 sec 4 Immediately add the Tag DNA polymerase to the master mix then return the tube to the cooler on ice 5 Vortex the master mix at high speed 3 times 1 sec each time 6 Pour the mix into the solution basin
193. te reproducibility and accuracy of genotype calls Deviation from Assay Protocol A problem in any step of the assay may lead to a decreased Call Rate The gel images produced before DNA digestion and before PCR cleanup the PCR yield after cleanup and a gel image after fragmentation can be used to identify problematic steps Consult Chapter 7 Troubleshooting for further information At a minimum a PCR negative control water instead of DNA template should be incorporated into each group of samples processed The Reference Genomic DNA 103 is included in the assay kit as a positive process control 168Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Oligonucleotide Controls The oligonucleotide control reagent contains oligonucleotide B2 and 4 hybridization control oligonucleotides B2 Oligo Performance The B2 oligo is a component of the Oligo Control Reagent 0100 OCR It is spiked into each hybridization cocktail and is highlighted on the image by the following The alternating pattern of intensities on the border The checkerboard pattern at each corner Figure 6 20 on page 168 and throughout the array The array name located in the lower left corner of the array Figure 6 21 on page 168 B2 Oligo serves as a positive hybridization control and is used by the software to place a grid over the image Variation in B2 hybridization intensities across the array is normal and does not indicate variation in hybri
194. teps for Stages 1 through 10 of the 48 sample protocol Keep all tubes on ice or in a cooling chamber on ice Keep all plates in cooling chambers on ice 26 Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Preparing the Work Area for Each Stage Many of the stages in the Genome Wide SNP 5 0 Assay must be performed rapidly and on ice to carefully control enzyme activity and temperature transitions Therefore we recommend that you set up all of the equipment consumables and reagents except for the enzymes prior to beginning each stage Below is an illustration of the setup for Stage 1 Sty Restriction Enzyme Digestion Pipettes and tips are not shown Set of strip tubes AccuGene water NEBuffer 3 BSA and Eppendorf tube Plate of genomic DNA Double cooling labeled Sty chamber Figure 4 2 Example of Work Area Preparation chapter 4 Affymetrix Genome Wide Human SNP Nsp Sty 5 0 Assay 48 Sample Protocol 27 Thermal Cyclers Plates and Plate Seals The Genome Wide SNP 5 0 Assay has been optimized using the following thermal cyclers reaction plates and adhesive film IMPORTANT Use only the PCR plate adhesive film and thermal cyclers listed in Table 4 1 Using other PCR plates and film that are incompatible with these thermal cyclers can result in crushed tubes loss of sample or poor results Table 4 1 Thermal Cyclers PCR Plate and Adhesive Film Optimized for Use With this
195. ter is specified be sure to use the AccuGENE water listed in Appendix A Using in house ddH5O or other water can negatively affect your results The enzymatic reaction in Stage 9 Fragmentation is particularly sensitive to pH and metal ion contamination If you run out of master mix during any of these procedures a volume error has been made or the pipettes are not accurate We recommend that you stop and repeat the experiment Reagent Handling and Storage Follow these guidelines for reagent handling and storage Keep dedicated equipment in each of the areas used for this protocol To avoid contamination do not move equipment between the Pre PCR Area the PCR Staging Room and the Main Lab Unless otherwise indicated keep all reagents except enzymes on ice in a cooling chamber that has been chilled to 4 C when working on the bench top Always leave enzymes at 20 C until immediately prior to adding them to master mixes When removed from the freezer immediately place in a cooler that has been chilled to 20 C and placed on ice Store the reagents used for the restriction digestion ligation and PCR steps in the Pre PCR Clean Area Consult the appropriate MSDS for reagent storage and handling requirements Do not re enter the Pre PCR Clean Area after entering the PCR Staging Room or the Main Lab Aliquot each of the reagents in the Pre PCR Clean Area before starting the rest of the experiment When performing the s
196. the center of the plate at high speed for 3 sec B Spin down the plate at 2000 rpm for 30 sec C Label the plate Lig D Place back in the cooling chamber on ice 5 To prepare the reagents A Vortex at high speed 3 times 1 sec each time except for the enzyme B Pulse spin for 3 sec C Place in a cooling chamber Preheat the Thermal Cycler Lids Main Lab Have someone in the Main Lab power on the thermal cyclers to be used for PCR to preheat the lids The lids must be preheated before loading samples leave the blocks at room temperature If you are preparing the plates for PCR it is best not to go from the Pre PCR Room or Staging Area to the Main Lab and then back again Aliquot Sty Ligated DNA to the PCR Plates To aliquot Sty ligated DNA to the PCR plates 1 Working one row at a time and using a 12 channel P20 pipette transfer 10 uL of each Sty ligated sample to the corresponding well of each PCR plate Example Figure 4 4 Transfer 10 uL of each sample from Row A of the Sty Ligation Stage Plate to the corresponding wells of row A on the plates labeled P1 P2 and P3 2 Seal each plate with adhesive film and leave in cooling chambers on ice 52 Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual 6 6 9 6 6 O6 0 Ligation stage plate with Sty ligated samples Lig 900000002020029906 9999999999906 l 6006000000006 PIC EZ P3 Pla
197. this end the GeneChip Mapping 500K array set early access version has recently been used for a comprehensive view of CNVs among 270 HapMap samples Greater than 1 000 copy number variable regions were found spanning a broad size range from less than 1kb to over 3Mb 92 93 Importantly the genetic correlation between CNVs and SNPs has also been studied In the case of biallelic CNVs and common deletion polymorphisms there is evidence of linkage disequilibrium with neighboring SNPs but this relationship 4 Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual is not nearly as strong in the case of complex CNVs 92 94 96 Thus whole genome SNP based association studies should benefit from the capability to type CNVs directly rather than relying on LD with SNP markers The fifth generation product in the mapping portfolio the Affymetrix Genome Wide Human SNP Array 5 0 also uses the WGSA assay that has been the hallmark characteristic of all previous mapping arrays This single array interrogates nearly 500 000 SNPs by combining the Nsp I and Sty I PCR fractions prior to the DNA purification step and through a reduction in the absolute number of features associated with each individual SNP on the array This array also contains 420 000 non polymorphic probes designed to interrogate CNVs in the genome 100 000 of these probes interrogate previously identified CNVs while the remaining 320 000 are distributed across the genome for im
198. tific www usascientific com VWR vwr com 206 Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Appendix THERMAL CYCLER PROGRAMS This appendix includes the thermal cycler programs required for the Genome Wide Human SNP 5 0 Nsp Sty Assay Before you begin processing samples enter and save these programs into the appropriate thermal cyclers GW5 0 Digest GW5 0 Digest Program Temperature Time 37 C 120 minutes 65 C 20 minutes 4 C Hold GW5 0 Ligate GW5 0 Ligate Program Temperature Time 16 C 180 minutes 70 C 20 minutes 4 C Hold 208 Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual GW5 0 PCR For the GeneAmp PCR System 9700 You must use GeneAmp PCR System 9700 thermal cyclers with silver or gold plated silver blocks Do not use GeneAmp PCR System 9700 thermal cyclers with aluminum blocks Ramp speed Max Volume 100 uL GW5 0 PCR Program for GeneAmp PCR System 9700 Temperature Time Cycles 94 C 3 minutes 1X 94 C 30 seconds 60 C 45 seconds 30X 68 C 15 seconds 68 C 7 minutes 1X 4 C HOLD Can be held overnight For the MJ Tetrad PTC 225 and Tetrad 2 Use Heated Lid and Calculated Temperature Volume 100 uL GW5 0 PCR Program for MJ Tetrad PTC 225 and Tetrad 2 Temperature Time Cycles 94 C 3 minutes 1X 94 C 30 seconds 60 C 30 seconds 30X 68 C 15 seconds 68 C 7 minut
199. to 20 to 24 in Hg and allow the beads to dry an additional 10 minutes Turn off the vacuum remove the filter plate and blot the bottom of the plate again on Kimwipes to remove any excess EtOH chapter 4 Affymetrix Genome Wide Human SNP Nsp Sty 5 0 Assay 48 Sample Protocol 91 Elute the Purified Reactions To elute the purified reactions 1 Attach the elution catch plate to the bottom of the filter plate using 2 strips of lab tape The filter and elution plate assembly is now referred to as the plate stack Figure 4 9 IMPORTANT Do not completely seal with tape Product will not elute if sealed Tape on two Y opposing sides of the plates Filter plate Elution catch plate Figure 4 9 Attaching the Elution Catch Plate to the Filter Plate Pour or pipette 3 mL of Buffer EB to a solution basin Using a 12 channel P200 pipette add 55 uL of Buffer EB to each well For accurate pipetting pre wet pipette tips with EB before dispensing Dispense as close to the beads as possible without touching them Buffer EB should be applied directly on top of the beads see Figure 4 10 and Figure 4 11 on page 92 Tap the plate stack to move all Buffer EB onto the filter at the bottom of the wells Using an adhesive film tightly seal the filter plate on the plate stack Place the plate stack on a Jitterbug for 10 minutes at setting 5 92 Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Ridge on Ra
200. tte 12 channel P20 accurate to within 5 1 Pipette 12 channel P200 As needed Pipette tips for pipettes listed above full racks 1 Plate optical For example the Greiner UV Star Transparent 96 well Use the optical plate recommended for use with your plate reader 1 Plate 96 well reaction 1 Plate centrifuge 5 Plate seal 1 Spectrophotometer plate reader 1 Solution basin 100 mL 1 Vortexer Reagents Required The following reagents are required for this stage Refer to Appendix A for vendor and part number information The amounts listed are sufficient to process 48 reactions Table 4 38 Reagents Required for Stage 8 Quantitation Quantity Reagent 15 mL AccuGENE water molecular biology grade chapter 4 Affymetrix Genome Wide Human SNP Nsp Sty 5 0 Assay 48 Sample Protocol 97 Important Information About This Stage To help ensure the best results carefully read the information below before you begin this stage of the protocol IMPORTANT e The accuracy of the OD measurement is critical Carefully follow this procedure and be sure the OD measurement is within the quantitative linear range of the instrument 0 2 to 2 0 OD The spectrophotometer plate reader should be calibrated regularly to ensure correct readings This protocol has been optimized using a UV spectrophotometer plate reader for quantitation The NanoDrop will give different quantitation results This protocol
201. turn to previous windows Upon completion of the QC analysis the QC Results window is displayed The application automatically begins to perform the genotyping using the files that pass the defined QC Call Rate threshold chapter 6 Data Analysis 157 B Bi MM Analysis Tool OC Results Jie QC Report Date 12 8 2006 1 56 34 PM Report File Name C Documents and Settings jburri My Documents D ata genotype data BAT Output 20061208_135235 qe report txt Batch 20061208_135235 Probe Array Type GenomeWideSNP_5 Analysis method1 analysis_name GC call rate all analysis dm group_name all_qe as percentage 1 dm cutoff 0 33 dm hetMult 1 25 precision 4 QC call rate read QC call rate QC call rate Gender Nsp ovale Sty all female 93 83 96 49 89 21 9431 NADESS1 Hw 3 female 93 57 95 5 88 89 93 55 NAD6S93_ Hw 3 male 91 52 95 07 89 05 92 32 NAO06383 Hw 3 male 3794 9834 8501 97 55 NA06933 Hw 3 male 3537 3661 3038 9514 NA06993_H 328 94 27 88 41 92 69 946 96 12 88 57 9418 NA07022 Hw 3 male 92 54 96 24 89 05 9381 Figure 6 10 OC Results Window Once the QC Results window is closed it cannot be reopened from BAT 2 0 The information displayed in this window is stored as a file in the Batch subfolder It is a standard tab delimited text file that can be opened in Notepad or Microsoft Excel 158Affymetrix Genome Wide Human SNP 5 0 Ns
202. uL 45 uL T4 DNA Ligase 400U uL 2uL 120 uL Total 5 25 uL 315 uL Add Sty Ligation Master Mix to Reactions To add Sty Ligation Master Mix to samples 1 Using a single channel P100 pipette aliquot 25 uL of Sty Ligation Master Mix to each tube of the strip tubes on ice Using a 12 channel P20 pipette aliquot 5 25 uL of Sty Ligation Master Mix to each reaction on the Sty Digestion Stage Plate The total volume in each well is now 25 uL chapter 4 Affymetrix Genome Wide Human SNP Nsp Sty 5 0 Assay 48 Sample Protocol 45 Sty Digested DNA 19 75 uL Sty Ligation Master Mix 5 25 uL Total 25 uL Contains ATP and DTT Keep on ice Seal the plate tightly with adhesive film Vortex the center of the plate at high speed for 3 sec Spin down the plate at 2000 rpm for 30 sec Ensure that the thermal cycler lid is preheated 3 4 5 6 7 Table 4 13 GW5 0 Ligate Thermal Cycler Program GW5 0 Ligate Program Load the plate onto the thermal cycler and run the GW5 0 Ligate program Temperature Time 16 C 180 minutes 70 C 20 minutes 4 C Hold 46 Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual Dilute the Samples IMPORTANT It is crucial to dilute the ligated DNA with AccuGENE water prior to PCR To dilute the samples 8 Place the AccuGENE water on ice 20 minutes prior to use 1 When the GW5 0 Ligate program is finished remove the plate
203. ukhim R Milner DA Granter SR Du J et al Integrative genomic analyses identify MITF as a lineage survival oncogene amplified in malignant melanoma Nature 2005 436 117 22 Sebat J Lakshmi B Troge J Alexander J Young J Lundin P Maner S Massa H Walker M Chi M et al Large scale copy number polymorphism in the human genome Science 2004 305 525 8 Sharp AJ Locke DP McGrath SD Cheng Z Bailey JA Vallente RU Pertz LM Clark RA Schwartz S Segraves R et al Segmental duplications and copy number variation in the human genome 4m J Hum Genet 2005 77 78 88 Tuzun E Sharp AJ Bailey JA Kaul R Morrison VA Pertz LM Haugen E Hayden H Albertson D Pinkel D et al Fine scale structural variation of the human genome 2005 37 727 732 12 Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual 85 86 87 88 89 90 9 92 93 94 95 96 Iafrate AJ Feuk L Rivera MN Listewnik ML Donahoe PK Qi Y Scherer SW Lee C Detection of large scale variation in the human genome Nat Genet 2004 36 949 5 Sharp AJ Cheng Z Eichler EE Structural Variation of the Human Genome Annu Rev Genomics Hum Genet 2006 Feuk L Carson AR Scherer SW Structural variation in the human genome Nat Rev Genet 2006 7 85 97 Feuk L Marshall CR Wintle RF Scherer SW Structural variants changing the landscape of chromosomes and design of
204. uman SNP Nsp Sty 5 0 Assay 48 Sample Protocol and Appendix C E gels for more information and instructions Following fragmentation run samples on a gel Successful fragmentation is confirmed by the presence of a smear of less than 200 bp in size See Chapter 4 Affymetrix Genome Wide Human SNP Nsp Sty 5 0 Assay 48 Sample Protocol and Appendix C E gels for more information and instructions Run controls in parallel with each group of samples Substitute water for DNA at the PCR step as a negative control The absence of bands on your PCR gel for this control confirms no previously amplified PCR product has contaminated your samples Use Reference Genomic DNA 103 as a positive control included in the reagent kit These controls are effective troubleshooting tools that will help you confirm the successful completion of each stage of the assay Oligonucleotide controls are included in the reagent kit These controls are added to the target samples prior to hybridization and act to confirm successful hybridization washing staining and sensitivity of the array The oligonucleotide control reagents contain oligo B2 which is used for grid alignment For greater efficiency we recommend using a team approach to sample processing This approach is described About Using Controls on page 35 Regularly calibrate all multichannel pipettes Check that your spectrophotometer is accurately calibrated and be sure the OD measurement is within the quanti
205. uman genome sequence it was clear that sites of genetic variation could be used as markers to identify disease segregation patterns among families This approach successfully led to the identification of a number of genes involved in rare monogenic disorders 1 Now that the genome sequence has been completed and is publicly available 2 3 attention has turned to the challenge of identifying genes involved in common polygenic diseases 4 5 The markers of choice that have emerged for whole genome linkage scans and association studies are single nucleotide polymorphisms SNPs Although there are multiple sources of genetic variation that occur among individuals SNPs are the most common type of sequence variation and are powerful markers due to their abundance stability and relative ease of scoring 6 Current estimates of the total human genetic variation suggest that there are over 10 million SNPs with a minor allele frequency of at least 5 7 The ongoing international effort to characterize human haplotypes HapMap Project in four major world populations will identify a standard set of common allele SNPs that are expected to provide the framework for new genome wide studies designed to identify the underlying genetic basis of complex diseases pathogen susceptibility and differential drug responses 8 9 chapter 1 Overview 3 Genome wide association studies which are based on the underlying principle of linkage disequilib
206. vanced Figure 6 7 on page 154 Block size refers to the number of probesets to process at once This parameter is useful when memory is limited If set to 0 default BAT 2 0 attempts to determine the available RAM and set it appropriately Refer to the apt probeset genotype manual for more information on adjusting this parameter Probeset ID File allows you to restrict analysis to a subset of SNPs Run time is directly proportional to the number of SNPs specified Using this option can greatly speed up the run time if you are interested in only a subset of SNPs 154 Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual x NOTE If the Probeset ID File option is used CHP files are not created Results are in a tab delimited text file B BRLMM P Advanced Analysis Options aax Block size O Probeset Id File Browse Figure 6 7 Advanced Analysis Options for the OC and Genotyping Workflow The use of advanced analysis options is indicated in the main Algorithm Options window by the text Advanced options specified Figure 6 8 BRLMM Analysis Tool E Algorithm Options for BRLMM P Parameters Score Threshold 0 05 Advanced options specified QC Call Rate Threshold 86 Select the QC report path C Documents and SettingsNiburriMy DocumentsND ata genotype data BAT Output 20061208_140238 qc r Output Options Output CHP Files AGCC Format C Output text files C Output tables
207. x llle 68 Add Nsp Ligation Master Mix to Reactions 005 68 Dilute the Samples sseseee es 70 What T D Next s v a Duaya pseudo pes QE SE d di ue iude 70 iv Affymetrix Genome Wide Human SNP 5 0 Nsp Sty Assay Manual stage GaNsp PGRs us tst o ht tle eee N nha At ts fee QA saa AA 71 About this Stage kk kk kk kK kK ee KI KK eee 71 Locationrandi Duration 22 ende RR led C E a 71 Input Required from Previous Stage KK RR RR RR KK 71 Equipment and Materials Required AVK KK 72 Reagents Required vk kk kk kK KK KK KK es 73 Gels and Related Materials Required WA RR RR RR 73 Important Information About This Stage 00 20 e ee 74 Prepare the Reagents Consumables and Other Components 74 Aliquot Nsp Ligated DNA to the PCR Plates 75 Prepare the Nsp PCR Master Mix KK K KK KIRI RI KII II ee II 76 Add Nsp PCR Master Mix to Samples 00 0000 77 Load Nsp PCR Plates Onto Thermal Cyclers 0 78 Running Gels cise PER PP RM 80 What TODO NEX iius la ded eet de a dal eo ele as 81 Stage 7 PCR Product Pooling and Purification 00 82 About THIS Stage A kirh eese e rest E br l da E SR ded act 82 Locatonand Duration n st E Y Ne PUO ER S toties 82 Input Required from Previous Stage 0 0 ce eee eee 82 Equipment and Consumables Required lll 83 Reagents Required v

Genome-Wide Human SNP Nsp/Sty Assay 5.0 User Manual

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