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SNPlex Genotyping System, 48

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1. Figure 5 4 Raw data view of allelic ladder showing pull down peaks Solution To prevent spectral calibration problems from occurring in future runs 1 Inspect the raw data from each capillary for pull up or pull down peaks 2 If you see pull up or pull down peaks from Several capillaries Rerun the spectral calibration using Dye Set S Only a few capillaries You can override the spectra in those capillaries with spectra from neighboring capillaries To override the spectra 1 Start the Data Collection software for the Applied Biosystems 3730 DNA analyzer 2 In the plate schematic click the well s that you want to override then click Override Spectral 3 From the drop down list select the nearest capillary with an acceptable spectral then click OK 4 Click Save then rename the calibration file The Data Collection software uses the newly created spectral calibration file as the default Refer to the Applied Biosystems 3730 3730xl DNA Analyzer Getting Started Guide for more information about running and troubleshooting spectral calibrations Troubleshooting Example lon Fronts An ion front typically affects a single peak or pair of peaks resulting in narrower and taller peaks than the
2. 1 24 SNPlex Genotyping System 48 plex User Guide Laboratory Design Preventing Amplicon Contamination Laboratory Design Preventing Amplicon Contamination Product Design Preventing amplicon contamination from previous PCR runs is especially important in protocols that use universal primers for all amplifications To help prevent amplicon contamination the SNPlex System kits are designed to be used in a linear workflow dedicated components used in each step of the assay are not reintroduced in subsequent steps of the workflow In addition the OLA master mix contains uracil N glycosylase UNG UNG acts on single and double stranded dU containing DNA to cause the release of uracil creating an alkali sensitive apyrimidic site in the DNA UNG has no activity on RNA or dT containing DNA Two Laboratory In order to minimize the risk of downstream PCR products contaminating upstream Areas OLA reactions SNPlex System experiments should be conducted in two separate laboratories OLA and PCR OLA Laboratory In this laboratory you perform OLA purify OLA products and assemble the PCR reactions You may prepare gDNA samples in the OLA laboratory or in a separate amplicon free area Observe the following precautions e Limit access to the OLA lab As much as possible a person who enters the PCR lab should not enter the OLA lab for the rest of the day Wear safety goggles lab coats gloves and
3. xvi Si E AMA xvil SDS WAS SUIS ass rassure EORR CUERO dd xvili Biologipal Hazard SAS TT exe send 40 RUERUR 3H EA EE oda Oe xix Workstation Safety auda aceanschon dod dede o dee ich RR dee Rr d XX SNPlex Genotyping System 48 plex User Guide XV Safety Information Safety Conventions Used in This Document XVI Safety Alert Words Four safety alert words appear in Applied Biosystems user documentation at points in the document where you need to be aware of relevant hazards Each alert word IMPORTANT CAUTION WARNING DANGER implies a particular level of observation or action as defined below Definitions IMPORTANT Indicates information that is necessary for proper instrument operation accurate chemistry kit use or safe use of a chemical AN Aue Indicates a potentially hazardous situation that if not avoided may result in minor or moderate injury It may also be used to alert against unsafe practices rene Indicates a potentially hazardous situation that 1f not avoided could result in death or serious injury N PYiicldid Indicates an imminently hazardous situation that if not avoided will result in death or serious injury This signal word is to be limited to the most extreme situations Except for IMPORTANTS each safety alert word in an Applied Biosystems document appears with an open triangle figure that contains a hazard symbol These hazard symbols are identical to t
4. 3 26 Abour Results GFOUpS x Iw bra iaa 3 26 Setting Up Results Groups 3 26 ADout Plate Records iu sia 9 2 2 06cm e de D RE os an od Re DUAE ddl S 3 28 Creating Plate Records 3 29 Using GeneMapper Software 3 30 Creating Plate Records by Importing Formatted Text Files 3 31 Creating Plate Records Manually 3 32 SNPlex Genotyping System 48 plex User Guide vii Chapter 4 Chapter 5 viii Loading and Running the Sample Plates 3 33 l Iate JASSelTIDIy o ia oe RUE mga Iob ge Saeed Alb wade ibus Reg ays 3 33 Required Materials 53923 2i tweed ee ew hoo a wh eria a Raid ote S aT 3 33 Loading Sample Plates 25 255 3 dida bed ewe Po ee auod rir b ed 3 33 PrereQuISITOS iz rat rad id aid 3 33 RUNNING the Plates siii sd ar etn Pur 3 34 Analyzing Data Using GeneMapper Software GB yuca ao aos AS Ge eae a 4 2 Installing GeneMapper Software V3 7 4 3 Importing SNPlex System Panels and Bins 4 3 About SNPlex System Panels and Bins 4 3 Importing SNPlex System Panels and Bins 4 3 IMPORT AE E cC ECCLE DEN 4 5 About Assay Information Files for the SNPlex System
5. 0 22 Chemistry Problems escitas CR 5 22 Troubleshooting Positive Hybridization Controls 5 22 Troubleshooting Negative Hybridization Controls 5 25 Troubleshooting Cluster Plots 5 26 SNPlex Genotyping System 48 plex User Guide Appendix A Using the SNPlex System Control Set Appendix B Index lroddcEDescHpblOoll 3 9 9 0 arse Bd E uberem dao n a Aeg Ar A reci ew dob A 2 About the Control Pool SNPs 2 A 2 About the Dried gDNA Plate llle Ie A 4 Using the GOntfol POOL aso aet os CR EORR D AA AAA ERES A 5 EXDected Results vs RA EE Rar Rp Ead NS ade eae A 5 GeneMapper Software v3 7 Analysis Methods for SNPlex oystem Assays Analysis Methods for SNPlex System Assays B 2 OMV a sucer uut lm ELEME LM O etu S a LE B 2 Selecting a Clustering Algorithm B 2 Terms Used in Clustering Analysis B 3 SNIPISx RUNES 37 90 erro ad Deo Nob rte ac Re MO EN n eto is o Re eo eh Oe een B 4 How the Rules Algorithm Works B 4 Settings of the SNPlex Rules 3730 Method B 5 Selecting an Allele Calling Method B 7 Modifying the Allele Cut off Value B 7 Modifyi
6. LSO Linker TT gDNA Exonuclease SNPlex System Lambda Exonuclease SNPlex System Exonuclease Buffer SNPlex System emmmmmmm 5 a a Unligated Probes Amplification Master Mix SNPlex System Amplification Primers SNPlex System Denature ZipChute Dilution Buffer SNPlex System ZipChute Mix 48 plex SNPlex System Hn Remove supernatant Size Standard 48 plex SNPlex System Allelic Ladder 48 plex SNPlex System 3 Applied Biosystems 3730 3730xl E Data Collection Software v2 0 or higher GeneMapper Software Sample Loading Reagent SNPlex System Legend Universal PCR primer sequence Universal reverse PCR priming site Genome equivalent region Mw ZipCode sequence li E oon P 5 phosphate LSO Locus specific oligo ASO Allele specific oligo A1 ASO 2 Allele specific oligo A2 ASO ASO linker L1 ASO 2 ASO linker L2 Universal reverse PCR primer biotinylated Universal forward PCR primer ZipChute probe Fluorescent dye label Mobility modifiers m ZipCode sequence X SNPlex Hybridization Plate Figure 1 3 Summary of the SNPlex System Assay process SNPlex Genotyping System 48 plex User Guide Chemistry Overview Preparing and Fragmenting Genomic DNA About Purification Kits for Purifying Genomic DNA Whole Genome Amplification About Fragmentation To prepare the
7. 4 5 IDiperting lb Sie LR as una a ee asie dd sas 4 5 Importing SNPlex System Data into GeneMapper Software 4 6 Required Fields EX LRU ls a oe oa ae caf oa dte e De n 4 6 Setting Analysis Method Size Standard and Panel Automatically 4 7 Importing Sample Files ss 93 a te o Da ed RR A 4 8 Analyzing SNPlex System Data 4 8 Reviewing Results 452 0 da do e AA B deb enfe cde ge d as 4 9 Exporting SNPlex System Data 4 11 Troubleshooting Troublestiootilig PFOCSSS 5 daa a ai e RE HR ee cb cd a dog eun ek 5 2 Troubleshooting Raw Data a Eater bus di ro iE S 5 3 Viewing Raw Data siii ERROR RI vec AERA eerte os acd a oy dae ie da 5 3 Electrophoresis Related Problems 5 4 Troubleshooting Signal Strength 2 5 4 Troubleshooting Resolution leeren 5 6 Troubleshooting Spectral Calibration 5 6 TrOUDISSNOQUNO Ion FONS sc oc Coe bout Lu DR chee tie Unes 5 7 Troubleshooting GeneMapper Software Analysis 5 10 GeneMapper Software Setup Problems 5 10 Troubleshooting Sizing Quality 5 10 Troubleshooting Allelic Ladders 5 18 Troubleshooting Analyzed Data
8. File Edit View Tools Alleles Help Insi l N23 LADDER 084 2004 0504f ja LADDER SNPlex 4plex Pand vl 084 10 M5 1_ MI5 1 M5 1 M5 M5 1 MEL M5 1_ 5 1008 M5 1 M5 M5 M5 1 ME M5 1 MS M5 1_ M5 1 M5 1 M5 1_0 M5 1 MS M5 1 M5 1 M5 1_ M5 1 M5 1 0 M5 1 0 M5 1 0 M5 1 028 20 30 40 50 50 70 80 30 100 110 2 1800 1600 1400 1200 1000 800 600 400 200 odo T LT de P Ao ih P si Each bin contains a single peak except for the following bins which should be empty Blue ladder gray bin for M5 1_CTL_002_PHC e Blue ladder pink bin for M5 1 CTL 001 NHC SNPlex Genotyping System 48 plex User Guide 4 9 Chapter 4 Analyzing Data Using GeneMapper Software 4 10 l0 x File Edit View Tools Alleles Help an RIEN reef E Lf 1H E fuc 3 PU sf RT ds i t Insi lt amp N23 LADDER 084 2004 05 04 fsa LADDE Y SNPlex 4 plex Pand vl i 0 4 miw efen MTCC O_O MONO MELUIID ME idi Ej gt X 28 53 Y 2317 Y NHC Blue Ladder Green ladder gray bin for M5 1_CTL_004_PHC e Green ladder pink bin for M5 1 CTL 003 NHC nix l File Edit View Tools Alleles Help Plot Setting SNPlex v2 1 v E Panes fi l DR um eu SE ha RUE fi fe bae LT ds fi 44 Samp
9. For a description of how to run the protocol on the ABI PRISM 7000 7700 or 7900HT Sequence Detection Systems refer to Human DNA Sample Quantification Protocol Using the RNase P Kit PN 4342582 PicoGreen Kits IMPORTANT If you use PicoGreen Kits to quantify DNA quantify the DNA before fragmenting it The PicoGreen dsDNA Quantitation Reagents and Kits from Molecular Probes are also available for double stranded DNA quantitation Note that fluorescence based assays are more sensitive to the quality of the gDNA preparation and consequently poor DNA quality can lead to falsely high values for gDNA concentration To achieve tight genotype clusters with the SNPlex System assay consider increasing the amount of input gDNA Molecular Item Probes Part Number PicoGreen dsDNA Quantitation Kit P 7589 PicoGreen dsDNA Quantitation Kit special packaging P 11496 PicoGreen dsDNA Quantitation reagent P 7581 PicoGreen dsDNA Quantitation reagent special packaging P 11495 SNPlex Genotyping System 48 plex User Guide 1 17 Chapter 1 Introduction Phosphorylating and Ligating Probes to gDNA OLA Protocol The SNPlex System Oligonucleotide Ligation Kit allows you to perform several Summary reactions of the SNPlex System assay workflow simultaneously see Phosphorylating and Ligating Probes to gDNA OLA on page 3 9 Step Description Prepare the Thaw a multiplexed set of SNPlex System ligation
10. Size PHC NHC Standar Potential Cause Possible Solution d OK OK OK DNA samples Repeat experiment with contaminated with DNA of uncontaminated DNA a different genotype Decontaminate pipettors or robotic tips with 1096 bleach solution Off scale peaks result in Too much PCR product spectral pull up in a bin of transferred to post PCR a different color causing step Adjust amount of angle distortion in the SNP PCR product transferred to clusters See Figure 5 21 streptavidin plates for further explanation Heduce injection time for capillary electrophoresis Additional dilution of samples with sample loading reagent containing size standard OLA probes are not SNP may not be specific to a unique assayable locus two different regions may be genotyped by one probe set Note Strong possibility if nonhuman SNPs are assayed and no genome screen is available during probe design Used plate cover that is Use recommended plate incompatible with the covers see Table 1 3 on SNPlex System assay page 1 10 High High OK Insufficient washing after Reoptimize washing steps signal signal ZipChute probe ensuring that all liquid is hybridization removed from wells at each step High High High Incorrect CE protocol Verify that correct signal signal signal injection too long electrophoresis protocol was used Decrease injection time Run allelic ladder plate and reoptimize electrophoresis performance
11. _ M UM M5 MEL M M M ML M ML ME M M5 M ML M ME _ MB M5 MB 20 60 70 60 90 100 110 120 Chinese SNPlex 4 plex Pand vl E 10 M5 MB 1_0 MB MB M5 ML ML M M5 ML M M5 M MB M ML MB Figure 5 11 Good allelic ladder sample with even signal and accurate binning Viewing Allelic Ladders in Plot View To inspect the allelic ladders for a run open them in Plot View Analysis gt Display Plots then select the SNPlex System plot settings from the Plot Setting menu As shown in Figure 5 11 the green and blue SNPlex System allelic ladders should be displayed independently and bins should be superimposed over the ladder peaks Note If peaks are not within bins establish proper binning before any further analysis Solution 1 Confirming That Allelic Ladders Are Injected With Each Set of Samples Because the GeneMapper software uses information from allelic ladders to compensate for run to run variations allelic ladders need to be included with each injection of samples to calculate and apply the necessary bin offset to all samples SNPlex Genotyping System 48 plex User Guide Troubleshooting GeneMapper Software Analysis If an allelic ladder is not included or omitted from a run the software attempts to use the default bin settings without any bin offsets when analyzing the samples If the peak positio
12. Hybridize ZipChute probes Purify OLA products Exonuclease Purification Kit Elute ZipChute probes Dilute purified OLA products Prepare sample plates for electrophoresis Assay Standards Kit Prepare PCR reactions Amplification Kit Create results groups and plate records Load and run sample plates Analyze data in GeneMapper software v3 7 Figure 1 8 Division of procedures between OLA and PCR labs SNPlex Genotyping System 48 plex User Guide 1 27 Chapter 1 Introduction 1 28 SNPlex Genotyping System 48 plex User Guide oetting Up Applied Biosystems 3 30 3 30xl DNA Analyzers for P SNPlex System Experiments This chapter covers CM Le oque d iaa ri as eds 2 2 Importing SNPlex System Files into the Data Collection Software 2 3 Performing Spatial and Spectral Calibrations 2 6 Validating Instrument Performatiee csi a Fais eut 2 8 SNPlex Genotyping System 48 plex User Guide 2 1 Chapter 2 Setting Up Applied Biosystems 3730 3730xl DNA Analyzers for SNPlex System Experiments Overview setting up the Applied Biosystem 3730 3730x DNA Analyzers for use with the SNPlex Genotyping System involves Importing SNPlex System modules into the Data Collection software Note Systems upgraded to Data Collection v3 0 have the SNPlex System instrument files pre installed PrebatchModule txt Module HTSNP36 POP7 V3 Dye Set S Prec
13. SNPlex Genotyping System 48 plex User Guide To dispense the allelic ladder continued Preparing Samples for Electrophoresis 5 Load the Allelic Ladder Note The Allelic Ladder 1s part of the SNPlex System Standards Kit If using 96 well plates pipette 1 25 uL of Allelic Ladder 48 plex plus 10 uL of Sample Loading Mix into the wells designated below If using 384 well plates pipette 1 0 uL of Allelic Ladder 48 plex plus 7 5 uL of Sample Loading Mix into the wells designated below 96 capillary array 96 well plate o 10 uL from hybridization plate 6006006000 600600600600 60000000 6000600000 60000000 600060000600 00000000 6006000600 6006006000 00000000 00000000 1 25 uL Allelic Ladder 10 uL Sample Loading Mix eeoo0000 48 capillary array 96 well plate o 10 uL from hybridization plate o w gt T m 1 25 uL i 0 00000000 00000000 900000000 00000000 000000090 60000000 00000000 00000000 00000000 00000000 60000000 60000000 E Allelic Ladder 10 uL Sample Loading Mix 96 capillary arrays 384 well plate Y7 5 uL from VOLE TAS TL 7M eo D gt 0000000000000000 0000000000000000 0000000000000000 0000000000000000 0000000000000000 0000000000000000 00000000000000003 0000000000000000 gt 0000000000000000o 00000000000000003 0000000000000000 000
14. amp 660600090 90006600 0668608860 960600060060 8068060 0606006000 0000060009 O TI m x SNPlex Genotyping System 48 plex User Guide Designing the Sample Plate Layout 3730 Instrument An instrument running a 48 capillary array injects twice from each of the quadrants 48 capillary of a 384 well plate performing eight separate runs 384 wells pa 1 pp a Injection 2 Injection 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 4 T i e i i i N 1 wm 14 15 16 17 18 19 20 21 22 23 24 8 9 A 00 00 12899 1100776807100 es OO BOO 49 OOD amp HOO 1 HOO BO DOO d d 65 amp 5 E 65 d 49 49 6 6 65 65 9 19 6 6 65 65 DS 85 69 69 4 4 9 49 6 6 65 65 9 5 6 61 65 6 amp 5 OO 1909 54 DO so so HED e CO ce 2090 50 50 PELE o 4 60 60 69 69 66 66 9 6b 66 OO 19 DO ss 35 OO st 100 7 67 909 6002656600 90200 OO 299 26 36 MO 2600 68 9 es 4000500669 0 0 62 60 60 6 66 6 OO 21890977090 ss 200 o 6 58 53 21 amp 9 29 6 6 5 45 65 69 6 6 69 69 p DD 2 EYED se e OO 1 00 70 70 54 54 BO 0 BBO do 6 62 6o 62 0 10 b C969 200 39 DO 5 55 OO 1 10 65 65 25 6 6 69 65 4 65 EE 65 9 O G9 69 OO o 4o 99 se 56 632 2 72 G9 65 69 e6066025206900006605000006 Injection 4 Injection 8 123 45 6 7 11 12 Tr UO O UJ 0 00 0 0 0 0 0 0 Q QsQO s0r0 0 0 0 AER 0 0
15. y a EH i C rese jw m Numbers recommended for troubleshooting Ea EXC EN ES pese m Ni ses m m SNPlex Genotyping System 48 plex User Guide Chapter 5 Troubleshooting To switch sizing quality views in GeneMapper software ll Select Tools gt Options then select the Analysis tab 2 Under Quality Metrics Display select Numbers En me General Add Samples Analysis Automatic Analysis Ie Automatically bring errors to the top of the table Gualty Metrics Display C Symbols Number If only one labelled allele in a genotype then duplicate the label Duplicate homozygous alleles Click OK to apply your settings Determining Sizing Quality of Size Standards If the sizing quality of a sample 1s less than 0 98 check the size standard for the sample using the Size Match Editor select Analysis gt Size Match Editor For size standards to produce good sizing quality all standard peaks should Have signal strength gt 50 RFU e Have relatively uniform signal strengths Becorrectly identified by GeneMapper software Example Good Sizing Quality Figure 5 7 shows a size standard with good sizing quality Note the even peak height and low background SNPlex Genotyping System 48 plex User Guide Troubleshooting GeneMapper Software Analysis g Size Match Editor File Edit View Tools EG Ins
16. SNPlex Genotyping System 48 plex User Guide Troubleshooting Analyzed Data 104 31 1 M 81 32 83 34 Figure 5 21 Off scale peaks cause spectral pull up As illustrated in Figure 5 21 blue off scale peaks in M5 1 023 cause a background signal due to the spectral pull up in M5 1 044 As a result angle differences occur during clustering for this marker producing data points that are smeared along the Y axis SNPlex Genotyping System 48 plex User Guide 5 35 Chapter 5 Troubleshooting 5 36 SNPlex Genotyping System 48 plex User Guide Using the SNPlex System Control oet This chapter covers Product DesctiPU ado soe en ose qu THRERI S DP RIEN d NX IE dV TIS A 2 Aboul Ihe Control Pool SNPS iremos RO ERRARE AA 054090040085 A 2 LED ISA DION S a4 24522 9279 45 4 9 1 308 389 EROR EUEROR D RADAR DRE pe RR A 5 PUB MA A 5 SNPlex Genotyping System 48 plex User Guide A 1 Appendix A Using the SNPlex System Control Set Product Description The SNPlex System Control Pool PN 4362635 is a set of ligation probes that can be used together with the SNPlex System Dried gDNA Plates Kit PN 4362637 to evaluate the performance of the SNPlex System When used with SNPlex System kits and reagents the ligation probes in the control pool detect 48 human SNPs in a single mutliplex reaction About the Control Pool SNPs The Control Pool SNPs Were selected from a list of SNPs for which val
17. Universal PCR primer sequence PCR reaction Universal reverse PCR priming site Amplification Master Mix SNPlex System Amplification Primers SNPlex System Genome equivalent region aww ZipCode sequence Universal reverse PCR primer biotinylated GeneAmp PCR System 9700 Thermal Cycler plc TOMATO PE M primier Thermal cycle a O Figure 1 7 PCR amplification procedure SNPlex Genotyping System 48 plex User Guide 1 21 Chapter 1 Introduction Hybridizing PCR Products to ZipChute Probes and Performing Electrophoresis 1 22 Protocol Summary The hybridization reagents binding buffer wash buffer ZipChute dilution buffer ZipChute kit and the SNPlex System Standards Kit use fluorescently labeled ZipChute probes and size standards to analyze the results of the assay see Hybridizing PCR Products to ZipChute Probes on page 3 18 Table 1 7 SNPlex protocol summary Step Prepare the hybridization plates then bind the PCR products to the hybridization plates Description Wash the streptavidin coated plates and combine the Binding Buffer SNPlex System with the Positive Hybridization Control SNPlex System Add binding buffer containing positive hybridization control to the hybridization plate then transfer PCR reactions to the hybridization plate Remove unbound material Wash the wells to remove unbound material Add denaturant to separate the strands The double stranded amp
18. P ertermirig POR 32 xenon uet piod d desir Gea OSA d ede oe CR CR dE ERR ae tee ana ak 3 16 Amplification Kit Components 3 16 Required Materials 3 16 Preparing the PCR Master Mix 3 16 Assembling and Running the PCR Reaction 3 16 Hybridizing PCR Products to ZipChute Probes 3 18 Reagents Required for Hybridization 3 18 Required Materials 3 18 Preparing the Hybridization Plates 3 18 Binding PCR Products to the Hybridization Plate 3 19 Isolating Biotinylated Strands on the Hybridization Plate 3 19 Hybridizing the ZipChute Probes 3 21 Eluting ZipChute Probes ovio rca E RE on dat eee eee 3 22 Standards Kit Components 3 22 Required Materials sia A or e SOR Kerr oM GE EROR 3 22 Preparing the Sample Loading Mix 3 22 Eluting the ZipChute Probes 3 23 Preparing Samples for Electrophoresis 3 24 Creating Results Groups and Plate Records 3 26 Starting Data Collection Software
19. Read from the Sample Set 310 377 Matrix to for all samples Read from the Sample r Set Panel to ex 48plex Panel 3730 for all samples O Read from Data collection Comment Panel field Set Sample Type to Q for all samples Read from Data collection Info field The size standard and panel should be the same for all analyses However you can set the analysis method to either SNPlex_Model_3730 or your samples For more information about analysis methods refer to Appendix B SNPlex Rules 3730 depending on which method you are using to analyze 3 Make sure that you set e 310 377 Matrix to Read from the Sample e Sample Type to Read from Data collection info field SNPlex Genotyping System 48 plex User Guide 4 7 Chapter 4 Analyzing Data Using GeneMapper Software Importing Sample GeneMapper software retains the folder structure of imported sample files That is if Files the Data Collection software stores each run in a separate folder GeneMapper software also creates one folder for each run For GeneMapper software to correctly analyze data all sample files from a run must be stored in the same run folder Additionally an allelic ladder sample from the same run must also be included in each run folder For 384 well plates sample files should be stored in four run folders for 96 capillar
20. See Design sample plate layout See Run PCR See Prepare hybridization plates and bind PCR product to plates See Prepare gDNA PCR Laboratory OLA Laboratory Phosphorylate and Phosphorylate and ligate probes to ligate probes to gDNA OLA dry gDNA OLA wet Add denaturant isolating biotinylated strand on hybridization plate Oligonucleotide Ligation Kit See Hybridization Reagents and Kits Purify OLA products Hybridize ZipChute probes Exonuclease Purification Kit Elute ZipChute probes Dilute purified OLA product i p See Assay Standards Kit Prepare sample plates for electrophoresis See Prepare PCR reactions Amplification Create results groups and plate records Load and run sample plates See Analyze data in GeneMapper software v3 7 Figure 3 1 SNPlex System experiment workflow SNPlex Genotyping System 48 plex User Guide Before You Begin Designing the Sample Plate Layout At this point you should already have ordered your SNPlex System probe pools If you have not done so refer to the SNPlex Genotyping System Assay Ordering Guide PN 4357460 for information about designing and ordering SNPlex System probe pools Designing the Sample Plate Layout Purpose Assumptions 3 30xl Instrument 96 capillary 96 wells To analyze SNPlex System data GeneMapper software requires that each run ncludes
21. samples use the standard analysis method to analyze all other samples Solution 4 Removing an Allelic Ladder from a Project GeneMapper software averages all allelic ladders from a run when determining binning offsets A misbinned ladder can throw off the average for the entire run When you cannot correct problems in an allelic ladder you may have to remove the ladder from the project to achieve correct binning To remove an allelic ladder go to the Samples View click the defective ladder then select Edit gt Delete From Project Reanalyze the samples after removing the problematic ladder Note Removing all ladders from a run can cause serious binning problems Make sure to examine samples for correct binning SNPlex Genotyping System 48 plex User Guide 5 21 Chapter 5 Troubleshooting Troubleshooting Analyzed Data Chemistry Problems Troubleshooting 5 22 Positive Hybridization Controls Problems that are not attributable to the electrophoresis or GeneMapper software set up processes may be related to the assay chemistry Troubleshooting SNPlex System assay chemistry involves inspecting assay controls cluster plot patterns and electropherograms Use Figure 5 13 to troubleshoot problems related to SNPlex System assay chemistry Evaluate Positive Hybridization Control PHC Is the signal intensity of marker Marker M5 1 CTL 002 PHC at least 150 RFU For more information r
22. 1 0 l CTL 004 PHC 85 86 Allele 2 A2 i 300 0 2000 700 0 12000 17000 22000 27000 Allele 1 A1 Figure 5 14 PHC signals from a single 96 capillary run A Electropherograms of the two PHC peaks B PHC signals from all samples in polar plot view C PHC signals from all samples in Cartesian plot view SNPlex Genotyping System 48 plex User Guide 5 23 Chapter 5 Troubleshooting Step 1 Evaluating Overall Median Signal Intensity Variations in signal intensity between runs or instruments can be expected The typical overall median signal intensity ranges from 150 2400 RFU Overall median signal intensities significantly lower than these values may indicate a problem in the post PCR steps of the assay For example incorrect dilution of PHC in binding buffer using binding buffer instead of ZipChute hybridization buffer during the ZipChute probe elution step or incomplete removal of hybridization wash buffer before ZipChute probe elution can cause overall signal intensity to drop Step 2 Evaluating Variation in Signal Intensity Across the Plate Variations in PHC signal intensity between runs or instruments can also be expected Variations up to an eightfold difference are acceptable Anything more indicates possible problems with post PCR assay procedures typically related to pipetting inaccuracies If you notice large variations in signal intensity across a plate inspect and recalibrate pipettes and robots as ne
23. 16 0 24 0 34 0 36 Chr 12 16 550 429 M5 1 012 hCV8879897 0 43 0 46 0 44 0 45 Chr 10_12 356 433 M5 1 013 hCV7505765 TSCO265430 rs961495 0 26 0 19 0 20 0 08 Chr 14 100 278 705 M5 1 014 hCV1637791 TSC0016017 rs 29673 0 45 0 40 0 24 0 20 Chr 18_3 057 735 M5 1 015 hCV1691378 TSCO265475 rs1115261 0 48 0 46 0 18 0 28 Chr 13 27 191 865 A 2 SNPlex Genotyping System 48 plex User Guide About the Control Pool SNPs Table A 1 48 SNPs in the SNPlex System Control Pool continued Celera SNP Minor Allele Frequency Zip Discovery Consortium dbSNP ID System ID ID rs AfA Cauc Chin Japn vs hCV TSC Mb c d e Location M5 1 016 hCV3194437 TSCO301076 rs984071 0 20 0 29 0 17 0 17 Chr 9 10 596 485 M5 1 017 hCV7571632 TSCO243654 rs927221 0 16 0 15 0 28 0 45 Chr 14_66 832 202 M5 1 018 hCV7537265 TSCO129483 rs 48573 0 32 0 22 0 33 0 33 Chr 2 45 728 104 M5 1 019 hCV7589926 0 30 0 11 NA NA Chr 9 3 834 392 M5 1 020 hCV8845932 TSCO324505 rs1156404 0 27 0 47 0 17 0 17 Chr 6 70 690 465 M5 1 021 hCV2179737 0 33 0 20 0 43 0 31 Chr 8 1 599 764 M5 1 022 hCV8792022 TSCO213380 rs8 9253 0 21 0 45 0 21 0 08 Chr 5 14 827 607 M5 1 023 hCV2489240 TSC0098582 rs1016146 0 12 0 35 0 48 0 27 Chr 6 35 446 932 M5 1 024 hCV2025116 TSCO757622 rs1507213 0 12 0 48 0 48 0
24. 30 db 50 30 310 40 50 10 29 5640 60 3 5HPCyS621778R5p22 J SNP CVBa7z47570 R 10 20 14 40 54 IB 20 10 4D 54 10 20 20 ab 50 10 30 20 49 50 ib 2B oh i B iB dh 30 4B di ih zB 36 di db iB ih 30 d di 34 SHPCVwTGBBD2 F 5pio 349 SNP Cva536350 R5p r AT 5MHPCv2962785F A E 13 E 15 E j E DE 3 1 i in Db XB 40 Si ib 20 30 ap 30 in 18 Xp 40 EP 14 z0 30 ab 50 if 30 14 40 44 ib 20 30 40 40 14 30 30 db 55 ig 30 30 40 40 Figure A 1 Cluster plots of the 48 SNPs in the Control Pool A 6 SNPlex Genotyping System 48 plex User Guide GeneMapper Software v3 7 Analysis B Methods for SNPlex System Assays This chapter covers Analysis Methods for SNPlex System Assays B 2 ANT Rues A perisset stener 899 ee ek B 4 SNPlex Model 3730 SNPlex Genotyping System 48 plex User Guide B 1 Appendix B GeneMapper Software v3 7 Analysis Methods for SNPlex System Assays Analysis Methods for SNPlex System Assays Overview Selecting a Clustering Algorithm B 2 An analysis method is a set of algorithm parameters that you apply to samples in a project before analyzing data The GeneMapper software v3 7 uses analysis methods for peak detection allele calling peak quality quantification and quality of results reports GeneMapper software v3 7 includes two default methods for analyzing SNPlex system data SNPlex Rules 3730 Uses the Rules clustering algorithm to calculate the SNP qu
25. 5 6 r4 8 9 10 123 45 6 7 8 9 10 11 12 13 14 15 16 1 19 20 21 22 23 24 100000000000 5 0000000000000000 0000 19860009 gt 0000000000000000 0000 POO OO OOO 0000000000000000 0000 SODODOODOOODOQO 10000000000000000 0000 99099400900 1 0000000000000000 C OOOO OOOOO L 0000000000000000 NIG 00 OOO Di0 00 0000000000000000 0000 E QOOOOOOOOO P 0000000000000000 0000 C Control DNA NTC No Template Control L Allelic Ladder For information about proper sample plate layout refer to Designing the sample Plate Layout on page 3 3 2 Into each of the control DNA wells add e 1 26 uL IX TE buffer pH 8 0 e 0 74 uL Control DNA x37 ng from the SNPlex System Assay Control Kit PN 4349363 Note Control DNA is already fragmented when shipped 3 Into each of the NTC wells add 2 uL of 1X TE buffer pH 8 0 4 Leave the wells for the allelic ladder empty For information on setting up the allelic ladder wells refer to Preparing samples for Electrophoresis on page 3 24 5 Dispense 2 uL of the fragmented gDNA from step 8 on page 3 7 into the remaining wells of the plate then briefly centrifuge the plate to ensure that the contents are collected at the bottom of the wells 6 Allow the plate to air dry for three days in a dark amplicon free location Cover the plate with a lint free tissue while air drying 7 Verify that all the liquid has evaporated 8 Seal the plates and
26. 50 Chr 12 85 457 089 M5 1 025 hCV1653240 TSCO136611 rs 51340 0 42 0 37 0 24 0 18 Chr 9 125 143 032 M5 1 026 hCV357822 TSC0783613 rs1520483 0 10 0 41 0 48 0 48 Chr 3 46 329 443 M5 1 027 hCV8686971 TSC0465947 rs1570903 0 23 0 44 0 45 0 26 Chr 13 94 986 498 M5 1 028 hCV706864 TSC0071873 rs288423 0 42 0 37 0 43 0 38 Chr 15 95 755 875 M5 1 029 hCV3017144 TSC0084538 rs1007106 0 19 0 34 0 45 0 45 Chr 8 124 612 406 M5 1 030 hCV9621778 TSCO318440 rs995178 0 41 0 49 0 49 0 45 Chr 5 22 445 803 M5 1 031 hCV8747570 TSC0679949 rs1597695 0 35 0 39 0 50 0 44 Chr 2 105 883 662 M5 1 032 hCV8862622 TSCO825772 rs1334334 0 15 0 24 0 27 0 17 Chr 1 87 717 343 M5 1 033 hCV8946637 TSC0809047 rs 94108 0 46 0 38 0 29 0 37 Chr 6 164 517 177 M5 1 034 hCV1358402 TSCO463216 rs1569244 0 42 0 17 0 41 0 37 Chr 6 164 517 144 M5 1 035 hCV7500677 TSCO296508 rs238196 0 12 0 10 0 28 0 30 Chr 20_48 536 640 M5 1 036 hCV9589619 TSC0984433 rs1925643 0 42 0 30 0 42 0 42 Chr 10 100 359 810 M5 1 037 hCV8921382 TSCO910879 rs1713423 0 42 0 50 0 31 0 41 Chr 14 18 850 202 M5 1 038 hCV1688032 0 22 0 31 0 16 0 23 Chr 1 184 612 692 M5 1 039 hCV9636350 0 16 0 37 0 36 0 37 Chr 12 122 620 930 M5 1 041 hCV2962785 0 40 0 11 0 17 0 24 Chr 22 25 219 892 M5 1 042 hCV2780152 TSCO851851 rs1861606 0 49 0 31 0 29 0 36 Chr 12 22 349 685 M5 1 043 hCV2569743 TSCO430769 rs1548543 0 22 0 34 0 31 0 29 Ch
27. Biosystems thermal cyclers use the following thermal cycling parameters Thermal cycling parameters are identical for 384 and 96 well plates Step Step Type Temperature C Time 1 HOLD 48 30 min 2 HOLD 90 20 min 3 25 Cycles 94 15 sec 57 7 min 4 HOLD 99 10 min 5 HOLD 4 00 2 When thermal cycling is complete remove the OLA reaction plate from the thermal cycler 3 Briefly spin the OLA reaction plates to collect the liquid in the bottom of the wells 4 For best results proceed directly to exonuclease digestion of OLA products as described in Purifying Ligated OLA Reaction Products on page 3 14 Alternatively you can store the OLA reactions at 20 C for up to 21 days SNPlex Genotyping System 48 plex User Guide 3 13 Chapter 3 SNPlex System Protocols Purifying Ligated OLA Reaction Products For a summary of the steps in the purification procedure refer to Purifying Ligated OLA Reaction Products on page 1 20 Purification Kit The components in the SNPlex System Purification Kit PN 4349357 are listed in Components the table below The kit contains enough reagent for 5 000 reactions Component Storage Temperature C Lambda Exonuclease SNPlex System 15 to 25 Exonuclease SNPlex System 15 to 25 Exonuclease Buffer SNPlex System 15 to 25 Required Refer to Required Non Kit Materials on page 1 10 for a complete list
28. Collection software In GeneMapper software preset Add Samples option for all import data Analysis Method Panel Size Standard Add sample files to Add sample files to Add sample files to GeneMapper software GeneMapper software GeneMapper software To Sample Information add Sample Type Analysis Method Panel Size Standard SNP Set Analyze data with Analyze data with Analyze data with GeneMapper software GeneMapper software GeneMapper software Figure 3 3 Three possible methods for creating plate records SNPlex Genotyping System 48 plex User Guide 3 29 Chapter 3 SNPlex System Protocols Using Another consideration you need to make when creating plate records is which GeneMapper GeneMapper software application you intend to use Software When GeneMapper software is installed on a computer that has Data Collection software two applications are available in the Automated Processing tab of the Results Group Editor GeneMapper Generic Generates sample files but does not perform autoanalysis e GeneMapper Znstrument Name Performs autoanalysis Table 3 2 indicates the required fields for creating plate records using each application Table 3 2 Minimum required fields for fragment analysis data collection Field GeneMapper Generic 2 eee es 3730 3730xI Analyzer Fields Sample Name required required Results Group required required In
29. DNA NTC No Template Control L Allelic Ladder For information about proper sample plate layout refer to Designing the sample Plate Layout on page 3 3 2 Pipette 5 0 uL of OLA reaction mix see step 4 on page 3 10 into each well of the plate IMPORTANT Do not add reaction mix into the allelic ladder wells For information on setting up the allelic ladder wells refer to Preparing samples for Electrophoresis on page 3 24 3 Cover 384 well reaction plates containing the SNPlex OLA reactions with one of the recommended plate covers see Table 1 3 on page 1 10 and an optical cover compression pad Note If you are using 96 well plates use MicroAmp Full Plate Covers to seal the plate a IMPORTANT Applied Biosystems has found that certain plate covers negatively affect the performance of the SNPlex System assay If you use covers other than the recommended plate covers test them using the SNPlex System Control Set see Appendix A SNPlex Genotyping System 48 plex User Guide Chapter 3 SNPlex System Protocols Assembling the You can set up the reaction at room temperature OLA Reaction Wet gDNA To prepare the OLA reaction when using wet gDNA 1 Retrieve and label the appropriate number of reaction plates 96 capillary array 96 well plate 96 capillary arrays 384 well plate gt Er o EF un oou Y nm 0 QOQOL A A T Ox OOO eee ee vesseess CHOCO e
30. Description Number of clusters must be no less than If the software detects fewer clusters than specified in this rule the SNP is failed Sigma separation must be no less than Hardy Weinberg p value must be no less than To determine this value the software measures cluster resolution fuziness and distance between the two clusters It reports a single value that encompasses both cluster width and separation r intensity Applied Biosystems recommends that you do not set this value to 6 because doing so decreases the accuracy Raising the value causes a slight increase in accuracy but also causes more SNPs to fail If set to n n96 of the time a valid SNP is failed For example if set to 0 01 196 of valid SNPs are failed Angle between clusters must be no larger than Call rate must be no less than Signal median value must be no less than Applies only to SNPs with only two clusters In these cases this rule ensures that the software does not call two homozygotes for a single SNP Percentage of genotypes that must be called for a SNP in order for the SNP to pass For example if Call Rate is 0 8 8096 of the genotypes for a SNP must be called in order for the SNP to pass Median value 5096 of the signals for a SNP fall below this value If you modify this value Applied Biosystems recommends that you raise it Do not lower the median value SNPlex Genotyping System 48 plex
31. If you are using the SNPlex Model 3730 analysis method or any method that uses the Model clustering algorithm or any method that uses analyses by project you must import the AIF file into GeneMapper software If you attempt to run the analysis without importing the AIF you will lose information about markers that are associated with SNPs The software assumes that all markers are associated with SNPs For more information about analysis methods refer to Appendix B Each SNPlex System probe pool order is accompanied by a SNPlex Genotyping System Ligation Probes CD which contains the files listed in Table 4 2 Table 4 2 Files in the SNPlex Genotyping System Ligation Probes CD File Description SNPlex nnnnnnnn nnnnnnn xml Assay information file that contains information about the probe pool including SNP set names where nnnnnnnn nnnnnnn is the design ID Do not modify this file ablogo gif Applied Biosystems logo file aiftypes v1 1 xsd Defines the structure of AIF types Do not modify this file Probes Insert DOC Limited license for the SNPlex System ASO LSO probe sets SNPIexAIF v1 1 xsd Defines the structure for the AIF file Do not modify this file SNPlexStylesheet v1 O xslt Contains instructions for displaying the SNPlex System data sheet Do not modify this file Importing AlFs To import AIFs into GeneMapper software 1 Select Tools gt GeneMapper Manager 2 In the SNP Sets
32. RSP instrument to automate the OLA portions of the SNPlex System assay SNPlex Genotyping System 4358100 e Explains how to set up the Tomtec Downloadable from the 48 plex Automating PCR Using Quadra 3 instrument for automating SNPlex System Web site the Tomtec Quadra 3 Getting the post PCR portion of the SNPlex and Documents on Started Guide System assay Demand Web site e Provides the step by step automation protocols for using the Tomtec Quadra 3 instrument to automate the post PCR portions of the SNPlex System assay Related Documentation Applied Biosystems 4331468 Provides information about using the Downloadable from the 3730 3730xI DNA Analyzers 3730 3730x instrument Documents on Demand Getting Started Guide Web site GeneMapper Software v3 7 NA Describes the analysis software and Installed with Online Help provides procedures for common tasks GeneMapper software a http www allsnps com b http docs appliedbiosystems com search taf c http myscience appliedbiosystems com snplex snplexStart jsp SNPlex Genotyping System 48 plex User Guide 1 7 Chapter 1 Introduction SNPlex System Kits and Reagents Overview _ The reagents you need to perform a SNPlex System assay are provided in six parts Kits and Reagents 1 8 e A core reagents kit which contains reagents required to perform the SNPlex System assay e A starter kit which contains additional reagents required by first time SNPlex System u
33. S A NOTICE TO PURCHASER DISCLAIMER OF LICENSE No license to use the Oligonucleotide Ligation Assay OL A Ligation Detection Reaction LDR and or Polymerase Chain Reaction PCR methods covered by patents owned or licensable by Applied Biosystems is conveyed expressly by implication or by estoppel to the purchaser by the purchase of this product Further information on purchasing licenses to practice the patented methods may be obtained by contacting the Director of Licensing Applied Biosystems 850 Lincoln Centre Drive Foster City California 94404 U S A SNPLEX SYSTEM ASSAY STANDARDS KIT 4349351 SNPlex System Assay Standards Kit is covered under one or more of U S Patent Nos 5 654 419 5 707 804 5 688 648 6 028 190 5 869 255 6 177 247 6 544 744 5 728 528 U S patent application 10 288 104 and corresponding foreign patents and patent applications licensed from the University of California NOTICE TO PURCHASER DISCLAIMER OF LICENSE This product is optimized for use in the DNA sequencing or fragment analysis methods covered by patents owned or licensable by Applied Biosystems No license under these patents to use the DNA sequencing or fragment analysis methods is conveyed expressly or by implication to the purchaser by the purchase of this product A license to use the DNA sequencing or fragment analysis methods for certain research and development activities accompanies the purchase of certain Applied Biosystems reagents when
34. System Ligation Probes 2 Thaw the OLA Master Mix at 4 to 8 C then invert several times to mix If a precipitate forms with the OLA Master Mix place the tube briefly in a heating block set to 37 C Note You can store the OLA Master Mix at 4 to 8 C for up to one month or frozen at 20 C for up to 1 year 3 Vortex then quick spin the tubes SNPlex Genotyping System 48 plex User Guide 3 9 Chapter 3 SNPlex System Protocols To prepare the OLA reactions continued 4 Prepare an OLA reaction mix by scaling the volumes indicated below to the desired number of OLA reactions You can set up the reactions at room temperature IMPORTANT Prepare extra volume to account for losses that may occur during pipetting Volume per Reaction uL Component Dried gDNA Method Wet gDNA Method Nuclease free water 2 30 0 30 Oligonucleotide Ligation 2 50 2 50 Master Mix SNPlex System Universal Linkers 48 plex 0 05 0 05 SNPlex System Ligation 0 10 0 10 Probes dATP 100X SNPlex System 0 05 0 05 Total 5 00 3 00 Note Once prepared you can keep the OLA reaction mix for up to 6 hours at room temperature before use without a loss in performance After 6 hours you may store the OLA reaction mix for up to 4 days at 4 C for later use You may also prepare a large quantity of the OLA reaction mix and store it at 4 C or at 20 C for up to 4 days 5 Depending on
35. User Guide SNPlex Rules 3730 Thresholds for the Model Algorithm IMPORTANT Applied Biosystems recommends that you do not modify these settings The Model algorithm rejects samples according to the following threshold values Well signal threshold Represents the mean signal from both alleles of a well It is the sum of all the peaks divided by the number of SNPs Well template threshold Represents how far the well s behavior is from an ideal well 0 5 The software rejects wells that have values that fall below this threshold SNPlex Genotyping System 48 plex User Guide B 9 Appendix B GeneMapper Software v3 7 Analysis Methods for SNPlex System Assays SNPlex Model 3730 How the Model Algorithm Works fsa files from a single run Bin offsets calculated based on allelic ladder First clustering Heject individual samples based on sample performance e f number of passing samples 24 no calls are made e f number of passing SNPs 18 no calls are made Second clustering on only passing samples Call Genotypes e Assign per point quality values e Calculate SNP quality value e Calculate Genotype quality values Filter bad samples based on well quality PQV Filter failing SNPs samples based SNPQ Filter failed genotypes based on genotype CV Figure B 2 Summary of the Model Algorithm B 10 Figure B 2 summarizes how the Model algorithm calculates the SNP quality For fa
36. allele quality values AQ of the two alleles in the SNP and the weighted values of each PQV The GeneMapper software online help provides detailed information about calculating the GQ For the Rules method the GQ is zeroed out if GQs fall below the CV For the Model method the GQ is not set to zero if the value falls below the CV Well Quality WQ Quality flag that represents the mean confidence value CV for all SNPs within a well The software fails wells whose CVs fall below the pass range specified for WQ SNP Quality Value serving as a preliminary indicator of the quality of an analyzed SNPQ SNP For rules based methods 1 indicates a passing SNP 0 indicates a failing SNP For model based methods the SNPQ is reported as a value between 0 and 1 The following sections describe the default methods for analyzing SNPlex System data SNPlex Genotyping System 48 plex User Guide B 3 Appendix B GeneMapper Software v3 7 Analysis Methods for SNPlex System Assays SNPlex Rules 3730 How the Rules Figure B 1 summarizes how the Rules algorithm calculates the SNP quality Algorithm Works fsa files from a single run Allelic ladder assigned Bin offsets calculated First clustering Reject individual samples All points for that sample uncalled Based on Well quality CV unaffected GQ 0 Second clustering on only passing samples Produce SNP characteristics Filter based on Rules if any one
37. analysis method to minimize ion fronts 1 Start the GeneMapper software v3 7 or higher 2 Select Tools GeneMapper Manager then select the Analysis Methods tab 5 8 SNPlex Genotyping System 48 plex User Guide Troubleshooting Raw Data To create an alternative analysis method to minimize ion fronts continued 3 Create a new analysis method based on the current method a From the list of analysis methods select the current analysis method that you are using to analyze SNPlex System data b Click Save As c Enter a name for the new method d Click OK The new method appears on the list of analysis methods e Click Done 4 Select the new analysis method then click Open 5 Inthe Analysis Method Editor select the Peak Detector tab 6 Change the Polynomial Degree to 7 Default is 5 7 Change the Peak Window Size to 9 Default is 11 8 Click OK to save the analysis method 9 Click Done to close the GeneMapper Manager then reanalyze the samples using the new analysis method Inspect the allelic ladder samples for correct binning SNPlex Genotyping System 48 plex User Guide 5 9 Chapter 5 Troubleshooting Troubleshooting GeneMapper Software Analysis GeneMapper Software Setup Problems Troubleshooting 5 10 Sizing Quality The second step in the troubleshooting process is to rule out nonchemistry related problems that m
38. and a final volume between 12 5 and 150 uL Note The starting concentration of DNA affects the fragment size achieved after boiling For more dilute DNA samples you may need to concentrate the DNA or reduce the duration of heating The duration of heating is determined empirically a 1X TE 10mM TrisHCl pH 8 0 and 1mM EDTA b Heat fragmentation is equally effective when you dilute purified DNA in nuclease free water 0 5X TE pH8 0 2X TE pH8 0 1X TE pH 7 5 1X TE pH 7 0 Gentra s PureGene DNA Hydration Solution or Qiagen s FlexiGene Hydration Buffer 3 6 SNPlex Genotyping System 48 plex User Guide Preparing Genomic DNA Fragmenting the To fragment the gDNA gDNA 1 Program the thermal cycler as follows Step Step Type Temperature C Time 1 Hold 4 1 min 2 Hold 99 10 min 3 Hold 4 00 2 Chill a 96 well aluminum block on ice then place a compatible reaction plate onto it 3 Dispense up to 150 uL well of the prepared gDNA onto the chilled reaction plate 4 Cover the reaction plate 5 Run the program to boil the gDNA a Start the thermal cycler b Pause the program after the thermal cycler block reaches 4 C c Insert the chilled reaction plate containing the prepared gDNA d Resume the program 6 After the program is complete remove the reaction plate and place it on the chilled aluminum block 7 If the same sample was divided into multi
39. are data tables in the instrument database that store information about the plates and the samples they contain Some plate record fields that are required for 3730 3730x analyzer operation and sample file generation must be completed before a run Depending on the needs of your laboratory you can either Complete the plate record manually adding information at the appropriate time in the workflow Or e Partially or fully automate the plate record creation process by importing information from LIMS or text editor generated files SNPlex Genotyping System 48 plex User Guide Creating Results Groups and Plate Records Creating Plate There are several ways to create plate records Figure 3 3 illustrates three possible Records methods manual partially automated and fully automated Manual Method Partially Automated Method Fully Automated Method Create plate record on Import plate record text file into Import plate record text file into the 3730 3730x Data Collection Data Collection software Data Collection software PC containing The following information is The following information is completed completed Sample Name Results Group Sample Name Sample Name Instrument Protocol Results Group Results Group Instrument Protocol Instrument Protocol Sample Type Sample Type SNP Set SNP Set Analysis Method Panel Size Standard Run Data Collection software Run Data Collection software Run Data
40. at least one allelic ladder sample which allows GeneMapper software to perform sizing bin adjustments on a per run basis greatly reducing binning errors Hasaunique run folder set up in the Data Collection software All samples from a run must be saved in a unique run folder Coordinating the layout of your sample plates with the structure and naming of Data Collection software run folders allows the software to organize SNPlex System data into folders grouped by probe pool and run IMPORTANT Combining sample plate layout with the proper run folder naming convention explained in Setting Up Results Groups on page 3 26 allows the Data Collection software to organize data into folders grouped by probe pool and instrument run This organization is the required data structure for GeneMapper software to perform clustering analysis This section describes recommended plate layouts Refer to Setting Up Results Groups on page 3 26 for information on setting up results groups for SNPlex System experiments The following illustrations provide examples of sample layouts for 384 well and 96 well plates The setups assume that there are four probe pools per 384 well plate and one probe pool per 96 well plate The number of gDNA samples controls NTCs and allelic ladders differs between 96 capillary and 48 capillary instruments An instrument running a 96 capillary array injects once picking up contents from each of the 96 wells of the
41. description of the parameters in the Peak Quality tab Search on the topic SNPlex Signal level Peak Quality MEE Although the software calculates values for Heterozygous min peak height the parameters in this tab the peak quality parameters do not affect the Genotype Heterozygate balance Quality because the Quality Flags are set Min peak height ratio to O Peak morphology Max peak width basepairs Pull up peak Pull up ratio Pull up scan Allele number Max expected alleles Cross talk peak Cross talk ratio 0 05 Quality Refer to the online help for a detailed Flags OS description of the parameters in the Peak Quality tab Search on the topic SNPlex Quality weights are between O and 1 Quality Flag See For SNPlex System default analysis Se Ua ECT DUAE methods all quality flags are set to O ui redada Consequently the software does not use BUD E these flags to pass or fail a SNP Rcs E However the PQVs are used to calculate the EN genotype quality as shown in Figure B 1 on page B 4 Sizing Quality From 0 38 to 1 0 From 0 0 to Genotype Quality From 0 75 to 1 0 From 0 0 to Well Quality From 0 95 to 1 0 From 0 0 to SMP Quality From 0 75 to 1 0 From 0 0 to B 6 SNPlex Genotyping System 48 plex User Guide SNPlex Rules 3730 Selecting an GeneMapper Software v3 7 provides two allele calling methods Allele Calling Method Filtering The filtering method allows you to analyze sample da
42. for SNPlex System Experiments 2 10 SNPlex Genotyping System 48 plex User Guide SNPlex System Protocols This chapter covers i o o m 3 2 Designing the Sample Plate Layout corirsoriarrisirar rivera 3 3 Fropanng ce DA UNA er csi RARA AAA AR 3 6 Phosphorylating and Ligating Probes to gDNA OLA 3 9 Purifying Ligated OLA Reaction Products 3 14 Per TL dl MAP A OO Eg 3 16 Hybridizing PCR Products to ZipChute Probes 3 18 Blutine Zt nue PrODBR Lada dnd x3 EORR UR RCRRHECAN arias 3 22 Preparing Samples Tor Electrophoresis assa ox CORRER E RREGH HE eR 3 24 Creating Results Groups and Plate Records 3 26 Loading and Running the Sample Plates 3 33 SNPlex Genotyping System 48 plex User Guide 3 1 Chapter 3 SNPlex System Protocols Overview Protocols in This Guide 3 2 This chapter provides all the protocols necessary to manually perform SNPlex System experiments using 96 or 384 well plates on the 3730 3730x analyzers All volumes are for single reactions and need to be scaled up appropriately The SNPlex Genotyping System 48 plex General Automation Getting Started Guide PN 4358099 provides modified protocols for automating the SNPlex System assay using robotics Figure 3 1 illustrates the workflow for SNPlex System experiments Design and order SNPlexru ligation probes
43. in electrophoresis or GeneMapper controls software setup they may be caused by chemistry related issues such as DNA quality liquid handling or thermal cycler programming Inspecting assay controls cluster plot patterns and electropherograms can help pinpoint the cause IMPORTANT By performing these steps in the suggested sequence you systematically rule out potential causes of a problem This chapter presents tables that guide you through the troubleshooting process when using 3730 3730xl instruments Additionally this chapter includes explanations of the potential problems cited in the flowcharts Some commonly used terms and their synonyms are e Signal strength peak height e Allelic ladder SNPlex System ZipChute Mix 5 2 SNPlex Genotyping System 48 plex User Guide Troubleshooting Raw Data Troubleshooting Raw Data Viewing Raw Data The first step in the troubleshooting process is to rule out nonchemistry related problems that may be caused by the capillary electrophoresis process Viewing electrophoresis data allows you to identify problems that can lead to ambiguous results Use Figure 5 1 to troubleshoot problems related to the electrophoresis process Check Signal Are peak heights in allelic ladder wells between 1500 and 5000 RFU p 5 4 Signal 1500 Signal gt 5000 Yes Verify that the proper volume of allelic ladder was added according to protocol If
44. optional Application Select GeneMapper Generic Plate setup Select 384 Well or 96 Well Plate sealing Select Septa or Heat Seal if using heat sealed plates Owner name Enter your owner name Operator name Enter your operator name Click OK In the Plate Editor dialog box for each row of the plate record table enter a A sample name b Comments for each well of the plate optional For each row of the Plate Record table a Select or create a results group b For the instrument protocol pair the Run Module HTSNP36 POP7 V2 with Dye Set S Note Refer to Importing SNPlex System Files into the Data Collection Software on page 2 3 1f the run module and dye set are not available on your system In the Description field enter a description of the plate record optional 10 Click OK The data collection software saves the plate record to the database If running more than one plate repeat steps 3 to 9 Note At this point you have specified only the Sample Name Results Group and Instrument Protocol fields as shown in the manual workflow in Figure 3 3 on page 3 29 You must specify the rest of the information in the plate record specifically in the GeneMapper Sample Table after adding sample files before you can analyze the data using GeneMapper software SNPlex Genotyping System 48 plex User Guide Loading and Running the Sample Plates Loading and Runni
45. plex and Sample Loading Reagent at room temperature If precipitates form place the tubes briefly in a heating block set to 37 C 2 Vortex then quick spin the tubes 3 Prepare a sample loading mix by scaling the volumes listed below to the desired number of samples Note Prepare extra volume to account for losses that may occur during pipetting Note Prepare fresh sample loading mix daily Component Volume per Reaction uL Size Standard 48 plex SNPlex System 0 6 Sample Loading Reagent SNPlex System 16 9 Total Volume 14 9 SNPlex Genotyping System 48 plex User Guide Eluting ZipChute Probes Eluting the To elute the ZipChute probes ZipChute Probes l After the 60 min incubation period see page 3 21 remove the supernatant from the hybridization plates IMPORTANT For best results do not let plates sit at room temperature for extended periods of time before removing supernatant Wash each well four times with 100 uL Wash Buffer diluted 1 10 Note Dilute the 10X Wash Buffer 1 10 with deionized water IMPORTANT The ZipChute probes may be stripped off the plate under the following conditions Rapidaspiration of the ZipChute Mix or Wash Buffer supernatant when using a plate washer Contact between the plate washer tips and the well surfaces Applied Biosystems recommends that you set the aspiration tip depth so that 15 to 20 uL of Wash Buffer remains in each
46. probes specific for up to pooled 48 SNPs ne Three probes are used to interrogate each SNP ligation e Two of the probes are allele specific oligos ASOs These are designed probe pool specifically for the polymorphism by having the discriminating nucleotide on the 3 end Each ASO probe sequence also contains a unique ZipCode sequence for ZipChute probe binding e Thethird probe is a locus specific oligo LSO Its sequence is common to both alleles of a given locus and anneals adjacent to the SNP site on its target DNA The LSO probe contains a partial binding site for a universal reverse primer In a 48 plex reaction there are 96 ASOs and 48 LSOs for a total of 144 SNP specific oligos Prepare the Thaw the universal linkers The same pool of linkers is compatible with all universal ligation probe pools linkers i Three linkers are used for each SNP e Two ofthe linkers anneal to the two ASOs These linkers contain A PCR primer sequence corresponding to the universal forward primer UA sequence A partial cZipCode sequence e Thethird linker anneals to the LSO and has a universal sequence that is compatible with all LSOs The sequence includes a partial binding site for a universal reverse primer In a 48 plex reaction there are 96 ASO linkers and a single LSO linker which anneals to all LSOs regardless of sequence for a total of 97 linkers Prepare the Thaw the OLA master mix and dATP and c
47. rule is failed SNP is failed SNPQ set to 0 Genotypes are not called CV unaffected GQ 0 SNP passes SNPQ set to 1 l If individual lt clustering Filter failed genotypes based genotype is uncalled on per point genotype CV CV unaffected GQ 0 Per point CV unaffected all genotypes unaffected GQ value not changed but the GQ is flagged as a fail in the Ul if it falls below the defined GQ threshold Figure B 1 Summary of the Rules Algorithm B 4 SNPlex Genotyping System 48 plex User Guide SNPlex Rules 3730 Settings of the Table B 1 lists the default settings of the SNPlex Rules 3730 SNPlex Rules 3 30 Method Table B 1 Default settings for the SNPlex Rules 3730 Tab Default Settings Modifying the Default Settings General Name SNPlex Rules 3730 Allele You can modify the following settings in the Analysis Method Editor SHPlex Allele tab e Bin Set Make sure that the bin set Bin Set matches the panel used for the analysis prs For SNPlex System experiments the bin nn set is always SNPlex 48plex Bin 3730 O Filtering e Allele Calling Method Refer to Filtering Selecting an Allele Calling Method on lec aue 03 page B 7 for information about selecting an allele calling method Qus Clustering If selected you can set Clustering i O the clustering threshold and specify mu d ee how data is clustered You can also Clustering Threshold 0 95 modify values of the rules that the
48. s Manual The SNPlex System Matrix Standard kit consists of one tube of matrix standard which is sufficient for a minimum of eight array runs on the 3730x analyzer and 16 runs on the 3730 analyzer The SNPlex System Matrix Standard contains five specific sizes of DNA fragments labeled with a unique fluorescent dye label This standard is formulated in buffer and is stable for one year when stored at 2 C to 8 C Do not freeze Avoid exposure to light SNPlex Genotyping System 48 plex User Guide Performing Spatial and Spectral Calibrations Anne CHEMICAL HAZARD Formamide Exposure causes eye skin and respiratory tract irritation It is a possible developmental and birth defect hazard Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves To prepare the matrix standard for the 3730 or 3730x analyzers l Thaw and thoroughly mix the contents of the tube and spin briefly in a microcentrifuge Prepare a spectral calibration sample by combining 900 uL Hi Di Formamide PN 4311320 e 100 uL DS 40 Spectral Calibration Standard PN 4349365 Heat the mixture at 95 C for 5 min to denature the DNA fragments then immediately place on ice Note Make samples fresh for each run Samples can be stored refrigerated for up to 8 hours Discard excess material Dispense 5 uL 384 well plates or 10 uL 96 well plates of the spectral calibration sample int
49. software v3 7 Install GeneMapper software v3 7 Import SNPlex system panels and bins Import assay information files Import SNPlex system data Analyze the data Review analyzed data Export analyzed data One time set up only Figure 4 1 Analyzing SNPlex System data with GeneMapper software v3 7 4 2 SNPlex Genotyping System 48 plex User Guide Installing GeneMapper Software v3 7 Installing GeneMapper Software v3 7 Install the GeneMapper Software v3 7 according to instructions provided in the GeneMapper Software v 3 7 Installation Guide PN 4359289 Importing SNPlex System Panels and Bins About SNPlex GeneMapper software uses the same analysis parameters for all 48 plex SNPlex System Panels System experiments All the parameter files required to perform analysis of the and Bins SNPlex System assay chemistry by GeneMapper software are installed on your computer when you install GeneMapper software v3 7 See Table 4 1 on page 4 3 Importing the parameter files into GeneMapper software is a one time setup step Note Check for updates on the Applied Biosystems Web site at http www appliedbiosystems com support software Table 4 1 Parameter files for analyzing SNPlex System data on GeneMapper software v3 7 File Name as Displayed After Import File Name into GeneMapper Software Description SNPlIex 48plex 3730 Bins txt SNPlIex 48plex Bin 3730 Contains bins for SNPlex System allelic ladd
50. tab click Import 3 Insert the SNPlex Genotyping System Ligation Probes CD then select the AIF indicated by SNPlex nnnnnnn nnnnnnn xml When the import is complete GeneMapper software displays a number of new SNP sets each corresponding to a single SNPlex System ligation probe pool The name of each pool is identical to the name on the tube label SNPlex Genotyping System 48 plex User Guide 4 5 Chapter 4 Analyzing Data Using GeneMapper Software Importing SNPlex System Data into GeneMapper Software 4 6 Required Fields The data import process consists of two steps Completing required plate record fields mporting SNPlex System sample files The following plate record fields must be completed before GeneMapper software can analyze data Sample Name Sample Type SNP Set Analysis Method Panel and Size Standard Of these fields e Sample Name and Sample Type are completed in the Data Collection software prior to the electrophoresis run These fields can be completed manually or imported from formatted text files see Creating Results Groups and Plate Records on page 3 26 SNP Set names can be imported from the AIF into GeneMapper software The SNP Set names in the Data Collection plate record must match those in the GeneMapper file IMPORTANT For GeneMapper software to read SNP Set names from the plate record you must import the AIF into GeneMapper software before importing the SNPlex
51. well after each aspiration preventing the tips from touching the bottom of the wells IMPORTANT For consistent results after the last wash spin the plate upside down at 1000 rpm for 60 sec on a stack of clean paper towels to remove all remaining buffer Immediately add 17 5 uL of Sample Loading Mix containing size standard to each well Cover the plate containing the size standard with one of the recommended plate covers see Table 1 3 on page 1 10 Incubate the plate in a 37 C oven for 30 min on a rotary shaker Note To avoid possible overheating do not place the plate directly on the floor of the oven a For more information about configuring plate washers refer to the SNPIex Genotyping System 48 plex General Automation Getting Started Guide or the SNPIex Genotyping System 48 plex Automation Guide Automating PCR Using the Tomtec Quadra 3 Getting Started Guide SNPlex Genotyping System 48 plex User Guide 3 23 Chapter 3 SNPlex System Protocols Preparing Samples for Electrophoresis To dispense the allelic ladder 1 Remove the hybridization plates from the oven and mix 2 Briefly spin the plates to collect the liquid at the bottom of the wells 3 Label a new reaction plate 4 e Ifusing 384 well plates transfer 7 5 uL from each well into the wells of the new plate e If using 96 well plates transfer 10 uL from each well into the wells of the new plate
52. 0 0 0 0 gt 2565650050 s6sc065s56s0650502 05 CO RA Z e 0 20 P3 RO RO O 8 8 16 46 24 24 32 62 40 40 0000000000000000 O Yellow Probe Set A Quadrant 1 Blue Probe Set C Quadrant 3 1 to 88 gDNA samples N NTC O Green Probe Set B Quadrant 2 C Orange Probe Set D Quadrant 4 C Control DNA L Allelic Ladder SNPlex Genotyping System 48 plex User Guide 3 5 Chapter 3 SNPlex System Protocols Preparing Genomic DNA IMPORTANT Perform all gDNA fragmentation procedures in an amplicon free environment Review Preparing and Fragmenting Genomic DNA on page 1 15 for important information about this process Preparing Prepare the purified gDNA for fragmentation Purified gDNA l Purify your gDNA see page 1 15 for recommended kits 2 Determine the concentration of your DNA Refer to About Quantification on page 1 16 Optional but recommended Run an aliquot of each quantified DNA sample on a 0 8 agarose gel e Ifthe sample appears as a solid high molecular weight band continue with the procedure e Ifthe sample appears smeared across the lane omit the heat fragmentation step This figure shows high molecular weight gDNA before heat fragmentation A and after 10 minutes fragmentation in 1X TE pH 8 0 at 99 C B 1 kB Using 1X TE pH 8 0 5 dilute the purified DNA to a final concentration of between 50 and 200 ng uL
53. 0000000000000s 000000000000000028 00000000000000002 0000000000000000z686 0000000000000000s8 00000000000000003 00000000000000003 00000000000000003 00000000000000003 eeeeoooooooooooo 1 uL Allelic Ladder 7 5 uL Sample Loading Mix 48 capillary array 384 well plate 12 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 2 1A000000000000000000000000 5 uL from 8000000000000000000000000 aa cO000000000000000000000000 D 000000000000000000000000 ybridization E 000000000000000000000000 blate F000000000000000000000000 6000000000000000000000000 HOOOOOOOOOOOOOOOO000000000 000000000000000000000000 J000000000000000000000000 KOOOOOOOOOOOO0O000000000000 LOOOOOO0000000000000000000 vOOOOOOOOOOOOOOOO0O0OO0Oo0G00 NOOOOOOOOOOOOOOOOOOOO0000 uL o0000000000000000000000090 POOOOOOOOOOOOOOO0000000000 Allelic Ladder Sample Loading Mix a See Preparing the Sample Loading Mix on page 3 22 Note After transferring the sample loading mix make sure there are no air bubbles trapped at the bottom of the wells If there are briefly spin the plate Note For information about proper sample plate layout refer to Designing the Sample Plate Layout on page 3 3 IMPORTANT If you are not going to immediately use the plates for analysis seal the plates and store at 20 C Note Consider the plate seal options for use with the 3730 and 3730xl instruments While both septa andheat seal film are available the septa do
54. 00090 060060006060 OOOOOOOG 686000000 00000600 06006006000 O O Control DNA Allelic Ladder 1 to 88 gDNA samples N NTC Probe Set C Quadrant 3 O Orange Probe Set D Quadrant 4 o _ N An instrument running a 96 capillary array injects once from each of the four quadrants of a 384 well plate performing four separate runs Injection 2 1234567289 1011 12 13 14 15 16 17 18 19 20 21 22 23 24 A 1 9 17 7 25 2 33 89 41 41 49 49 57 67 65 65 73 13 81 61 69 69 B EL Ph d LS eH e HH es C D 4 4 4 945 494 9474 dh th pop ph de E FO O1 O O77 OOO O Or Or OO G 4 b 12 42 20 80 22 28 36 86 44 44 52 62 60 60 68 68 76 76 64 D 92 62 H D 4 e ded de ed s J0 0 0007 Or OO 007050 K 6 14 44 22 22 30 60 38 88 46 46 54 6 62 62 70 70 76 8 86 86 N MW L 479 444 494 9499 4 9 47 474 9 4 9 ph 4 40 48 48 56 86 64 64 P O O O2 OL Ov Or Ou 3726969 Ot Injection 4 12 34 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 A 1 9 17 dp 25 G5 33 89 41 49 49 49 67 67 65 65 73 19 81 61 69 69 P 000000000000000000000000 N NTC L Allelic Ladder 1 to 92 gDNA samples C Control DNA An instrument running a 48 capillary array injects twice from a 96 well plate picking up contents from half of the wells 48 wells per injection and performing Injection 2 N o 0O U gt 60000000 9606066660 6606000000 amp oo0909009090 6060600000
55. 4 zd HMM Universal reverse PCR priming site 5 VVV dATP SNPlex System Genome equivalent region amp Note Phosphorylation Dieses Linkers APP m ZipCode sequence occurs during the Ligation Probes SNPlex gt OLA reaction System Spacer P 5 phosphate S LSO Locus specific oligo S EE ASO Allele specific oligo A1 lt GeneAmp PCR System 9700 Thermal Cycler ASOA Allele specific oligo A2 2 ASO ASO linker L1 E ASO 2 ASO linker L2 gt c DNA M NN 9 ASO po LSO ASO m m Ed LSO Linker Figure 1 5 OLA procedure SNPlex Genotyping System 48 plex User Guide Chapter 1 Introduction Purifying Ligated OLA Reaction Products Protocol The SNPlex System Purification Kit uses two exonucleases to digest portions of the Summary _ ligated OLA reaction products unligated and partially ligated oligonucleotides and gDNA see Purifying Ligated OLA Reaction Products on page 3 14 Step Description Perform the Prepare a 2X Exonuclease mix exonuclease Add the 2X Exonuclease mix to the OLA reaction to digest the digestion appropriate DNA sequences including e Unligated oligonucleotides e Ligated products not protected by linker spacers e 5 portion of ASO linker of correct OLA product to permit primer annealing e 3 portion of LSO linker of correct OLA product to permit primer annealing e gDNA Spacers protect a complete ligation product from exonuclease digestion D
56. 5 Kits for Purifying Genomic DNA 1 15 Whole Genome Amplification 1 15 SNPlex Genotyping System 48 plex User Guide V ADOUL Eragrmentallobl s cmo o Ooh deut tov lutte e be ee eee te on oe dod 1 15 About Quantification 1 16 Phosphorylating and Ligating Probes to gDNA OLA 1 18 Protocol Summa seire bbei ence ghee od Wale Pe ae hh Cei keh ee a 1 18 Tie OLACPIOOSQUIGS icio ga E ee on E ta pet E OR 1 19 Purifying Ligated OLA Reaction Products 1 20 Protocol Summary 235 2 a ee RC WAR NCC i CORR eoe e d oie Ce Bae 1 20 The Purification Procedure 1 20 PCR Amplifying Ligated OLA Reaction Products 1 21 Protocol SUMMA esua ras cita ded o o qa M ee Ee dio d A sd 1 21 Th PG Procedure saure ic RE Am Sco DA o Ra E EUR C n 1 21 Hybridizing PCR Products to ZipChute Probes and Performing Electrophoresis 1 22 PrOtOCO SUMMA ocio dn wi ohne ERO e lcu o 1 22 Hybridization and Loading 1 24 Laboratory Design Preventing Amplicon Contamination 1 25 Product DESIO o 3 ea xsv bat hie eate Sabe at RA Ee S RE EM Rd 1 25 WO aDo rato ZA OBS tradi aov tue 1 25 SNPlex System Assay Workflow 1 27 Chapter 2 Setting Up A
57. 5 6 phosphorylation kit 3 9 pooling probes 3 9 plate records creating 3 26 plates hybridization 3 18 pooling SNPlex Ligation Probes 3 9 Positive Hybridization Control PHC troubleshooting 5 22 prebatch files for SNPlex system 3 33 PrebatchModule txt file replacing 2 3 prerequisites for running plates 3 33 probes allele specific 1 3 ASO LSO pool 1 3 concentration of SNPlex Ligation Probes 3 9 locus specific 1 3 OLA probe set 1 3 pooling OLA probe set 3 9 SNP specific 1 3 universal linkers 1 3 protocol for SNPlex System 2 4 protocols instrument 3 32 purification diluting reactions 3 15 kit 1 8 3 14 Lambda Exo reaction preparing 3 14 overview diagram 1 20 storing digests 3 15 thermal cycling conditions 3 15 Purification kit 1 8 3 14 Q quantifying gDNA 1 16 R radioactive waste handling xix reaction plate ligation 3 11 3 12 records plate 3 26 replacing PrebatchModule txt 2 3 required materials 2 2 required system files 2 3 SNPlex Genotyping System 48 plex User Guide resolution troubleshooting 5 6 Results Group setting up run folder 4 8 Rules method six rules B 8 run folder setting up Results Group 4 8 software requirements 4 8 run folder naming convention and sample plate layout 3 3 running SNPlex plates on 3730 xl analyzer 3 34 S safety alert words xvi chemical xvii conventions xvi ergonomic xx workstation xx sample plate layout and run folder naming convention 3 3 sam
58. 5613 36 cm 48 capillary array 3730 Applied Biosystems 4331247 instrument 36 cm 96 capillary array 3730xl Applied Biosystems 4331244 See your Applied Biosystems representative for information Block Module Reaction MicroAmp Optical 96 Well Reaction Applied Biosystems N8010560 Plates Plate ABI PRISM 384 Well Clear Optical Applied Biosystems 4309849 Reaction Plate with Barcode 50 plates ABI Prism 384 Well Optical Reaction Applied Biosystems 4326270 Plate with Barcode 500 plates 384 Well Plate Base heat sealed Applied Biosystems 4334877 384 Well Plate Retainer septa sealed Applied Biosystems 4334868 96 Well and 384 Well Plate Retainer Applied Biosystems 4334865 heat sealed Heat Seal Film Applied Biosystems 4337570 96 Well Sample Plates w barcode Applied Biosystems 4306737 96 Well Plate Septa Applied Biosystems 4315933 SNPlex Genotyping System 48 plex User Guide Required Non Kit Materials Table 1 3 Required equipment and consumables continued GeneMapper Software v3 7 Item Vendor NU Reaction 96 Well Plate Base septa sealed Applied Biosystems 4334873 nea 96 Well Plate Base heat sealed Applied Biosystems 4334875 96 Well Plate Retainer septa sealed Applied Biosystems 4334869 384 Well Plate Septa Applied Biosystems 4315934 384 Well Plate Base septa sealed Applied Biosystems 4334874 React
59. 92008 SNPLEX SYSTEM PURIFICATION KIT 4349357 NOTICE TO PURCHASER DISCLAIMER OF LICENSE No license to use the Oligonucleotide Ligation Assay OL A Ligation Detection Reaction LDR and or Polymerase Chain Reaction PCR methods covered by patents owned or licensable by Applied Biosystems is conveyed expressly by implication or by estoppel to the purchaser by the purchase of this product Further information on purchasing licenses to practice the patented methods may be obtained by contacting the Director of Licensing Applied Biosystems 850 Lincoln Centre Drive Foster City California 94404 U S A SNPLEX SYSTEM ZIPCHUTE KIT 48 PLEX 4349361 NOTICE TO PURCHASER LIMITED LICENSE SNPLEX SYSTEM ZIPCHUTE KIT is covered by composition claims of U S Patent 5 777 096 owned by Applied Biosystems or counterpart claims in patents and applications outside the U S The purchase price of SNPlex System Zipchute Kit includes a limited non transferable non exclusive license without the right to resell repackage or sublicense to use only this amount of the product solely for the purchaser s own research and development activities No other rights are granted expressly by implication or by estoppel under these or any other patent rights owned or licensable by Applied Biosystems Further information on purchasing licenses may be obtained by contacting the Director of Licensing at Applied Biosystems 850 Lincoln Centre Drive Foster City CA 94404 U
60. Clustering Threshold the Cluster By Project option Method Model O Rules Refer to Modifying the Clustering Parameters on page B 7 for more information E auia cl Men recommends that you accept the default settings for the Model threshold values Peak Same as the settings for the Rules method Refer to Refer to Peak Detector on page B 5 Detector Peak Detector on page B 5 Peak Same as the settings for the Rules method Refer to Refer to Peak Quality on page B 6 Quality Peak Quality on page B 6 Quality Refer to the online help for a detailed Flags AS description of the parameters in the Peak Quality weights are between O and 1 Quality Flag Settings Spectral Pull up Control Concordance Broad Peak Low Peak Height Qut of Bin Allele Off scale Cross Talk Peak Height Ratio Paw Thresholds Sizing Quality From to 1 0 Genotype Quality From 0 95 to 1 0 From 0 0 to From 0 0 ta Well Quality From 0 98 to 1 0 From 0 0 to SMP Quality From to 1 0 Fram 0 0 to Factory Defaultz Quality tab Search on the topic SNPlex Quality Flag For SNPlex System default analysis methods all quality flags are set to O Consequently the software does not use these flags to pass or fail a SNP However the PQVs are used to calculate the genotype quality as shown in Figure B 2 on page B 10 SNPlex Genotyping System 48 plex User Guide Appendix B GeneMapper
61. DNA Analyzers Getting Started Guide PN 4331468 Provides information about using the 3730 3730x1 instrument GeneMapper Software v3 7 Online Help Describes the analysis software and provides procedures for common tasks Note For additional documentation see How to Obtain Support on page xiii Send Us Your Applied Biosystems welcomes your comments and suggestions for improving its Comments user documents You can e mail your comments to techpubs appliedbiosystems com xii SNPlex Genotyping System 48 plex User Guide How to Obtain Support How to Obtain Support For the latest services and support information for all locations go to http www appliedbiosystems com then click the link for Support At the Support page you can Search through frequently asked questions FAQs Submit a question directly to Technical Support Order Applied Biosystems user documents MSDSs certificates of analysis and other related documents Download PDF documents Obtain information about customer training Download software updates and patches In addition the Support page provides access to worldwide telephone and fax numbers to contact Applied Biosystems Technical Support and Sales facilities SNPlex Genotyping System 48 plex User Guide xiii Preface xiv SNPlex Genotyping System 48 plex User Guide oafety Information This section includes the following topics Safety Conventions Used in This Document
62. For example if you have 10 runs out of 96 in which a single data point is shown as Heterozygote 2 Het 2 by clustering the 10 runs in the project you obtain 10 points displayed as Het 2 Consequently it is easier to determine if the call 1s accurate or if it is an experimental artifact Note Regardless of the clustering method that you select bin offsets are calculated by run that is by grouping all samples in a folder into a single run SNPlex Genotyping System 48 plex User Guide B 7 Appendix B GeneMapper Software v3 7 Analysis Methods for SNPlex System Assays B 8 Setting the Clustering Threshold The clustering threshold value 1s the minimum confidence value of the sample in a cluster Forthe Rules method if the confidence value for an individual data point in a particular cluster is 70 95 the point is labeled with the genotype for that cluster If the CV falls below the threshold the genotype is zeroed out e For the Model method the genotype for the data point is always displayed whether or not the sample passes the threshold An exception is when a sample is removed from analysis none of its data points will have a CV Modifying Values of Individual Rules Rules Method Only An important point to remember about the Rules method is that the SNP needs to fail only one of the six rules for the software to fail the SNP The following table lists the six rules according to which SNPs are assessed Rule
63. L of gDNA from step 8 on page 3 7 nto each control well wells labeled C in diagrams above add 2 0 uL of control DNA see step 2 on page 3 4 Cover 384 well reaction plates containing the SNPlex OLA reactions with one of the recommended plate covers see Table 1 3 on page 1 10 and an optical cover compression pad Note If you are using 96 well plates use MicroAmp Full Plate Covers to seal the plate 5 Transfer the reaction plates to a thermal cycler a IMPORTANT Applied Biosystems has found that certain plate covers negatively affect the performance of the SNPlex System assay If you use covers other than the recommended plate covers test them using the SNPlex System Control Set see Appendix A 3 12 SNPlex Genotyping System 48 plex User Guide Phosphorylating and Ligating Probes to gDNA OLA Running the OLA To thermal cycle the OLA reactions Reactions on the Thermal Cycler l If you use an Applied Biosystems thermal cycler program the thermal cycler as follows Thermal cycling conditions are the same for 384 and 96 well plates except for the ramp as indicated in the following table Step Step Type Temperature C Time 1 HOLD 48 30 min 2 HOLD 90 20 min 3 25 Cycles 94 15 sec 60 30 sec 51 30 sec 3 ramp 4 HOLD 99 10 min 5 HOLD 4 00 a Use a 2 ramp with standard or maximum setting for 96 well plates If you use non Applied
64. Performance To prepare the test sample plate continued 3 Prepare the test sample by mixing e 920 uL SNPlex Sample Loading Reagent from the SNPlex System Assay Standards Kit PN 4349351 40 uL of SNPlex Size Standard from the SNPlex System Assay Standards Kit PN 4349351 40 uL of the diluted ZipChute Mix 4 Vortex thoroughly 5 Dispense 10 uL of the spectral calibration sample into the appropriate number of wells of a MicroAmp Optical 96 Well Reaction Plate 48 wells for a 3730 analyzer e 96 wells for a 3730x analyzer 6 Complete the plate record selecting the instrument protocol you created for SNPlex System experiments Creating an Instrument Protocol for SNPlex System Experiments on page 2 4 7 Start the run Evaluating the To evaluate the sample run review the sample data from each well of the sample SNPlex System plate using the History View of the Data Collection software Run All 11 size standard peaks shown in orange should be approximately the same height and width Figure 2 1 shows examples of acceptable and poor resolution Good Resolution Poor Resolution M d CUR Y thun la 1000 1200 1400 1600 1800 2000 2200 2400 1000 1200 1400 1600 1800 2000 2200 2400 Figure 2 1 Example of acceptable and poor resolution SNPlex Genotyping System 48 plex User Guide 2 9 Chapter 2 Setting Up Applied Biosystems 3730 3730xl DNA Analyzers
65. SNPlex Genotyping System 48 plex User Guide Applied Biosystems O Copyright 2005 Applied Biosystems All rights reserved For Research Use Only Not for use in diagnostic procedures Information in this document is subject to change without notice Applied Biosystems assumes no responsibility for any errors that may appear in this document This document is believed to be complete and accurate at the time of publication In no event shall Applied Biosystems be liable for incidental special multiple or consequential damages in connection with or arising from the use of this document SNPLEX SYSTEM ASSAY CONTROL KIT 4349363 NOTICE TO PURCHASER DISCLAIMER OF LICENSE No license to use the Oligonucleotide Ligation Assay OL A Ligation Detection Reaction LDR and or Polymerase Chain Reaction PCR methods covered by patents owned or licensable by Applied Biosystems is conveyed expressly by implication or by estoppel to the purchaser by the purchase of this product Further information on purchasing licenses to practice the patented methods may be obtained by contacting the Director of Licensing Applied Biosystems 850 Lincoln Centre Drive Foster City California 94404 U S A SNPLEX SYSTEM OLIGONUCLEOTIDE LIGATION KIT 4362268 NOTICE TO PURCHASER LIMITED LICENSE The purchase price of SNPLEX6 SYSTEM OLIGONUCLEOTIDE LIGATION KIT includes a limited non transferable non exclusive license without the right to resell repackage or su
66. Software v3 7 Analysis Methods for SNPlex System Assays B 12 SNPlex Genotyping System 48 plex User Guide Index Numerics 48 capillary array injection scheme 384 well plate 3 5 96 well plate 3 4 96 capillary array injection scheme 384 well plate 3 4 96 well plate 3 3 A AIF importing 4 5 allele calling methods described B 7 Allele Cutoff Value for Filtering method B 7 allele specific oligos 1 3 1 18 allelic ladder 1 5 1 23 3 8 adjusting green or blue thresholds 5 20 binning failure 5 19 file type assignment 5 19 peak misidentification 5 20 removing 5 21 troubleshooting 5 18 Amplification kit 1 8 3 16 analysis method defaults B 2 defined B 2 setting 4 7 which to use B 2 analysis methods for SNPlex System data 4 8 analyzed data troubleshooting 5 22 Applied Biosystems contacting xiii customer feedback on documentation xii Information Development department xii Services and Support xiii Technical Support xiii ASOs See allele specific oligos Assay Information File AIF importing 4 5 B biohazard warning xix biohazardous waste handling xix blue threshold adjusting 5 20 Bold 1 xi SNPlex Genotyping System 48 plex User Guide C calibration standards 1 10 capillary array preconditioning 2 5 CAUTION description xvi chemical safety guidelines xviii cluster plots troubleshooting 5 26 clustering analysis term definitions B 3 Clustering method by run or by project B 7 clustering threshold v
67. System sample files fsa Depending on the way you set up your plate record see Creating Results Groups and Plate Records on page 3 26 Analysis Method Size Standard and Panel information can be completed n the plate record before electrophoresis using Data Collection software typically by importing a formatted text file containing this information Automatically upon sample file import See Setting Analysis Method Size Standard and Panel Automatically on page 4 7 Manually using GeneMapper software to edit the plate record Note Applied Biosystems recommends using either of the first two methods Manually entering the information by editing the plate record in GeneMapper software can be time consuming and error prone SNPlex Genotyping System 48 plex User Guide Importing SNPlex System Data into GeneMapper Software Setting Analysis To set Analysis Method Size Standard and Panel automatically Method Size Standard and l Inthe GeneMapper main window select Tools gt Options then select the Panel Add Samples tab Automatically 2 Specify the Analysis Method Size Standard and Panel as shown in the following figure Options p KA General Add Samples Analysis When adding new samples automatically Set Analysis Method to SMPlex Model 3730 v for all samples Read from the Sample BORDURE o O M SMPlex 48 plex v1 Y for all samples
68. UAR i a uses to calculate SNP Refer to Modifying the Clustering Parameters on page B 7 for more information Peak LS Refer to the online help for a detailed Detector rcm eae es description of the parameters in the Peak Peak Detection Algorithm e Analysis Full Range Sizing All Sizes hull B l1 G Smoothing and Baselining Mone CO Light Q Heavy Baseline Window pts Smoothing Size Calling Method Q 2nd Order Least Squares 3rd Order Least Squares Q Cubic Spline Interpolation Local Southern Method CO Global Southern Method Peak Detection Peak Amplitude Thresholds o R Min Peak Half cite Polynomial Degree Peak Window Size Factory Defaults Detector tab Search on the topic SNPlex Peak Detector e For SNPlex System experiments the Peak Detection Algorithm must be Advanced e The Peak Amplitude Thresholds should be low default is 10 for B G Y and R and 50 for O f set below 10 background noise may lengthen and interfere with analysis If set above 10 clustering is affected SNPlex Genotyping System 48 plex User Guide Appendix B GeneMapper Software v3 7 Analysis Methods for SNPlex System Assays Table B 1 Default settings for the SNPlex Rules 3730 Tab Default Settings Modifying the Default Settings Peak olores Refer to the online help for a detailed Quality
69. ZipChute probes diluted properly you may need to decrease injection time on instrument Verify that the proper volume of allelic ladder was added according to protocol If ZipChute probes diluted properly you may need to increase injection time on instrument Poor sample quality Is the capillary array new Verify proper storage of SLR Allelic Ladder and Size Standard Verify that correct electrophoresis module was used Verify that polymer and buffer have not expired For more information see section on troubleshooting signal Check Signal Are the peak heights in the allelic ladder relatively even across each sample p 5 17 Yes For more information see section on troubleshooting signal p 2 5 Verify that the array was preconditioned with Array Conditioning Buffer before installation p 2 5 Is the capillary array new Yes Check Resolution for all Samples Does the peak resolution look normal Are the peaks distinct and sharp p 5 5 Is resolution poor in Is resolution poor in only a few capillaries all capillaries Possible injection problem Verify that array use has not on select capillaries exceeded 300 runs If resolution Reinject samples is poor for 300 runs check resolution for sequencing standard Run sequencing standard sample to verify array performance is acceptable Verify that correct injection module was used Improper spect
70. adjacent peaks in the allelic ladder as illustrated in Figure 5 5 SNPlex Genotyping System 48 plex User Guide 5 7 Chapter 5 Troubleshooting 1000 1200 1400 1600 1300 2000 2200 lon front A I i ME Figure 5 5 Raw data of allelic ladder showing a tall narrow peak resulting from an ion front Ion fronts typically occur because of polymer aging in the instrument or the use of expired polymer lots The affected peak s can cause binning problems for an entire run because the ZipChute probe peaks in the front region do not migrate as expected by the software Solution If you observe an ion front in your raw data e Verify that the Data Collection software is using run module HTSNP POP7 V2 or later This module incorporates a short prerun that generally eliminates the ion front Replace the polymer and buffer on the instrument Do not use old or expired lots of polymer and buffer e If you cannot rerun the samples with the correct module or with fresh buffer and polymer or if the ion front persists after the rerun you can analyze the data in GeneMapper software using the alternative analysis method described in the following section Note Although the settings in the alternative analysis method can help minimize the effects of ion fronts in some cases you should not use these settings for regular analyses When possible replace the polymer and buffer and use an appropriate module To create an alternative
71. ality according to a set of custom rules SNPlex Model 3730 Uses the Model clustering algorithm which compares the behavior of alleles to an ideal or model to calculate the SNP quality The parameters of an analysis method are specified in the tabs of the Analysis Method Editor The software online help provides detailed information about each of these parameters Note that analysis parameters for SNPlex System applications are different from those for other applications such as AFLP kits or microsatellites To view the descriptions for SNPlex System analysis parameters in the software online help click the Workflows tab Under Applications specific Workflows click SNPlex Analysis Workflow then click Creating Analysis Methods Of all the parameters the clustering algorithm determines how the analysis method analyzes data The default analysis methods for SNPlex System data are named after the clustering algorithm used in the method The following table explains how to decide which method to use for your data Use the SNPlex Model 3730 Method Use the SNPlex Rules 3730 Method For high throughput experiments with 40to e Forlow frequency SNPs 94 samples per run e When you want to apply the filters such e Samples must contain DNA Positive as the Hardy Weinberg filter used by the controls are included in the sample Rules algorithm Refer to Modifying count Values of Individual Rules Rules Method Onl
72. alue B 8 settings B 7 Control kit 1 8 control pool results with gDNA plate A 5 SNP contents A 2 using A 5 controls hybridization 3 19 conventions for describing menu commands xi IMPORTANTS xi in this guide xi Notes xi safety xvi user attention words xi customer feedback on Applied Biosystems documents xii D DANGER description xvi data analysis required plate record fields 4 6 Data Collection v2 0 1 2 data review guidelines 4 9 documentation related xu Dye Set S installing 2 4 dye sets 1 10 3 32 E electrophoresis plate assembly 3 33 ergonomic safety xx exporting data 4 11 Index 1 F files prebatch 3 33 required for system 2 3 Filtering method using Allele Cutoff Value B 7 G gDNA dispensing into reaction plates 3 8 drying down 3 7 fragmenting 3 7 preparing for fragmentation 3 6 gDNA plate contents A 4 results with control pool A 5 GeneMapper software v 3 7 1 2 genomic DNA guidelines for purification 1 15 purification kits 1 15 quantification 1 16 green threshold adjusting 5 20 guidelines chemical safety xviii gDNA preparation 1 15 waste disposal xix H hazardicons xvi HTSNP36 POP7 V2 module importing 2 3 hybridization binding PCR products 3 19 denaturing with NaOH 3 20 eluting ZipChute probes 3 23 master mix 3 21 overview diagram 1 24 positive control 3 19 preparing plates 3 18 sample loading mix preparing 3 22 ZipChute probes 3 21 IMPORTANT descript
73. ation Control PHC 5 22 resolution 5 6 signal intensity variation across plate 5 24 signal strength 5 4 sizing quality 5 10 spectral calibration 5 6 three step process overview 5 2 U user attention words described xi W WARNING description xvi waste disposal guidelines xix workstation safety xx Z ZipChute probes description 1 5 eluting 3 23 hybridizing 3 21 ZipCode sequences 1 3 Index 4 SNPlex Genotyping System 48 plex User Guide Worldwide Sales and Support Applied Biosystems vast distribution and service network composed of highly trained support and applications personnel reaches 150 countries on six continents For sales office locations and technical support please call our local office or refer to our Web site at www appliedbiosystems com Applera is committed to providing the world s leading technology and information for life scientists Applera Corporation consists of the Applied Biosystems and Celera Genomics businesses Headquarters 850 Lincoln Centre Drive Foster City CA 94404 USA Phone 1 650 638 5800 Toll Free In North America 1 800 345 5224 Fax 1 650 638 5884 06 2005 Applied Biosystems Part Number 4360856 Rev B
74. ations of amplified DNA during the ligation step To produce the most consistent results for SNPlex System genotyping fragment the DNA by boiling Before fragmenting DNA verify that all samples have comparable quality Fragmenting degraded samples leads to over fragmented DNA which in turn leads to poorly clustered genotypes To check the quality of the DNA run an aliquot of each sample on a 0 8 agarose gel see Figure 1 4 on page 1 16 High quality DNA appears as a solid high molecular weight band Degraded DNA appears as a smear If DNA is already degraded before fragmentation omit the heat fragmentation step SNPlex Genotyping System 48 plex User Guide 1 15 Chapter 1 Introduction About Quantification 3 kB 1 kB Figure 1 4 High molecular weight gDNA before heat fragmentation A and after 10 minutes fragmentation in 1X TE pH 8 0 at 99 C B Applied Biosystems recommends quantifying the concentration of human gDNA using the TagMan RNase P DNA Quantification Kit which provides both quantitative and qualitative assessment of DNA Fluorescence or absorbance based assays such as the PicoGreen assay are more sensitive to the DNA preparation process and can result in falsely high calculations of gDNA concentration In such cases Applied Biosystems recommends using double the gDNA concentration during the ligation step as recommended when quantifying the gDNA with the TaqMan RNase P Quantification A
75. ay be caused by incorrectly setting up GeneMapper software Errors in sample sheet setup file type assignment size standard peak assignment or allelic ladder binning can cause analysis to fail unless they are corrected Use Figure 5 6 to troubleshoot problems related to the software set up process Evaluate Positive Hybridization Control PHC Is the signal intensity of marker Marker M5 1 CTL 002 PHC at least 150 RFU Yes For more information refer to section on evaluating PHC p 5 22 No Is PHC signal intensity varied accross the plate PHC is OK Is the signal intensity of marker Marker M5 1 CTL 004 PHC at least 150 RFU Yes PHC is OK Some signal variation is expected and signals below this value are occasionally observed Some variation in PHC signal intensity is expected across Overall median signal intensity the plate 4 to 8 fold variation significantly lower than these values may indicate a systematic Higher signal variation usually problem in post PCR steps indicates pipetting inaccuracies of assay during the post PCR steps of assay See Troubleshooting PHC for more detail p 5 22 Evaluate negative hybridization control NHC Are peaks present in the NHC markers Refer to section on evaluating p 5 24 C Are peaks present in both bins for every marker including homozygous samples p 5 24 p 5 24 Confirm that allelic ladders a
76. bleshoot problems that can arise from the assay chemistry you can analyze information from Positive hybridization controls See page 5 22 Negative hybridization controls See page 5 25 Cluster plots See page 5 26 The Positive Hybridization Control PHC which is added to the binding buffer when PCR products are bound to streptavidin plates is used to monitor the efficiency and reproducibility of the ZipChute probe hybridization and elution steps It consists of two single stranded biotinylated oligonucleotides containing a ZipChute probe binding site SNPlex Genotyping System 48 plex User Guide Troubleshooting Analyzed Data When analyzed in GeneMapper software every well should include two PHC peaks one for each marker M5 1 CTL 002 PHC and M5 1 CTL 004 PHC Both peaks should fall into the second bin allele 2 and cluster plots for the PHC should display a single cluster associated with allele 2 Example Figure 5 14 shows PHC signals from a single 96 capillary run Some variation in signal intensity within a run is acceptable The signal intensity of the encircled sample is significantly lower than the rest of the samples indicating potential problems with post PCR assay steps A B C Marker M5 1 CTL 002 PHC ao ex Pand vl H 2 83 84 Allele 2 A2 308 9 CCC CD 20 0 l 5 200 0 2000 7000 12000 17000 22000 27000 log10 Intensity Marker M5 1 CTL 004 PHC
77. blicense under the method claims of U S Patents 4 988 617 and 5 830 711 or counterpart claims in patents and applications outside the U S to use only this amount of the product to practice the Oligonucleotide Ligation Assay OLA and Ligation Detection Reaction LDR methods described in said patents solely for the purchaser s own research and development activities when this product is used in conjunction with SNPlex System Ligation Probes or SNPlex System Control Pool No other rights are granted expressly by implication or by estoppel or under any other patent rights owned or licensable by Applied Biosystems Further information on purchasing licenses for the OLA and LDR methods and other applications may be obtained by contacting the Director of Licensing at Applied Biosystems 850 Lincoln Centre Drive Foster City CA 94404 U S A NOTICE TO PURCHASER LIMITED LICENSE This product is sold under licensing arrangements between Applied Biosystems and Invitrogen Corporation The purchase price of this product includes limited nontransferable rights under U S Patent 5 035 996 and foreign equivalents owned by Invitrogen Corporation to use only this amount of the product to practice the claims in said patents solely for activities of the purchaser within the field of research Further information on purchasing licenses under the above patents may be obtained by contacting the Director Corporate Development at Invitrogen Corporation 1600 Faraday Aven
78. bridization Binding Buffer SNPlex System Hybridization Wash Buffer 10X SNPlex System ZipChute Dilution Buffer SNPlex System SNPlex System ZipChute Kit 48 plex SNPlex System Standards Kit 48 plex SNPlex System Hybridization Plates 384 well or 96 well SNPlex System Matrix Standard DS 40 Dye Set S SNPlex System Array Conditioning Kit Additional Precautions Use filter tips for all pipetting steps Routinely decontaminate robotic equipment Refer to the manufacturer s directions for a procedure Routinely decontaminate thermal cyclers Refer to the manufacturer s directions for a procedure Routinely decontaminate laboratory work surfaces 1 If performing the electrophoresis runs in the PCR laboratory SNPlex Genotyping System 48 plex User Guide Laboratory Design Preventing Amplicon Contamination SNPlex System Figure 1 8 summarizes the procedures that you should carry out in each lab Assay Workflow Design and order SNPlex ligation probes Design sample plate layout J DTE i Run PCR Prepare hybridization plates Prepare gDNA PCR Laboratory and bind PCR product to plates OLA Laboratory Add denaturant isolating biotinylated strand on hybridization plate Phosphorylate and Phosphorylate and ligate probes and ligate probes and linkers to gDNA linkers to gDNA OLA dry OLA wet Oligonucleotide Ligation Kit Hybridization Reagents and Kits
79. cal Waste Nem HAZARDOUS WASTE Refer to Material Safety Data Sheets Hazard Chemical Waste Safety Guidelines XVIII and local regulations for handling and disposal AN IEEE CHEMICAL WASTE HAZARD Wastes produced by Applied Biosystems instruments are potentially hazardous and can cause injury illness or death AINTE CHEMICAL STORAGE HAZARD Never collect or store waste in a glass container because of the risk of breaking or shattering Reagent and waste bottles can crack and leak Each waste bottle should be secured in a low density polyethylene safety container with the cover fastened and the handles locked in the upright position Wear appropriate eyewear clothing and gloves when handling reagent and waste bottles To minimize the hazards of chemical waste e Read and understand the Material Safety Data Sheets MSDSs provided by the manufacturers of the chemicals in the waste container before you store handle or dispose of chemical waste e Provide primary and secondary waste containers A primary waste container holds the immediate waste A secondary container contains spills or leaks from the primary container Both containers must be compatible with the waste material and meet federal state and local requirements for container storage e Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals for example safety glasses gloves or protective clothing For addit
80. cessary SNPlex Genotyping System 48 plex User Guide Troubleshooting Analyzed Data Troubleshooting The Negative Hybridization Control NHC is used to monitor nonspecific Negative hybridization that may occur during the hybridization step It consists of two Hybridization ZipChute markers M5 1 CTL 001 NHC and M5 1 CTL 003 NHC whose Controls binding sites are not complementary to the products generated during the SNPlex System assay Example When analyzed in GeneMapper software each well should include the two NHC markers As shown in Figure 5 15 no peaks should be visible for either marker and signal intensity of the markers should be less than 150 RFU Higher RFU values may indicate a software binning problem or problems with the hybridization or washing steps of the assay NA171604 Figure 5 15 Electropherograms showing NHC markers Observation Peak Appears in NHC Peaks in NHC markers are almost always caused by binning errors If you observe peaks for NHC markers review the allelic ladders to confirm that all peaks are correctly binned Observation Peak Appears in NHC Even with Correct Binning True peaks in NHC markers indicate problems with the hybridization or wash steps of the assay When peaks appear in NHC markers it is likely that the peaks appear in each bin for all markers even for samples that are homozygous If you observe true peaks for NHC markers confirm that you are performing both the hybridiza
81. d reoptimize electrophoresis performance High salt in sample Remove all liquids from wells by centrifuging plates upside down before adding sample loading mix Note If the control DNA shows a signal that is significantly higher and outside one of the clusters you observe with your own DNAs assume that the quality of your DNA is low and or suboptimal concentrations of DNA were added see 3730xl Instrument 96 capillary 96 wells on page 3 3 73730xl Instrument 96 capillary 384 wells and 3730 Instrument 48 capillary 96 wells on page 3 4 3730 Instrument 48 capillary 384 wells on page 3 5 and Dispensing gDNA into Reaction Plates on page 3 8 SNPlex Genotyping System 48 plex User Guide 5 29 Chapter 5 Troubleshooting Example Tight Clustering of Data Points with Few Low Signal Intensity Outliers Figure 5 18 illustrates cluster plots in which Most data points fall into tight discernible clusters e Several data points fall outside clusters outliers e Clustered data points have signal intensity of log 73 e Outliers have signal intensity of log lt 3 In such cases GeneMapper software is unable to identify clusters because of the presence of outliers After the outliers are removed the software correctly identifies the clusters This pattern suggests that the assay chemistry 1s working but that errors are occurring in specific samples SEC Angle in Radta
82. d by the up front fee component Rights to the up front fee component must be obtained by the end user in order to have a complete license These rights under the up front fee component may be purchased from Applied Biosystems or obtained by purchasing an authorized thermal cycler No right to perform or offer commercial services of any kind using PCR including without limitation reporting the results of purchaser s activities for a fee or other commercial consideration is hereby granted by implication or estoppel Further information on purchasing licenses to practice the PCR process may be obtained by contacting the Director of Licensing at Applied Biosystems 850 Lincoln Centre Drive Foster City California 94404 or at Roche Molecular Systems Inc 1145 Atlantic Avenue Alameda California 94501 USA NOTICE TO PURCHASER LIMITED LICENSE This product is sold under licensing arrangements between Applied Biosystems and Invitrogen Corporation The purchase price of this product includes limited nontransferable rights under U S Patent 5 035 996 and foreign equivalents owned by Invitrogen Corporation to use only this amount of the product to practice the claims in said patents solely for activities of the purchaser within the field of research Further information on purchasing licenses under the above patents may be obtained by contacting the Director Corporate Development at Invitrogen Corporation 1600 Faraday Avenue PO Box 6482 Carlsbad California
83. e SO De 3 3 3730xl Instrument 96 capillary 96 wells 3 3 vi SNPlex Genotyping System 48 plex User Guide 3730xl Instrument 96 capillary 384 wells 3 4 3730 Instrument 48 capillary 96 wells 3 4 3730 Instrument 48 capillary 384 wells 3 5 Preparing Genoomi DNA ur oiu dC ru e x a MO Ede a eee a a xoa 3 6 Preparing Purified gDNA aii SL grae o o e xc coe Ri AAA 3 6 FragmentinG tre DONA sicario EIS ERE E SERERE xS 3 7 DAC DOWNODNA PET UU 3 7 Dispensing gDNA into Reaction Plates 3 8 Phosphorylating and Ligating Probes to gDNA OLA 3 9 Oligonucleotide Ligation Kit Components 3 9 About SNPlex System Ligation Probes 3 9 Preparing the OLA Reactions 3 9 Assembling the OLA Reaction Dried Down gDNA 3 11 Assembling the OLA Reaction Wet gDNA 3 12 Running the OLA Reactions on the Thermal Cycler 3 13 Purifying Ligated OLA Reaction Products 3 14 Purification Kil COmponents 2 uv de V As v a au 3 14 Required Materials 2 3 14 Preparing an Exonuclease Reaction 3 14
84. e Type Select 96 well or 384 well as appropriate Plate Seal Select Septa or Heat Seal as appropriate Owner Name Enter a name Operator Name Enter a name c Click OK A blank sample sheet appears d Complete the following fields Sample Name Instrument Protocol Select the instrument protocol that you created in step 6 on page 2 4 Place the plate with the spectral calibration samples into the In Stack Click Run For details on how to perform a spectral calibration refer to the Applied Biosystems 3730 3730xl DNA Analyzers Getting Started Guide PN 4331468 Validating Instrument Performance To assess signal intensity and resolution you must perform a mock run using a diluted solution of the SNPlex ZipChute Mix and an internal size standard Preparing the AN ZE CHEMICAL HAZARD SNPlex Sample Loading Reagent Test Sample Plate causes eye skin and respiratory tract irritation It is a possible developmental and birth defect hazard Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves To prepare the test sample plate l Dilute the ZipChute Mix by combining 2 uL ZipChute Mix 48 plex from the SNPlex System ZipChute Kit 48 plex PN 4349361 e 448 uL molecular biology grade deionized water Vortex thoroughly 2 8 SNPlex Genotyping System 48 plex User Guide Validating Instrument
85. e standard omitted from sample or not properly injected Solution 1 Checking the Signal Strength Use the Size Match Editor Analysis gt Size Match Editor to make sure that all size standard peaks have a signal of at least 50 RFU The default analysis method for analyzing SNPlex System data uses an orange threshold setting of 50 GeneMapper software does not identify any peaks that fall below this threshold and consequently sizing fails Consistently low size standard peaks across a single run may indicate incorrect dilution of size standard in sample loading reagent or incorrect injection settings Low signal strengths for size standards in a small number of capillaries in a single run may result from electrophoresis or pipetting errors Ifsize calling appears accurate for most capillaries but inaccurate for others the problem may be due to electrophoresis or pipetting errors If the problem occurs in the same capillary over multiple runs when different samples are used the capillary may be defective If a capillary consistently fails for the same sample the problem may be with the sample Try placing the sample on a different position in the plate to rule out a capillary problem e fpeaks are clearly distinguishable but fall below the orange threshold you can try to adjust the analysis method so that the orange threshold value is less than that of the shortest peak To lower the orange threshold in the SNPlex System analys
86. e the PCR master mix PCR Master Mix 1l Thaw the Amplification Primers 2 Vortex then quick spin the tube 3 Prepare a PCR master mix by scaling the volumes listed below to the desired number of PCR reactions Note Prepare extra volume to account for losses that may occur during pipetting Component Volume per Reaction uL Nuclease free water 2 4 Amplification Master Mix 2X SNPlex 5 0 System Amplification Primers 20X SNPlex System 0 5 Total Volume 7 9 Assembling and To assemble and run the PCR reaction Running the PCR Reaction 1 Into each well of a 384 or 96 well plate dispense 7 9 uL PCR master mix e 2 1 uL diluted OLA reaction product see step 10 on page 3 15 3 16 SNPlex Genotyping System 48 plex User Guide Performing PCR To assemble and run the PCR reaction continued 2 Cover 384 well reaction plates containing the SNPlex OLA reactions with one of the recommended plate covers see Table 1 3 on page 1 10 and an optical cover compression pad Note If you are using 96 well plates use MicroAmp Full Plate Covers to seal the plate Program the thermal cycler Step Step Type Temperature C Time 1 HOLD 95 10 min 2 30 cycles 95 15 sec 63 1 min 3 HOLD 4 00 Transfer the reaction plates to the thermal cycler and start the program When thermal cycling is complete remove the reaction p
87. each aspiration Low OK Low Capillary injection failure Reinject samples signal signal for for outliers Run allelic ladder plate and outliers optimize electrophoresis performance High salt in sample Remove all liquids from wells by centrifuging plates upside down before adding sample loading mix Example Tight Clustering of Data Points with Outliers at Different Angles Figure 5 19 illustrates cluster plots in which Most data points fall into tight discernible clusters e Several data points fall outside clusters outliers e All data points clustered and outlying have signal intensity of log gt 3 Outliers are present in different angles as measured in the polar plot The vertical axis in the polar plot represents the angle in radians between the x axis and the data points in the Cartesian plot Data points that extend at a different angle in the plot may indicate Sample contamination e Secondary mutation close to the SNP site on one allele for a subset of the DNA samples Allele duplication SNPlex Genotyping System 48 plex User Guide 5 31 Chapter 5 Troubleshooting SNP CV8870159 Zur x HT EET a aie a a OG OT 1 Angle in Radians 0 6 0 4 02 PRT TLL 0 8 1 0 2 0 340 30 50 log10 Intensity Figure 5 19 Polar plots with discernib
88. ed freeze thaw cycles Ordering SNPlex First time Orders System Kits and Reagents The first time you order SNPlex System reagents you must order Ligation probes A starter kit e A core reagents kit e A set of hybridization plates either 96 well or 384 well depending on your experiment A control pool e A dried gDNA plates kit SNPlex Genotyping System 48 plex User Guide 1 9 Chapter 1 Introduction Subsequent Orders As you consume the reagents you can order ligation probes core reagents hybridization plates control pools and dried gDNA plates kits as needed Note You can order components of the core reagents kit individually using the individual kit part numbers instead of the core reagent kit part number Required Non Kit Materials Equipment and This is a list of all of the required materials not provided in the kits Consumables table 1 3 Required equipment and consumables Item Vendor Part Number Applied Biosystems 3730 3730xl DNA Analyzer See your Applied Biosystems representative for information GeneAmp PCR Block Module or GeneAmp PCR instrument System 9700 Dual 384 Well Sample System 9700 Dual 96 Well Sample Consumables POP 7 Performance Optimized Applied Biosystems 4335615 Polymer DS 40 Spectral Calibration Standard Applied Biosystems 4349365 Kit Dye Set S 10x Running Buffer with EDTA Applied Biosystems 433
89. efer to section on evaluating PHC p 5 22 Is PHC signal intensity varied accross the plate PHC is OK Is the signal intensity of marker Marker M5 1 CTL 004 PHC at least 150 RFU PHC is OK Some signal variation is expected and signals below this value are occasionally observed Some variation in PHC signal intensity is expected across Overall median signal intensity the plate 4 to 8 fold variation significantly lower than these values may indicate a systematic Higher signal variation usually problem in post PCR steps indicates pipetting inaccuracies of assay during the post PCR steps of assay See Troubleshooting PHC for more detail p 5 22 Evaluate negative hybridization control NHC Are peaks present in the NHC markers Refer to section on evaluating p 5 24 C Are peaks present in both bins for every marker including homozygous samples p 5 24 Confirm that allelic ladders are binned correctly Incorrect peak may be appearing in NHC bin p 5 24 NHC is OK Nonspecific hybridization is occuring during the hybridization step or incomplete ZipChute probe removal is occuring during the wash step Verify that these steps are Evaluate cluster plots performed according to protocol and that all liquid is removed Refer to section on evaluating from wells during the wash step cluster plots p 5 25 Figure 5 13 Workflow for troubleshooting analyzed data To trou
90. efly shake the plates upside down on a clean paper towel IMPORTANT All excess liquid must be removed from the plate before adding new reagents However keeping the plates empty for extended periods of time negatively affects the performance of the SNPlex System assay Add 50 uL of 0 1 N NaOH then cover the plate with one of the recommended plate covers see Table 1 3 on page 1 10 Note Applied Biosystems recommends that you prepare the 0 1 N sodium hydroxide solution fresh every 4 weeks Incubate for 5 to 30 min at room temperature on a rotary shaker Carefully remove the supernatant from each well then wash each well five times with 100 uL of Wash Buffer diluted 1 10 Note Dilute the Wash Buffer 1 10 with deionized water Briefly shake the plates upside down on a clean paper towel IMPORTANT All excess liquid must be removed from the plate before adding new reagents However keeping the plates empty for extended periods of time negatively affects the performance of the SNPlex System assay SNPlex Genotyping System 48 plex User Guide Hybridizing PCR Products to ZipChute Probes Hybridizing the A anne CHEMICAL HAZARD Zipchute Dilution Buffer SNPlex ZipChute Probes System Exposure causes eye irritation Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Denaturant SNPlex System Exposure causes eye skin and respiratory tract irri
91. egend Universal PCR primer sequence Universal reverse PCR priming site Genome equivalent region LT I ZipCode sequence e Oligo Spacer LSO Locus specific oligo ASO Allele specific oligo A1 ASO Allele specific oligo A2 ASO ASO linker L1 ASO ASO linker L2 Figure 1 1 Interaction between SNP specific probes and universal linkers 1 4 SNPlex Genotyping System 48 plex User Guide Product Overview About ZipChute ZipChute probes are used for decoding the genotype information by functioning as Probe Based reporter probes Chemistry Each ZipChute probe has A ZipCode binding sequence This sequence binds to the single stranded cZipCode complementary ZipCode region of the PCR products Mobility modifiers Each ZipChute probe contains a different number of mobility modifiers which enable size separation during electrophoresis A fluorescent label The fluorescent dye allows the 3730 3730x DNA Analyzer to detect the ZipChute probe ZipChute probes are arranged in pairs each pair representing both alleles of a SNP The peaks of a pair observed after electrophoretic separation are used to identify the alleles of the corresponding SNP Because each allele within a locus 1s represented by the same color the SNPlex System uses both the size and color of the ZipChute probes to resolve alleles within a locus The SNPlex System Hybridization Kit contains a universal ZipChute m
92. em 48 plex User Guide SNPlex System Kits and Reagents Reagent Name Part Number SNPlex System Starter Kit 48 plex 4362267 SNPlex Genotyping Dried gDNA Plate Control Pool System CD 4366107 SNPlex Genotyping System 48 plex User Guide 4340856 SNPlex Genotyping System 48 plex Quick Reference Card 4340855 SNPlex Genotyping System 48 plex General Automation Getting 4363143 Started Guide SNPlex System 48 plex Support Files CD 4352129 SNPlex System Array Conditioning Kit 4352018 SNPlex System Control Pool 48 plex 4362635 SNPlex System Dried gDNA Plates 4362637 DS 40 Spectral Calibration Standard Kit Dye Set S 4349365 SNPlex System Control Pool Kit 4362639 e Control Pool 48 plex SNPlex System e SNPlex Genotyping Dried gDNA Plate Control Pool System CD SNPlex System Dried gDNA Plates Kit 4366135 e Dried gDNA Plate SNPlex System e SNPlex Genotyping Dried gDNA Plate Control Pool System CD SNPlex System Hybridization Plates 384 well 5 plates 4349369 SNPlex System Hybridization Plates 96 well 5 plates 4357279 SNPlex System Hybridization Plates 96 well 10 plates 4362933 SNPlex System Ligation Probes 4346978 a Each SNPlex System kit provides sufficient reagent to perform 5 000 reactions If you do not expect to consume all the reagents in a kit in a single use Applied Biosystems recommends that you aliquot the reagents to minimize repeat
93. er Consequently the first four bins in the ladder are incorrectly positioned 10 DIST TINY MEE INTEL DNE DGE DORE DE IM IM GEL MR ZipChute peak not in bin Threshold for Blue and Green 10 Tuvu 10000 2000 2000 Peak with 78 rfu B T A h I 1 H ambajajas las eri a nsh aile ajaja nl Ai A3 h wi ASK Aah Al AS UA A jai Ai Aa Alp Aap aia as E SE ash ashap wi as laa mijas aaja Aib aapi miles Aa as Ai Aa Threshold for Blue and Green 200 ae r D Epi Mey ay ny Manny gen EN ET Y ya ml VJ D a ag ny ly D D ne D D er q AZ Jai Alfa A asja Alpi az at aa far ah malle Az asi fal jas 1 aah ail wz Az mil Athi AIR aa Jar Aral Aah AIR waht aih AZ a 2 ail aa AIR aah AIR AI Az Jar sz jas aaja Ah mahi mas 22 ar wile aa Figure 5 12 Top Example with incorrect binning Bottom Example with correct binning In this example generating a separate analysis method for the allelic ladders can correct the problem Adjust the threshold for the color of the misidentified peak in this case green to a higher value to ignore the peak Adjusting the green threshold to 200 in the analysis method causes the software to ignore the first peak because it 1s less than 200 RFU and corrects the problem Note that this type of problem can occur in either the green or the blue allelic ladder To adjust the blue or green threshold in the SNPlex System analysi
94. er This file along with the panel file contains information about the expected sizes of the allelic ladder IMPORTANT Do not edit this file SNPlex 48plex 3730 Panels txt SNPlex 48plex Panel 3730 Contains panels for SNPlex System allelic ladder This file along with the bin file contains information about the expected sizes of the allelic ladder IMPORTANT Do not edit this file Importing SNPlex To import SNPlex System panels and bins System Panels and Bins 1 Start the GeneMapper software v3 7 2 Access the Panel Manager a Select Tools gt Panel Manager Ctrl J b Click 34 Panel Manager SNPlex Genotyping System 48 plex User Guide 4 3 Chapter 4 Analyzing Data Using GeneMapper Software To import SNPlex System panels and bins continued 3 Import the files a Import the panels Click TErane manager Click File gt Import Panels Ctri M navigate to the GeneMapper Files folder on the SNPlex System 48 plex Support Files CD then select SNPlex 48plex 3730 Panels txt b Import the bins Click caswes apex 3730 Click File gt Import Bin Set Ctrl Shift B navigate to the GeneMapper Files folder on the SNPlex System 48 plex Support Files CD then select SNPlex 48plex 3730 Bins txt Note If when you try to import the files the GeneMapper software displays an error message that tells you the settings already exist override the old settings 4 Click OK
95. ex User Guide 3 21 Chapter 3 SNPlex System Protocols Eluting ZipChute Probes Standards Kit Components Required Materials Preparing the Sample Loading MIX For a summary of the steps in the purification procedure refer to Hybridizing PCR Products to ZipChute Probes and Performing Electrophoresis on page 1 22 The components included in the SNPlex System Assay Standards Kit PN 4349351 are listed in the table below The kit contains enough reagent for 5 000 reactions Component Storage Temperature C Size Standard 48 plex SNPlex System 15 to 25 Sample Loading Reagent SNPlex System 15 to 25 Allelic Ladder 48 plex SNPlex System 15 to 25 a After the reagent is opened store at 4 C and minimize exposure to light b Minimize exposure to light IMPORTANT The effectiveness of the Allelic Ladder and Size Standard declines with increasing freeze thaw cycles Make aliquots as necessary Refer to Required Non Kit Materials on page 1 10 for a complete list of vendors and part numbers Nem CHEMICAL HAZARD Sample Loading Reagent SNPlex System Exposure causes eye skin and respiratory tract irritation SNPlex Sample Loading Reagent is a possible developmental and birth defect hazard Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves To prepare the sample loading mix 1 Thaw the Size Standard Allelic Ladder 48
96. f the SNPlex 48 plex Quick Reference Card 384 well protocols for the 3730 3730x Genotyping System analyzers as well as summaries of Starter Kit 48 plex troubleshooting and GeneMapper e Downloadable from the software analysis SNPlex System Web site and Documents on Demand Web site SNPlex Genotyping System 4363143 e Describes options for automating e Part of the SNPlex 48 plex General Automation the SNPlex System assay using Genotyping System Getting Started Guide liquid handling instruments Starter Kit 48 plex e Describes the laboratory set up for e Downloadable from the automation SNPlex System Web e Provides a sample automated site and O UMERE workflow on Demand Web site SNPlex Genotyping System 4360796 e Explains how to set up the Biomek Downloadable from the SNPlex System Web site and Documents on Demand Web site 1 6 SNPlex Genotyping System 48 plex User Guide Table 1 2 SNPlex Genotyping System 48 plex documentation continued Product Overview l Part PET Document Title Number Contents Availability SNPlex Genotyping System 4360790 e Explains how to set up the TECAN Downloadable from the 48 plex Automating OLA Using Genesis RSP instrument for SNPlex System Web site the TECAN Genesis RSP automating the OLA portion of the and Documents on Getting Started Guide SNPlex System assay Demand Web site e Provides the step by step automation protocols for using the TECAN Genesis
97. genomic DNA gDNA samples you need to Purify the DNA sample Determine the concentration of the DNA Fragment and dilute DNA e Optionally dry down the DNA IMPORTANT Perform procedures related to purifying fragmenting and determining concentration of DNA in an amplicon free environment see Laboratory Design Preventing Amplicon Contamination on page 1 25 Because most laboratories have their own methods for purifying samples Applied Biosystems offers only guidelines rather than a specific procedure for purifying genomic DNA gDNA gDNA purified for SNP analysis should have A concentration of DNA between 50 to 200 ng uL e A length of 212 kb before boiling e A low level of protein contamination Low levels of PCR inhibiting substances such as guanidine hydrochloride heme isopropanol and ethanol Applied Biosystems suggests the following kits for purifying genomic DNA from blood for the SNPlex System assay Qiagen Flexigene Kit PN 51206 Gentra Puregene Kit PN D 5000 Consider using whole genome amplification WGA if you encounter problems arising from insufficient quantities of gDNA When using WGA consider the Quality of gDNA Use only high quality gDNA for WGA nput quantity of gDNA Use at least 10 ng of gDNA to avoid allelic imbalance and under representation Ifthe gDNA is degraded low quality consider using higher input concentrations Consider using higher concentr
98. h GeneMapper software may incorrectly identify a small shoulder preceding a peak as the main peak shown in Figure 5 10 Because this shoulder peak does not occur at the correct position relative to the other peaks sizing fails size Matches Size Calling Curve Sizing Quality 0 0 Override 50 25 bp shoulder is incorrectly labeled as a peak Ok Cancel Apply Size Matches Size Calling Curve Sizing Quality 1 0 Ovenide 0 To examine a peak more closely click on a peak or shoulder to zoom in Figure 5 10 Size standard with shoulder incorrectly labeled as a peak By adjusting the analysis method so that the orange threshold value is greater than the height of the shoulder you may achieve good sizing To raise the orange threshold in the SNPlex System analysis method 1 Start the GeneMapper software 2 Select Tools gt GeneMapper Manager then select the Analysis Methods tab SNPlex Genotyping System 48 plex User Guide Troubleshooting GeneMapper Software Analysis To raise the orange threshold in the SNPlex System analysis method continued 3 Create a new analysis method based on the current method a Select the current analysis method that you are using to analyze SNPlex System data b Click Save As c Enter a name for the new method d Click OK The new method appears in the list of analysis methods e Click Done 4 Select the new analys
99. hair covers that are dedicated to the OLA lab Usea thermal cycler in the OLA lab that has been designated for OLA only Store the following kits and associated materials for example microtiter plates and pipette tips in the OLA lab SNPlex System Oligonucleotide Ligation Kit 48 plex SNPlex System Purification Kit 48 plex SNPlex System Amplification Kit 48 plex SNPlex System Assay Control Kit SNPlex System Ligation Probes SNPlex System Control Pool 48 plex SNPlex System Dried gDNA Plates Kit Placea sticky mat at the entrance of the OLA lab SNPlex Genotyping System 48 plex User Guide 1 25 Chapter 1 Introduction PCR Laboratory In this laboratory you thermal cycle the PCR reactions then proceed with the remaining steps in the SNPlex System assay You may perform the run on the 3730 3730x1 analyzers in this laboratory or in a separate data collection area IMPORTANT Never move equipment containers or other items from the PCR Laboratory or data collection area into the OLA laboratory OLA Lab PCR Lab OLA Lab PCR Lab Observe the following precautions Wear safety goggles lab coats gloves and hair covers that are dedicated to the PCR lab Usea thermal cycler in the PCR lab that has been designated for PCR amplification only e Store the following kits and associated materials for example microtiter plates and pipette tips in the PCR lab Hy
100. he hazard icons that are affixed to Applied Biosystems instruments Examples The following examples show the use of safety alert words IMPORTANT You must create a separate a Sample Entry Spreadsheet for each 96 well microtiter plate AN eue The lamp is extremely hot Do not touch the lamp until it has cooled to room temperature Nee CHEMICAL HAZARD Formamide Exposure causes eye skin and respiratory tract irritation It is a possible developmental and birth defect hazard Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves EN Pyles ELECTRICAL HAZARD Failure to ground the instrument properly can lead to an electrical shock Ground the instrument according to the provided instructions SNPlex Genotyping System 48 plex User Guide Chemical Safety Chemical Safety Chemical Hazard Nee CHEMICAL HAZARD Before handling any chemicals refer Warning to the Material Safety Data Sheet MSDS provided by the manufacturer and observe all relevant precautions gt MESUbIeI CHEMICAL HAZARD All chemicals in the instrument including liquid in the lines are potentially hazardous Always determine what chemicals have been used in the instrument before changing reagents or instrument components Wear appropriate eyewear protective clothing and gloves when working on the instrument Anne CHEMICAL HAZARD Four liter reagent and waste bottles can crack and lea
101. ical Hazard Sale arras donee XIX Workstation Safety 2a dus dete ira AE AE AA dU ep dd XX Chapter 1 Introduction Product OVeIVIOW vi a o V RET eR n tt OR RA GP ded Rc RC t Sl le 1 2 Product DescHDbLEHOD EUR uy bag A A PA etas Roos 1 2 About SNP Specific Probes 1 3 About Universal ASO LSO Linkers 1 3 About ZipChute Probe Based Chemistry 1 5 Supported Configurations 1 5 SNPlex System Documentation 1 6 SNPlex System Kits and Reagents 1 1 8 OVOIVIEW Lt ad a e a deca ag em EN er asie 1 8 ITS ANG Read emi cacas ds i Ca afe enh aca as aer ac des RUD mea 1 8 Ordering SNPlex System Kits and Reagents 1 9 Required Non Kit Materials 1 10 Equipment and Consumables 1 10 aco c PE 1 12 Other Required Materials 1 12 Optional Materials ints 2 cet ee Us ii AA 1 13 Chemistry Overview susci dd Ecko ded acer Re wc doce 1 14 COMPONESNTERTES 4 3745 da audi CO RE MAT e Co Doo c o ee e iet 1 14 ji fep I A AAA AA A AS 1 14 Preparing and Fragmenting Genomic DNA 1 15 ADOMCIPBEHIGA HOLD acd coe troc run mono ub D dg uu ar i ico nes laa 1 1
102. idated TaqMan assays were available Typically have a minor allele frequency of at least 0 1 in at least one of the following populations African American Caucasian Japanese Chinese Were validated by individually genotyping 180 DNA samples selected from the four major populations with TaqMan probe based 5 nuclease assay Table A 1 lists the 48 SNPs in the Control Pool Table A 1 48 SNPs in the SNPlex System Control Pool Celera SNP Minor Allele Frequency Zip 2 Discovery Consortium dbSNP ID System ID ID rs AfA Cauc Chin Japn hCV TSC Mb c d e Location M5 1 001 hCV2058031 TSC0630913 rs1425151 0 23 0 26 0 28 0 28 Chr 11 10 727 372 M5 1 002 hCV7547436 TSC0691193 rs1457947 0 39 0 42 0 49 0 44 Chr 6 77 426 938 M5 1 0083 hCV1901045 TSC0806211 rs1323881 0 29 0 20 0 38 0 37 Chr 13 100 315 097 M5 1 004 hCV7536854 TSCO556240 rs1388276 0 46 0 33 0 34 0 45 Chr 3 72 816 542 M5 1 005 hCV2597763 TSCO926934 rs1980408 0 12 0 23 0 48 0 44 Chr 21 40 775 977 M5 1 006 hCV2059319 0 43 0 31 0 15 0 08 Chr 11 19 245 581 M5 1 007 hCV8929459 TSCO339341 rs1035089 0 16 0 42 0 38 0 38 Chr 16 11 016 789 M5 1 008 hCV2986015 TSCO295775 rs 05681 0 21 0 45 0 22 0 22 Chr 1 6 999 213 M5 1 009 hCV8720462 TSCO165692 rs893613 0 30 0 48 0 41 0 40 Chr 15 87 994 667 M5 1 010 hCV349615 TSCO314577 rs992690 0
103. iled samples WQ set to O Per point CV set to 0 GQ set to 0 WQ unaffected Per point CV unaffected all genotypes for this sample set to no call GQ set to 0 SNPQ unaffected Per point CV unaffected all genotypes for failing SNPS marked no call GQ set to 0 Per point CV unaffected all genotypes below per point CV threshold marked no call All genotypes elow per point CV threshold have GQ set to 0 Per point CV unaffected all genotypes unaffected GQ value not changed but the GQ is flagged as a fail in the Ul SNPlex Genotyping System 48 plex User Guide SNPlex Model 3730 Settings of the Table B 2 lists the default settings of the SNPlex Model 3730 SNPlex Model 3 30 Method Table B 2 Default settings for the SNPlex Model 3730 Tab Default Settings Modifying the Default Settings General Name SNPlex Model 3730 Allele You can modify the following settings in the Analysis Method Editor SHPlex Allele tab e Bin Set Make sure that the bin set Bin Set matches the panel used for the analysis SNPlex For SNPlex System experiments the bin Allele Calling set is always SNPlex 48plex Bin 3730 O Filtering e Allele Calling Method Refer to Filtering Selecting an Allele Calling Method on al page B 7 for information about selecting an allele calling method M Clustering If selected you can set d EFIE I the clustering threshold and specify pm how data is clustered You cannot use
104. ilute purified ligation products with nuclease free water The Purification Procedure Legend emmmmmmm A 2o 4S J WWmmm mm Universal PCR primer sequence Purification Universal reverse PCR priming site ase pem e de ARO d Genome equivalent region MM ZipCode sequence d Spacer Figure 1 6 Purification procedure 1 20 SNPlex Genotyping System 48 plex User Guide Chemistry Overview PCR Amplifying Ligated OLA Reaction Products Protocol The SNPlex System Amplification Kit allows you to amplify the purified and Summary diluted OLA reaction products see Performing PCR on page 3 16 Step Description Prepare the PCR The Amplification Master Mix SNPlex System contains buffer and master mix enzyme The Amplification Primers SNPlex System contains two universal primers e The universal forward primer is unlabeled e The universal reverse primer is biotinylated Combine the Amplification Master Mix with the Amplification Primers to form the PCR master mix Assemble the PCR The diluted exonucelase digested OLA reaction products are reaction amplified when the universal primers bind and are extended in the presence of enzyme and adequate cycling conditions Thermal cycle The resulting product is a double stranded amplicon with one biotinylated strand The PCR Procedure gt Legend Assemble the
105. ion MicroAmp Full 96 Well Plate Cover Applied Biosystems N8010550 Plate Covers ABI Prism Optical Cover Compression Applied Biosystems 4312639 Pad e Heatseals Easy Peel 610 meter roll ABGene AB 3739 ee Easy Peel individual sheets ABGene AB 0745 UNISEAL AL Whatman 7704 0002 Plate Sealer ALPS 300 ABGene AB 0950 Thermo Sealer ABGene AB 0384 e Adhesive 384 Well Microplate Aluminum Sealing Corning 6569 seals Tape Adhesive PCR foil seal ABGene AB 0626 Silverseal Greiner 676 090 See your Applied Biosystems representative for information Data Collection Software v2 0 or higher See your Applied Biosystems representative for information a IMPORTANT Applied Biosystems has found that certain plate covers negatively affect the performance of the SNPlex System assay If you use covers other than the recommended plate covers test them using the SNPlex System Control Set see Appendix A b Modules for GeneMapper and Data Collection Software are available at http www appliedbiosystems com support software SNPlex Genotyping System 48 plex User Guide Chapter 1 Introduction Reagents Table 1 4 Required reagents Part Item Vendor Number Hi Di Formamide Applied Biosystems 4311320 Sterile 1X TE buffer 10 mM Tris base pH 8 0 and Fluka 93283 1 mM Na EDTA 0 1 N NaOH Major Laboratory Supplier MLS Nuclease free water Promega P119C Other Required Table 1 5 Othe
106. ion to establish a relationship between the signal emitted by each capillary and the position where that signal falls and is detected by the CCD camera Perform a spatial calibration after you nstall a new or used capillary array Remove the capillary array from the detection cell block even to adjust it Move the instrument even if the instrument was moved on a table with wheels To perform a spatial calibration 1 In the Data Collection software navigation pane select the Spatial Run Scheduler 2 Select the SpatialFill 1 module 3 Perform the spatial calibration as described in the Applied Biosystems 3730 3730xl DNA Analyzers Getting Started Guide PN 4331468 The SNPlex Matrix Standard DS 40 PN 4349365 is used to generate the multicomponent matrix required when analyzing 6FAM dR6G BigDye TAMRA BigDye ROX and LIZ labeled DNA fragments on the Applied Biosystems 3730 3730x DNA Analyzers The Data Collection Software for these instruments uses the multicomponent matrix to automatically analyze the five differently colored fluorescent dye labeled samples in a single capillary You do not need to run matrix standards with every set of sample injections However you do need to run the standards once in order to generate a matrix file that is then applied to samples run under similar conditions For more information on the use of matrix standards refer to the instrument User
107. ion xvi Information Development department contacting xii instrument protocol creating 2 4 ion front minimizing 5 8 troubleshooting 5 7 Italic 1 xi Index 2 kits Amplification 1 8 3 16 components 1 8 1 9 Control 1 8 Phosphorylation 3 9 Purification 1 8 3 14 Standards 3 22 L ladder allelic 1 5 1 23 3 8 ligation 3 11 3 12 linkers 1 3 1 18 locus specific oligos 1 3 1 18 LSOs See locus specific oligos M master mix hybridization 3 21 Lambda Exo reaction 3 14 oligo ligation assay with UNG 3 9 PCR 3 16 Purification 3 14 materials optional 1 13 required 2 2 matrix standard preparing 2 7 median signal intensity troubleshooting 5 24 menu commands conventions for describing xi mock run 2 8 evaluating 2 9 Model algorithm sample rejection B 9 module importing HTSNP36 POP7 V2 2 3 MSDSs description xvii obtaining xiii xvii referring to xviii N Negative Hybridization Control NHC troubleshooting 5 25 O oligo ligation assay master mix with UNG 3 9 preparing with UNG 3 9 thermal cycling conditions with UNG 3 13 orange threshold for data analysis 5 14 lowering 5 14 raising 5 16 SNPlex Genotyping System 48 plex User Guide P panel setting for analysis 4 7 panels and bins importing 4 3 parameter files 4 3 PCR Amplification kit 1 8 3 16 assembling reaction 3 16 master mix 3 16 overview diagram 1 21 thermal cycling conditions 3 17 peaks pull up or pull down
108. ional safety guidelines consult the MSDS e Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation for example fume hood For additional safety guidelines consult the MSDS Handle chemical wastes in a fume hood After emptying the waste container seal it with the cap provided SNPlex Genotyping System 48 plex User Guide Biological Hazard Safety Dispose of the contents of the waste tray and waste bottle in accordance with good laboratory practices and local state provincial or national environmental and health regulations Waste Disposal If potentially hazardous waste is generated when you operate the instrument you must e Characterize by analysis if necessary the waste generated by the particular applications reagents and substrates used in your laboratory Ensure the health and safety of all personnel in your laboratory Ensure that the instrument waste is stored transferred transported and disposed of according to all local state provincial and or national regulations IMPORTANT Radioactive or biohazardous materials may require special handling and disposal limitations may apply Biological Hazard Safety General WARNING BIOHAZARD Biological samples such as tissues body fluids Biohazard and blood of humans and other animals have the potential to transmit infectious diseases Follow all applicable local state provincial and or nationa
109. is method 1 Start the GeneMapper software 2 Select Tools gt GeneMapper Manager then select the Analysis Methods tab SNPlex Genotyping System 48 plex User Guide Troubleshooting GeneMapper Software Analysis To lower the orange threshold in the SNPlex System analysis method continued 3 Create a new analysis method based on the current method a Select the current analysis method that you are using to analyze SNPlex System data b Click Save As c Enter a name for the new method d Click OK The new method appears in the list of analysis methods e Click Done Select the new analysis method then click Open In the Analysis Method Editor select the Peak Detector tab Change the Peak Amplitude Threshold for orange to a value less than the signal intensity of the shortest size standard peak Do not alter other color settings Note Applied Biosystems recommends that you keep the orange threshold value at 50 Click OK to save the analysis method Click Done to close the GeneMapper Manager then reanalyze the samples using the new analysis method SNPlex Genotyping System 48 plex User Guide Chapter 5 Troubleshooting Solution 2 Verifying GeneMapper Software Size Calls Use the Size Match Editor to make sure that all size standard peaks are identified correctly by GeneMapper software When the signals of the size standard peaks are very hig
110. is method then click Open 5 Inthe Analysis Method Editor select the Peak Detector tab 6 Change the Peak Amplitude Threshold for orange to a value greater than the signal intensity of the shoulder peak 7 Click OK to save the analysis method 8 Click Done to close the GeneMapper Manager then reanalyze the samples using the new analysis method Solution 3 Checking Evenness of Signal Strength Use the Size Match Editor Analysis gt Size Match Editor to make sure that all size standard peaks have reasonably even signal strengths To rule out instrument related problems run a plate of ZipChute ladder with size standard as described in Preconditioning the Capillary Array on page 2 5 SNPlex Genotyping System 48 plex User Guide 5 17 Chapter 5 Troubleshooting Troubleshooting Allelic Ladders GeneMapper software determines the positions of allelic ladder peaks for each run then compensates for offset between the positions of the bins and those of the ladder peaks Through this process the software compensates for run to run variations that can occur in electrophoresis and ensures that experimental peaks are assigned to bins correctly Example Figure 5 11 shows an allelic ladder where all peaks have a relatively even signal and are binned correctly ic A fi age RT s s t Sample File Sample Name SQI os SQ Chinese M23 fsa Chinese SNPlex_4fplex_ Pand vl E 1 0 M M M
111. ition capillary arrays with Array Conditioning Buffer Otherwise the resulting data has poor resolution and inconsistent signal strength Figure 5 3 poor resolution for a non conditioned capillary 300 1100 1300 1500 1700 1500 2100 2300 Good resolution Poor uo SO p IW i Ace ae 1 pl i I1 M Fur rad Figure 5 3 Raw data view of an allelic ladder showing poor resolution and loss of signal Solution If your raw data displays poor resolution Ensure that all reagents in the instrument are fresh and that regular maintenance has been performed Refer to the Applied Biosystems 3730 3730xl DNA Analyzer Getting Started Guide PN 4359476 e Precondition arrays before filling them with polymer or running samples Refer to Preconditioning the Capillary Array on page 2 5 ncases where only one of several runs that were processed simultaneously shows poor resolution try reinjecting samples a second time Example Pull up or pull down peaks in raw data indicate problems with spectral calibration Figure 5 4 SNPlex Genotyping System 48 plex User Guide Troubleshooting Raw Data
112. ixture which can be used for all multiplex reactions The ZipChute probes constitute a library of reference alleles called an allelic ladder The master set of probes 1s used to normalize GeneMapper software parameters and aid in simplifying and automating allele scoring Figure 1 2 shows the functional parts of a ZipChute probe 3 Fluorescent dye label Mobility modifiers E ZipCode sequence Figure 1 2 Parts of a ZipChute probe Supported Table 1 1 lists the supported configurations for the SNPlex 48 plex system Configurations Table 1 1 Supported configurations for the SNPlex 48 plex System Instrument is e a de Automation Options 3 30 48 96 well None 384 well None 3730x 96 96 well None 384 well Yes a Chapter 3 provides the 96 and 384 well protocols for the 3730 3730x analyzers b Refer to the SNPlex Genotyping System 48 plex General Automation Getting Started Guide PN 4363143 for more information about automating the SNPlex System assay Also see the specific robotics Getting Started Guides under Related Documentation on page xii and SNPlex System Documentation on page 1 6 SNPlex Genotyping System 48 plex User Guide 1 5 Chapter 1 Introduction SNPlex System Table 1 2 lists the documentation available for the SNPlex Genotyping System Documentation 48 plex Table 1 2 SNPlex Genotyping System 48 plex documentation 48 plex Automating OLA U
113. k Each 4 liter bottle should be secured in a low density polyethylene safety container with the cover fastened and the handles locked in the upright position Wear appropriate eyewear clothing and gloves when handling reagent and waste bottles ANNEE CHEMICAL STORAGE HAZARD Never collect or store waste in a glass container because of the risk of breaking or shattering Reagent and waste bottles can crack and leak Each waste bottle should be secured in a low density polyethylene safety container with the cover fastened and the handles locked in the upright position Wear appropriate eyewear clothing and gloves when handling reagent and waste bottles About MSDSs Chemical manufacturers supply current Material Safety Data Sheets MSDSs with shipments of hazardous chemicals to new customers They also provide MSDSs with the first shipment of a hazardous chemical to a customer after an MSDS has been updated MSDSs provide the safety information you need to store handle transport and dispose of the chemicals safely Each time you receive a new MSDS packaged with a hazardous chemical be sure to replace the appropriate MSDS in your files Obtaining You can obtain from Applied Biosystems the MSDS for any chemical supplied by MSDSs Applied Biosystems This service 1s free and available 24 hours a day To obtain MSDSs 1 Go to https docs appliedbiosystems com msdssearch html 2 In the Search field type in the chemical name part number
114. l informatics appliedbiosystems com In Europe contact Informatics by e mail at InformaticsInfo eur appliedbiosystems com or by fax at 44 0 1925 282 502 In Asia Pacific contact Informatics by e mail at InformaticsAsiaPacific appliedbiosystems com by phone at 65 6896 1036 or by fax at 65 6896 2147 SNPlex Genotyping System 48 plex User Guide Product Overview About Each assay includes three SNP specific ligation probes SNP Specific Probes e Two of the probes are allele specific oligos ASOs These are designed specifically for the detection of polymorphisms by having the discriminating nucleotide on the 3 end Each ASO probe sequence also contains one of 96 unique ZipCode sequences for ZipChute probe binding In a multiplex reaction the universal ZipCode sequences on each ASO are unique Therefore in a 48 plex reaction there are 96 ASOs two for each SNP and 96 different ZipCode sequences The third probe is a locus specific oligo LSO Its sequence is common to both alleles of a given locus and anneals adjacent to the SNP site on its target DNA Each LSO also contains a partial universal PCR primer binding site In a 48 plex reaction there are 48 LSOs All 144 probes for a 48 plex reaction are shipped together as an ASO LSO probe pool It is this pool that confers genotyping specificity to the SNPlex System assay All other reagents are universal and not SNP specific Order SNP specific probes separa
115. l regulations Wear appropriate protective eyewear clothing and gloves Read and follow the guidelines in these publications US Department of Health and Human Services guidelines published in Biosafety in Microbiological and Biomedical Laboratories stock no 017 040 00547 4 http bmbl od nih gov Occupational Safety and Health Standards Bloodborne Pathogens 29 CFR 1910 1030 http www access gpo gov nara cfr waisidx 01 29cfr1910a 01 html Additional information about biohazard guidelines 1s available at http www cdc gov SNPlex Genotyping System 48 plex User Guide Xix Safety Information Workstation Safety XX Correct ergonomic configuration of your workstation can reduce or prevent effects such as fatigue pain and strain Minimize or eliminate these effects by configuring your workstation to promote neutral or relaxed working positions Nonis MUSCULOSKELETAL AND REPETITIVE MOTION HAZARD These hazards are caused by potential risk factors that include but are not limited to repetitive motion awkward posture forceful exertion holding static unhealthy positions contact pressure and other workstation environmental factors To minimize musculoskeletal and repetitive motion risks Use equipment that comfortably supports you in neutral working positions and allows adequate accessibility to the keyboard monitor and mouse Position the keyboard mouse and monitor to promote relaxed body and head p
116. lace the array into the solution filled cover Assemble the 20 mL syringe Luer adaptor and tubing supplied in the SNPlex System Array Conditioning Kit PN 4352018 Slip the open end of the tubing over the capillary bundle at the detection end of the array Using the syringe pull enough of the diluted Array Conditioning Buffer solution to fill the array Ensure that the array is filled by looking at the detection cell Capillaries filled with Array Conditioning Buffer solution appear dark Additionally a small volume of solution enters the attached tubing Allow the solution to incubate in the array for 30 min Make sure that the array tips are submerged in the solution Remove the syringe assembly then rinse the array bundle end briefly with deionized water Install the array on the 3730 3730x analyzer using the Array Install wizard You do not need to rinse the array interior before installing it on the instrument Select the optional extra array fill at the end of the wizard Change the buffer cup and tray water and waste reservoirs SNPlex Genotyping System 48 plex User Guide 2 5 Chapter 2 Setting Up Applied Biosystems 3730 3730xl DNA Analyzers for SNPlex System Experiments Performing Spatial and Spectral Calibrations Performing a Spatial Calibration Performing a Spectral Calibration 2 6 The 3730 3730x Data Collection Software uses images collected during spatial calibrat
117. lates If you use the PCR reaction products mmediately Proceed to Hybridizing PCR Products to ZipChute Probes on page 3 18 e Within 24 hours Store at 4 C Within 35 days Store at 20 C a IMPORTANT Applied Biosystems has found that certain plate covers negatively affect the performance of the SNPlex System assay If you use covers other than the recommended plate covers test them using the SNPlex System Control Set see Appendix A SNPlex Genotyping System 48 plex User Guide 3 17 Chapter 3 SNPlex System Protocols Hybridizing PCR Products to ZipChute Probes For a summary of the steps in the ZipChute hybridization procedure refer to Hybridizing PCR Products to ZipChute Probes and Performing Electrophoresis on page 1 22 Reagents Required for Hybridization Required Materials Preparing the Hybridization Plates The reagents required to complete the hybridization process are listed below Each reagent or kit contains enough volume for 5 000 reactions Component Storage Temperature C Or SNPlex System Hybridization Plates 96 well SNPlex System Hybridization Plates 384 well Ambient Denaturant SNPlex System e ZipChute Mix 48 plex SNPlex System Positive Hybridization Controls SNPlex System Hybridization Wash Buffer SNPlex System Ambient Hybridization Binding Buffer SNPlex System Ambient ZipChute Dilution Buffer SNPlex System Ambient SNP
118. le File Sample Name Pand SQI OS SQ Insi l N23 LADDER 084 2004 05 04 fsa LADDER SNPlex_d plex_Pand vl E jo ME 1 034 MET 035 MET 036 451 037 MELLE NET d 44 45 46 47 48 43 50 51 52 53 54 55 56 57 58 53 60 61 62 63 A M M M M ME lt gt gt LL NHC Green Ladder For more information about allelic ladders see Troubleshooting Allelic Ladders on page 5 18 Review the cluster plots Select Analysis gt Display Cluster Plots then observe if Clusters are tight Signal strength is consistent There are any calls that you want to edit For more information about cluster plots see Troubleshooting Cluster Plots on page 5 26 SNPlex Genotyping System 48 plex User Guide Exporting SNPlex System Data Exporting SNPlex System Data To export Genotype information Select the Genotype tab then select File gt Export Table e Sample information Select the Sample tab then select File gt Export Table Both genotype and sample information Select File gt Export Combined Table SNP Table Once the cluster plot has been selected select File gt Export Table Note When exporting both types of information you can select File gt Export Combined Table regardless of which tab you are viewing You can also use the Report Manager feature of GeneMapper Software v3 7 to generate multi column custom reports from the data in the sample and genotype tables For m
119. le clusters and outliers present at different angles Table 5 3 Troubleshooting cluster plots with discernible clusters and outliers present at different angles contaminated with DNA of a different genotype Size PHC NHC Standard Potential Cause Possible Solution OK OK OK DNA samples Repeat experiment with uncontaminated DNA Decontaminate pipettors or robotic tips with 10 bleach solution OLA cross contamination of some samples Evaluate and optimize pipetting accuracy in assay workflow Secondary SNP within genome equivalent region of OLA probe sequence Allele duplication Redesign probe set using alternative SNP Example Good Signal Intensity but Data Points Smeared Across Y Axis Angle Poor Cluster Formation Figure 5 20 illustrates cluster plots in which e All data points have signal intensity of log gt 3 e No discernible clusters are formed e Samples appear to be smeared or stretched vertically in the polar plot SNPlex Genotyping System 48 plex User Guide Troubleshooting Analyzed Data x L3 sort Angle in Radians se x os X 2 0 20 40 6 0 logit Intensity Figure 5 20 Cluster plots with good signal intensity but poor cluster formation SNPlex Genotyping System 48 plex User Guide 5 33 Chapter 5 Troubleshooting 5 34 Table 5 4 Troubleshooting cluster plots with good signal intensity but poor cluster formation
120. lex Genotyping System 48 plex User Guide Troubleshooting Raw Data 2 Ifthe allelic ladder is appropriately diluted adjust the injection time to compensate for instrument to instrument variation Note The default injection time for the SNPlex System module is set to 10 s at 1 kV Increasing the injection time increases the signal strength decreasing the injection time decreases the signal strength Note Applied Biosystems recommends that you keep injection times between 5 and 20 seconds Injection times outside this range result in poor resolution a Start the Data Collection software for the Applied Biosystems 3730 DNA Analyzer b Open the Module Manager then click New c In the Run Module Editor for Name Enter a name for the module Type Select Regular e Template For Data Collection v2 0 select HTSNP POP7 V2 For Data Collection v3 0 select HTSNP POP 7 V3 e Injection Time Enter a new injection time between 5 and 20 seconds d Click OK to save the module SNPlex Genotyping System 48 plex User Guide 5 5 Chapter 5 Troubleshooting Troubleshooting Resolution Troubleshooting Spectral Calibration e Open the Protocol Manager f Create a new protocol using the module you just created Alternatively edit an existing protocol by replacing the currently selected module with the module you just created Example For optimal results when running SNPlex System assay chemistry precond
121. lex System ZipChute Kit 48 plex 15 to 25 a Avoid exposure to light and minimize freeze thaw cycles Refer to Required Non Kit Materials on page 1 10 for a complete list of vendors and part numbers To prepare the hybridization plates 1 Remove the PCR reaction plates from storage see step 6 on page 3 17 2 Vortex then briefly spin the PCR reaction plates to collect the liquid in the bottom of the wells 3 Label the SNPlex Hybridization Plates making sure well A 1 is at the top left corner 4 Wash the wells of the SNPlex Hybridization Plates three times with 100 uL Wash Buffer diluted 1 10 Note Dilute the Wash Buffer 1 10 with deionized water SNPlex Genotyping System 48 plex User Guide Binding PCR Products to the Hybridization Plate Isolating Biotinylated Strands on the Hybridization Plate Hybridizing PCR Products to ZipChute Probes To prepare the hybridization plates continued 5 Briefly shake the plates upside down on a clean paper towel IMPORTANT For this and all subsequent washing steps all excess liquid must be removed from the plate before adding new reagents However keeping the plates empty for extended periods of time negatively affects the performance of the SNPlex System assay Prepare the Binding Buffer with the Positive Hybridization Control for the desired number of hybridization reactions For each reaction you need e 17 491
122. licon becomes single stranded The biotinylated strand remains bound to the streptavidin while the unbound strand is washed away Hybridize the ZipChute probes to the amplicon Prepare the hybridization master mix then add fluorescently labeled ZipChute probes which bind specifically to the single stranded cZipCode sequence of the bound biotinylated PCR strand Prepare the sample loading mix Combine Size Standard 48 plex and Sample Loading Reagent to form the sample loading mix To establish a sizing calibration curve that is used to identify ZipChute probes each well contains a fluorescently labeled Size Standard 48 plex Eleven size standard orange peaks appear in each lane of the electropherogram Elute the ZipChute probes Add the Sample Loading Mix to the wells and incubate the plate at 37 C to release the ZipChute probes from the biotinylated strand SNPlex Genotyping System 48 plex User Guide Chemistry Overview Table 1 7 SNPlex protocol summary continued Dispense the Allelic Ladder The allelic ladder is labeled with FAM and dR6G dyes 48 plex Dispense the ladder into the appropriate number of wells of the sample loading plate as specified in the following table Allelic Instrument Protocol Ladder Wells 3730 96 well 4 48 capillary 384 well 16 3 30xl 96 well 2 96 capillary 384 well 8 Load reactions onto the Load plates o
123. mit SNPs as IDs from common databases such as dbSNP or the Celera Discovery System online platform or as custom sequences containing your SNPs of interest For more information about the assay design process refer to the SNPlex Genotyping System 48 plex Assay Design and Ordering Guide PN 4357460 Validated SNP content Applied Biosystems SNPbrowser software provides annotated locus information generated by Celera Genomics allowing you to design assays based on polymorphisms in human DNA samples Genotyping analysis software The SNPlex System software suite consists of Data Collection v2 0 or higher and GeneMapper 3 7 software The suite allows you to collect and manage raw data process and analyze the data and finally store and manage the processed locus sample and called genotype information By working with Applied Biosystems Professional Services you can add sample tracking and consumables management capabilities to your software Electophoresis instruments and consumables The Applied Biosystems 3730 3730xl DNA Analyzers 3730 3730x analyzers enable the separation and detection of SNP specific reporter probes using standard capillary arrays electrophoresis buffers and polymers A unique matrix standard allows you to spectrally calibrate your electrophoresis instruments 1 Informatics Software and Services Applied Biosystems 3833 North First Street San Jose CA 95134 USA Phone 800 762 5467 E mai
124. n More Information Related The following related documents are available Documentation SNPlex Genotyping System 48 plex Assay Design and Ordering Guide PN 4357460 Describes the SNPlex Genotyping Systems assay design and ordering process the file formats to use for a successful submission and guidelines to maximize the assay design success rate SNPlex Genotyping System 48 plex Quick Reference Card PN 4360855 Provides condensed procedures for using the SNPlex Genotyping System 48 plex SNPlex Genotyping System 48 plex General Automation Getting Started Guide PN 4363143 Assists principal investigators and laboratory staff with using the SNPlex Genotyping System 48 plex with general robotics SNPlex Genotyping System 48 plex Automating OLA Using the Biomek FX Getting Started Guide PN 4360796 Explains how to set up the Biomek FX instrument for automating the OLA portion of the SNPlex System assay SNPlex Genotyping System 48 plex Automating OLA Using the TECAN Genesis RSP Getting Started Guide PN 4360790 Explains how to set up the TECAN Genesis RSP instrument for automating the OLA portion of the SNPlex System assay SNPlex Genotyping System 48 plex Automation Guide Automating PCR Using the Tomtec Quadra 3 Getting Started Guide PN 4358100 Explains how to set up the Tomtec Quadra 3 instrument for automating the post PCR portion of the SNPlex System assay Applied Biosystems 3730 3730xl
125. n using the SNPlex System control pool together with the SNPlex System dried gDNA plate Three genotype clusters should be observed for 45 48 SNPs Exceptions are hcv7505765 SNP 13 hcv7571632 SNP 17 and hcv2962785 SNP 41 which show only two clusters The precision or the measure of the reproducibility in genotype calls between identical gDNA samples should be 99 7 or better The accuracy or the measure of the genotype data achieved with the SNPlex System against reference genotypes should be 99 5 or better The call rate or the measure of the number of genotypes made relative to all possible calls should be 95 or better Note If you use your own gDNA plate together with the SNPlex System control pool the signal strength and cluster angle you observe for one SNP should be similar to what you observe with the dried gDNA plate Figure A 1 shows the cluster plots for each of the 48 SNPs Cluster plots are numbered Note that numbers 40 and 47 are not associated with a SNP in this figure SNPlex Genotyping System 48 plex User Guide A 5 Appendix A Using the SNPlex System Control Set D WEIREEIBERIENI 10 iP z60 20 40 54 if 30 30 ad 40 10 ib 13 40 40 14 30 30 40 44 14 306 30 4d ib 30 30 4b 30 if 30 30 40 4 if 30 30 a0 40 ig 18 14 40 40 14 30 26 40 14 19 20 30 9 690 1a 20 30 49 60 Lp 20 30 46 0 LD 20 30 48 30 1 70 30 40 50 10 20 10 43 5n is 30 40 50 10 30 3D 40 54 10 34 D 40 60 io r0
126. nalyzed by GeneMapper software without you having to edit the plate record Additionally these fields must be entered exactly as they are defined in GeneMapper software The most convenient way to create plate records 1s to import appropriately formatted text files that have been generated by a text editor or by a LIMS sysem The simplest way to get started is to export a working plate record using the Data Collection software then use it as a template to develop a plate record generation tool Plate records exported by the Data Collection software contain additional header information including Container Name Plate ID Description ContainerType AppType Owner Operator PlateSealing and SchedulingPref Again the simplest way to define these fields correctly is to use a working plate record as a guide Creating Plate Applied Biosystems recommends using a partially automated method to generate Records by plate records see Figure 3 3 Such a method helps eliminate problems arising from Importing data entry errors and can also greatly reduce the time spent setting up plate records Formatted Text Files To set up plate records by importing text files l Inthe Data Collection software open the Plate Manager 2 Click Import then navigate to the text file that you want to import 3 Select the file that you want to import then click Open The Data Collection software imports the contents of the file into a new
127. ng the Clustering Parameters B 7 SNPlex Model 3790 aia Bie ee eee ee Sends CUR bene C RC ek B 10 How the Model Algorithm Works B 10 Settings of the SNPlex Model 3730 Method B 11 SNPlex Genotyping System 48 plex User Guide ix SNPlex Genotyping System 48 plex User Guide Preface How to Use This Guide How to Use This Guide Purpose of This Guide Audience Text Conventions User Attention Words The Applied Biosystems SNPlex Genotyping System 48 plex User Guide provides information on how to use the SNPlex System kits with the Applied Biosystems 3730 3730xl DNA Analyzer This document includes new information about mproved protocols for 384 well and 96 well plates Control ligation probe pool and dried gDNA plates kit which you can use to evaluate the performance of the SNPlex System Use of GeneMapper Software v3 7 to analyze SNPlex System data This version of the software provides a new clustering algorithm Model which raises the accuracy of scoring The existing Rules clustering algorithm is also included IMPORTANT Chapters 1 and 3 describe significant changes in the assay setup This guide is intended for novice and experienced SNPlex Genotyping System 48 plex users who perform SNPlex System assays and analyze the data using GeneMapper software This guide uses the following conventio
128. ng the Sample Plates Plate Assembly Assemble the plates for loading onto the 3730 3730x analyzer The 384 well plate assembly shown below is similar to the 96 well plate assembly use sample plates plate septa and plate retainers for the 96 well format Plate retainer Plate septa Required Refer to Required Non Kit Materials on page 1 10 for a complete list of vendors Materials and part numbers Loading Sample To load the plates in the stacker Plates 1 Pull open the stacker drawer The stacker light flashes green 2 Open the metal door of the In Stacker tower 3 Place the plates in the stacker 16 maximum The bottom plate runs first IMPORTANT Ensure that the plate assembly fits flat in the stacker and that plate retainer clips are properly seated in the base 4 Close the metal In Stacker tower door 5 Close the stacker drawer Prerequisites If you are using Data Collection v2 0 verify that the default prebatch file has been replaced with the SNPlex System prebatch file Note If you are using Data Collection v3 0 there is no specific prebatch file and you may disregard the following paragraph Double click PrebatchModule txt typically in E AppliedBiosystems UDC DataCollection SupportFiles ga3730 Service Modules If the first line of the file is not SNPlex v2 0 Prebatch refer to Replacing the PrebatchModule txt File on page 2 3 for m
129. not provide an air tight seal Some gradual signal loss occurs over time when using the septa If the SNPlex plates will remain on the instrument in excess of 12 hours Applied Biosystems recommends using the pierceable heat seal option Heat Seal film PN 4337570 Be aware that after the heat seal 1s pierced by the instrument for sample injection the seal is no longer intact SNPlex Genotyping System 48 plex User Guide 3 25 Chapter 3 SNPlex System Protocols Creating Results Groups and Plate Records Starting Data Collection Software About Results Groups Setting Up Results Groups To start the 3730 Analyzer Data Collection Software 1 Select Start gt Programs gt Applied Biosystems gt Data Collection gt Run 3730 Data Collection v2 0 or higher 2 Wait as the Service Console dialog box starts the applications of the data collection software 3 When all applications are running the Data Collection Viewer opens Results Groups allow you to specify autoanalysis settings designate a data storage location and specify naming conventions for sample files and run folders By including Plate Quadrant as a parameter for naming run folders the Data Collection software automatically generates separate run folders for each plate quadrant Because the sample plate is set up so that each plate quadrant corresponds to a probe pool the data for each probe pool is stored in a separate f
130. ns i 30 3 0 4 a 1 0 in 30 4 log intensity logit imensi Figure 5 18 Cluster plots with discernible clusters and low signal intensity outliers Table 5 2 Troubleshooting cluster plots with discernible clusters and low signal intensity outliers PHC NHC nize Potential Cause Possible Solution Standard OK OK OK Insufficient DNA in wells Confirm that input quantity with low signal of DNA is adequate Incomplete or excessive Check distribution of DNA DNA fragmentation fragments on agarose gel DNA contains OLA Repurify DNA inhibitors Pipetting errors during Evaluate and optimize OLA PCR exonuclease pipetting accuracy in assay digestion or PCR transfer workflow for wells with low signal 5 30 SNPlex Genotyping System 48 plex User Guide Troubleshooting Analyzed Data Table 5 2 Troubleshooting cluster plots with discernible clusters and low signal intensity outliers PHC NHC mad d Potential Cause Possible Solution Low OK OK Streptavidin failure in wells Repeat experiment with signal with outliers new streptavidin plate for outliers Pipetting errors in post Evaluate and optimize PCH steps pipetting accuracy in post PCR assay workflow Bound ZipChute probes Reconfigure the plate stripped off plate washer Set the aspiration tip depth so that 15 to 20 uL of Wash Buffer remains in each well after
131. ns Bold indicates user action For example Type 0 then press Enter for each of the remaining fields e Italic text indicates new or important words and is also used for emphasis For example Before analyzing always prepare fresh matrix e A right arrow bracket gt separates successive commands you select from a drop down or shortcut menu For example Select File gt Open gt Spot Set Right click the sample row then select View Filter gt View All Runs Two user attention words appear in Applied Biosystems user documentation Each word implies a particular level of observation or action as described below Note Provides information that may be of interest or help but is not critical to the use of the product IMPORTANT Provides information that is necessary for proper instrument operation accurate chemistry kit use or safe use of a chemical SNPlex Genotyping System 48 plex User Guide xi Preface Examples of the user attention words appear below Note The size of the column affects the run time Note The Calibrate function is also available in the Control Console IMPORTANT To verify your client connection to the database you need a valid Oracle user ID and password IMPORTANT You must create a separate Sample Entry Spreadsheet for each 96 well plate Safety Alert Safety alert words also appear in user documentation For more information see Words Safety Alert Words on page xvi How to Obtai
132. ns of a sample differ from those of the default bin settings binning can fail This failure can be corrected by running an allelic ladder and measuring the required offset Reinject samples that have been run without an allelic ladder using an allelic ladder Solution 2 Confirming File Type Assignment The GeneMapper software requires that allelic ladder wells be designated as samples of type allelic ladder in the Sample Type column If allelic ladder wells are mistakenly designated as sample or positive control the software does not use this allelic ladder when calculating bin offsets If necessary assign the correct sample type to the allelic ladder then reanalyze the samples To reassign sample types l Locate the allelic ladder sample s in the Samples View 2 For each ladder sample verify that the sample type indicated in the Sample Type column is Allelic Ladder If the sample is labeled incorrectly click the sample type then select Allelic Ladder from the popup menu Reanalyze the sample SNPlex Genotyping System 48 plex User Guide 5 19 Chapter 5 Troubleshooting 5 20 Solution 3 Checking Accuracy of Peak Assignment Occasionally the GeneMapper software misidentifies a peak in the allelic ladder causing problems with binning offsets In the example in Figure 5 12 the software misidentified a 78 RFU peak as the first peak in the green ZipChute ladd
133. ntensity and Little to No Cluster Formation Figure 5 17 illustrates cluster plots in which e Most of the data points have log intensity lt 3 There are no clearly defined clusters SHP Cvand5932 SHP Cuv2639761 M x X X X A x x NX yx Pe TAA un e E m E E E di E x X By A 20 log10 intensity Figure 5 17 Cluster plots with low signal intensity and poor cluster information SNPlex Genotyping System 48 plex User Guide 5 27 Chapter 5 Troubleshooting 5 28 Table 5 1 Troubleshooting cluster plots with low signal and poor cluster information Size PHC NHC Potential Cause Possible Solution Standard OK OK OK Insufficient DNA Confirm that input quality of DNA is adequate Incomplete or excessive Check distribution of DNA DNA fragmentation fragments on agarose gel DNA contains OLA Repurify DNA inhibitors Used plate cover that is incompatible with the SNPlex System assay Use recommended plate cover see Table 1 3 on page 1 10 Concentration of OLA probe ligase or PCR primer is too low Verify that quantities and concentrations of components used are as specified in the protocol Incorrect OLA or PCR thermal cycling conditions Ensure that thermal cycling conditions used are as specified in the protocol Exonuclease step omitted Repeat experiment with exonuclease step Exonuclease mix kept at room tempe
134. nto the 3730 3730xl analyzer to generate 3730 3730x analyzer sample files Data analysis is conducted using GeneMapper Analysis Software v3 7 SNPlex Genotyping System 48 plex User Guide 1 23 Chapter 1 Introduction Hybridization and Loading Wash the Legend hybridization Tit 8 3 plates Y SNPlex Universal PCR primer sequence Hybridization Plate Universal reverse PCR priming site SNPlex System Hybridization Wash Buffer Genome equivalent region Add the PCR SNPlex System aw ZipCode sequence reaction and positive hybridization controls WN AB 8 Universal reverse PCR primer to the plates A e biotinylated i 0 1 N NaOH Universal forward PCR primer Wash the ZipChute probe E Fluorescent dye label and add Remove supernatant Mobility modifiers denaturant c G ZipCode sequence Denaturant SNPlex System uM ZipChute Dilution Buffer SNPlex System Hybridization Plate E ZipChute Mix SNPlex System Hybridize the Y ZipChute ES probes palo Prepare the size standard Size Standard 48 plex SNPlex System and elute the Sample Loading Reagent SNPlex System ZipChute probes Dispense the SNPlex wt Allelic Ladder Load reactions onto 3730 3730xl instrument Applied Biosystems 3730 3730xl DNA Analyzer E Data Collection Software Perform data analysis GeneMapper Software v3 7
135. o the appropriate number of wells 48 wells for a 3730 analyzer e 96 wells for a 3730x analyzer For instructions on setting up a plate for a 48 capillary array or a 96 capillary array refer to the Applied Biosystems 3730 3730xl DNA Analyzers User Reference Guide PN 4331468 Centrifuge the plate to ensure that the samples are at the bottom of the wells Create a spectral instrument protocol in the Protocol Manager as shown in the figure below Protocol Editor m Ea Mame swPlex Spectral Description Type SPECTRAL gt Polymer Pop7 Arey Length se Chemistry Matrix Standard Fun Module Spect36 MtxStd POP7 2 Edit Param o Cancel For details on setting up a run refer to the Applied Biosystems 3730 3730xl DNA Analyzers User Reference Guide PN 4331468 SNPlex Genotyping System 48 plex User Guide 2 7 Chapter 2 Setting Up Applied Biosystems 3730 3730xl DNA Analyzers for SNPlex System Experiments To prepare the matrix standard for the 3730 or 3730x analyzers continued Js Create a plate record for the spectral calibration as explained in the Applied Biosystems 3730 3730xl DNA Analyzers Getting Started Guide PN 4331468 Briefly a Select Plate Manager b Click New then complete the following fields Plate ID Enter an ID for the plate Plate Name Enter a name for the plate Application Select Spectral Calibration Plat
136. of vendors Materials and part numbers Preparing an To prepare an exonuclease reaction Exonuclease Reaction 1 Thaw the Exonuclease Buffer at room temperature If a precipitate forms place the tube briefly in a heating block set to 37 C 2 Vortex then quick spin the tubes 3 Prepare a 2X Exonuclease master mix on ice by scaling the volumes listed below to the desired number of OLA reactions Note Prepare extra volume to account for losses that may occur during pipetting Component Volume per Reaction uL Nuclease free water 4 2 Exonuclease Buffer 10X SNPlex System 0 5 Lambda Exonuclease SNPlex System 0 2 Exonuclease SNPlex System 0 1 Total 5 0 Note Prepare the 2X Exonuclease master mix on ice immediately before use Applied Biosystems does not recommend preparing a large volume of the 2X Exonuclease master mix for later use 4 Pipette 5 uL of 2X Exonuclease master mix into each well of the OLA reaction plate 5 Seal the plate with one of the recommended plate covers see Table 1 3 on page 1 10 If you are using 96 well plates use MicroAmp Full Plate Covers to seal the plate 3 14 SNPlex Genotyping System 48 plex User Guide Purifying Ligated OLA Reaction Products To prepare an exonuclease reaction continued 6 Vortex the plates and spin to collect liquid in the bottom of the wells s Program the thermal cycler Step S
137. older If you use a 48 capillary array including Run Sequence Number or Run Name as a parameter for naming run folders enables the Data Collection software to generate a separate folder for each run required to complete a sample plate see Designing the Sample Plate Layout on page 3 3 For example if you use a 48 capillary instrument two runs are required to run a single 96 well plate or one quadrant of a 384 well plate total of eight runs for a 384 well plate Similarly if you use a 96 capillary instrument a single run 1s required for a 96 well plate or one quadrant of a 384 well plate total of four runs for a 384 well plate Applied Biosystems recommends that you use the above Results Groups conventions in order to allow separate sizing bin adjustments to be applied to individual runs even if they originate from the same probe pool quadrant In the latter case you can cluster the runs individually or together as a project IMPORTANT For GeneMapper software to correctly process SNPlex System data run folder naming conventions and sample plate layout explained in Designing the Sample Plate Layout on page 3 3 must correspond to each other To set up results groups for SNPlex System experiments l Inthe Data Collection software double click Results Group to open the Results Group Editor Er fh a Instruments Il Results Group 2 Select the Naming tab SNPlex Genotyping System 48
138. ombine them with the SNPlex OLA reaction System ligation probes and universal linkers mix 1 18 SNPlex Genotyping System 48 plex User Guide Chemistry Overview Assemble Dispense the OLA reaction mix containing OLA Master Mix dATP ligation the OLA probes and universal linkers into the wells of a reaction plate that reaction contains either dried gDNA or wet gDNA Reserve the appropriate number of wells as indicated in the following table Plate Ctrl Allelic Total Total MS MUMmERI Type e DNA Ladder Controls Samples 3730xl 96 1 1 2 4 92 96 capillary Well 384 4 4 8 16 92X4 Well 3730 96 2 2 4 8 88 48 capillary Well 384 8 8 16 32 88 X 4 Well Thermal Under temperature controlled conditions enzyme phosphorylates the cycle the ASO and LSO linkers and ligation probes the linkers anneal with their OLA respective ligation probes and one or both of the 96 ASO probes and one reactions of the 48 LSO probes per locus bind to the gDNA sample Ligase promotes the ligation of linkers with their respective ligation probes and the ligation of ASO and LSO probes UNG enzyme present in the OLA master mix prevents the re amplification of dU containing accidental carryover PCR products The OLA a ASO LSO linker Procedure umo e E x MG ASO po ASO gt E E ON Legend O zs Universal PCR primer sequence A es
139. on Software Before the Data Collection software can process SNPlex System data you must load three files from the SNPlex System 46 plex Support Files CD or download them from the Applied Biosystems Web site PrebatchModule txt HTSNP36 POP7 V2 e S zip Note If you are using Data Collection v3 0 skip to Creating an Instrument Protocol for SNPlex System Experiments on page 2 4 Replacing the To replace PrebatchModule txt PrebatchModule txt File 1 On the computer running the Data Collection software navigate to E AppliedBiosystems UDC DataCollection SupportFiles ga3730 ServiceModules 2 Rename the existing PrebatchModule txt file For example OriginalPrebatchModule txt 3 Copy the PrebatchModule txt file from the Data Collection Files folder on the SNPlex System support CD 4 Verify that the first line of the file reads SNPlex v2 0 prebatch 5 Paste the file into the ServiceModules folder Importing the To import the HTSNP36 POP7 V2 module HTSNP36 POP7 V2 Module l Determine if the module is installed on your computer a Start the Data Collection software b Open the Module Manager window c Check the list of modules 2 If the module is not installed copy the the HTSNP36 POP7 V2 xml file from the Data Collection Files folder in the SNPlex System support CD 3 Paste the file into the following directory E AppliedBiosystems UDC DataCollection Suppo
140. on Molecular Probes P 11495 reagent special packaging 1 13 SNPlex Genotyping System 48 plex User Guide Chapter 1 Introduction Chemistry Overview Component Kits Workflow Preparing and Fragmenting Genomic DNA Phosphorylating probes and linkers Ligating probes and Linkers SNPlex System Oligonucleotide Ligation kit Purifying Ligated OLA Reaction Products Purification Kit PCR Amplifying Ligated OLA Reaction Products Amplification Kit Binding biotinylated amplicons to streptavidin coated plates Binding Buffer Wash Buffer ZipChute Dilution Buffer ZipChute Kit 48 plex Performing Electrophoresis Standards Kit Analysis 1 14 Several kits and reagents are required for the SNPlex System Assay as specified in Kits and Reagents on page 1 8 These kits and reagents include the associated enzymes master mixes and other components required to perform each step in the SNPlex System Assay Figure 1 3 summarizes the processes required to perform the SNPlex System Assay For simplicity the figure shows the assay for a single SNP allele IM as Note Phosphorylation occurs during the OLA reaction C e gt C ASO v LSO linker ww CASO py LSO mma Ge ASO po ASO 5 OLA Master Mix SNPlex System dATP SNPlex System Universal Linkers 48 plex SNPlex System Ligation Probes SNPlex System ASO po LSO ASO gt lt
141. onditioning the capillary array Performing spatial and spectral calibrations e Validating instrument performance All files referred to in this chapter are supplied on the SNPlex System 48 plex Support Files CD PN 4352129 You can also download the files from the Applied Biosystems Web site at http www appliedbiosystems com support software Supported The SNPlex Genotyping System is optimized for use with Configuration 5750 57301 analyzer Data Collection software v2 0 or higher POP 7 Performance Optimized Polymer e A 36 cm capillary array GeneMapper Software v3 7 or higher Required Materials Description Vendor Part Number POP 7 Performance Optimized Polymer Applied Biosystems 4335615 SNPlex System Array Conditioning Kit Applied Biosystems 4352018 DS 40 Spectral Calibration Standard Kit Applied Biosystems 4349365 Dye Set S 10x Running Buffer with EDTA Applied Biosystems 4335613 36 cm 48 capillary array 3730 analyzer Applied Biosystems 4331247 36 cm 96 capillary array 3730xl analyzer Applied Biosystems 4331244 Hi Di Formamide Applied Biosystems 4311320 SNPlex System Assay Standards Kit Applied Biosystems 4349351 MicroAmp Optical 96 Well Reaction Plate Applied Biosystems N8010560 2 2 SNPlex Genotyping System 48 plex User Guide Importing SNPlex System Files into the Data Collection Software Importing SNPlex System Files into the Data Collecti
142. opriate SNP set for each sample 2 Click B Analysis gt Analyze Samples 4 8 SNPlex Genotyping System 48 plex User Guide Reviewing Results Reviewing Results Use the following guidelines for reviewing your data e Review the bin offsets If the run was good offsets should be small lt 0 5 If offsets are large in one run but not in others inspect the failed run To view bin offsets for a run select the run folder then click View gt Bin Offsets Note The software calculates bin offsets by comparing sample data to the allellic ladder for that run and assumes that all sample files within a folder are from the same run For this reason it is crucial that you keep samples from a run in the same folder For more information about sizing quality refer to Troubleshooting Sizing Quality on page 5 10 Review the sizing quality SQ Any sample that fails sizing is not used in the analysis Select Analysis gt Size Map Editor then observe if the SQ values for size standards passed For more information about sizing quality refer to Troubleshooting Sizing Quality on page 5 10 Review the allelic ladders Allelic ladder samples that do not pass the well quality WQ parameter are not included in the analysis Check binning even for allelic ladder samples with passing WQs Select Analysis gt Display Plots then observe if Each bin contains a single allelic ladder peak
143. or other information that appears in the MSDS of interest Select the language of your choice then click Search 3 Find the document of interest right click the document title then select any of the following Open To view the document Print Target To print the document e Save Target As To download a PDF version of the document to a destination that you choose SNPlex Genotyping System 48 plex User Guide xvii Safety Information Chemical Safety Guidelines To minimize the hazards of chemicals Read and understand the Material Safety Data Sheets MSDS provided by the chemical manufacturer before you store handle or work with any chemicals or hazardous materials See About MSDSs on page xvii Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals for example safety glasses gloves or protective clothing For additional safety guidelines consult the MSDS Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation for example fume hood For additional safety guidelines consult the MSDS Check regularly for chemical leaks or spills If a leak or spill occurs follow the manufacturer s cleanup procedures as recommended on the MSDS Comply with all local state provincial or national laws and regulations related to chemical storage handling and disposal Chemical Waste Safety Chemi
144. ore information SNPlex Genotyping System 48 plex User Guide 3 33 Chapter 3 SNPlex System Protocols Running the To run the plates Plates l In the tree pane of the Data Collection Software double click GA Instruments gt ga3730 or ga3730x1 gt Instrument Name gt gt Run Scheduler 2 Inthe Input Stack group box of the Run Scheduler view click Search then click Find All 3 Select the plate record then click Add 4 Click the green arrow in the toolbar to begin the run Note As part of the prebatch function the instrument oven heats to temperature before the run begins As the data 1s collected you can view it in the Array Viewer SNPlex Genotyping System 48 plex User Guide Analyzing Data Using GeneMapper ooftware This chapter covers dori arrendadores ara AE 4 2 Installing GeneMapper Software v3 7 4 3 Importing SNPlex System Panels and Bins 4 3 EOS IUD eoa E rr si ad dao 4 5 Importing SNPlex System Data into GeneMapper Software 4 6 Analyzing SNPlex System DA uaa dd ERE dA ERE REOR RE CR OED 4 8 Reviewing Results 5 ARRE 4 9 Exporting SNPlex System Data ss ssa dex ER RA FER CR RC 4 11 SNPlex Genotyping System 48 plex User Guide 4 1 Chapter 4 Analyzing Data Using GeneMapper Software Overview Figure 4 1 summarizes the process of analyzing SNPlex System data with GeneMapper
145. ore information about exporting SNPlex System data refer to the GeneMapper software online help SNPlex Genotyping System 48 plex User Guide 4 11 Chapter 4 Analyzing Data Using GeneMapper Software SNPlex Genotyping System 48 plex User Guide Troubleshooting This chapter covers ls e MA 5 2 Troubleshooting Raw DA iua adire REOR ARA RAR 64805065 5 3 Troubleshooting GeneMapper Software Analysis 5 10 Troubleshooting Analyzed Data 1 oye dice edd eek a x br a A REN 5 22 SNPlex Genotyping System 48 plex User Guide 5 1 Chapter 5 Troubleshooting Troubleshooting Process An effective way to carry out SNPlex system troubleshooting is to follow the three step sequential process as explained in the following table See Step Potential Problems Page 1 Inspect raw Problems arising from capillary electrophoresis 5 3 electrophoresis data such as resolution or signal can be identified by studying raw electrophoresis data These problems may not be SNPlex System assay related and can cause ambiguous results even when the chemistry is working properly 2 Review Small errors in sample sheet setup file type 5 10 GeneMapper assignment size standard peak assignment or software analysis binning of allelic ladders can cause GeneMapper software analysis to fail or to produce ambiguous results 3 Study analyzed If problems do not seem to be caused by 5 22 results and assay mistakes
146. ostures SNPlex Genotyping System 48 plex User Guide Introduction This chapter covers li A 1 2 SNPlex System Kits and Reagents ss sek C rra A test 1 8 Kegmred Non Kit MSIE uua dg ace HORACE ROLE ERROR IR Et aes 1 10 Hace 1 14 Laboratory Design Preventing Amplicon Contamination 1 25 SNPlex Genotyping System 48 plex User Guide 1 1 Chapter 1 Introduction Product Overview Product Description The human genome contains between 6 million and 30 million single nucleotide polymorphisms SNPs Geneticists estimate that 300 000 or more SNPs may be required per individual to map complex diseases such as cancer and heart disease in a given population The SNPlex Genotyping System uses Applied Biosystems oligonucleotide ligation assay OLA combined with multiplex PCR technology to achieve allelic discrimination and target amplification The chemistry is made possible through the use of a set of universal core reagent kits and a set of SNP specific ligation probes The complete SNPlex Genotyping System contains the following components Universal SNPlex System Kits and reagents The SNPlex System Kits contain all the reagents needed to perform the SNPlex System Assay SNPlex System Kits and Reagents on page 1 8 provides details about each kit SNP specific ligation probes Applied Biosystems designs SNP specific probes also called assays based on the SNPs that you specify You can sub
147. plate and performing a single run o k N 000000000000 e 2 9 9 69 6 69 69 9 9 69 0000600000000 6 9 6 6 6 6 6 6 6 6 6 6 5 3 69 9 62 9 69 6 9 69 0 S 9 62 69 69 69 69 69 9 9 69 9 C 9 9 65 9 69 9 9 62 5 69 69 62 amp 9 69 69 69 3 6 9 1 to 92 gDNA samples C Control DNA N NTC L Allelic Ladder SNPlex Genotyping System 48 plex User Guide 3 3 Chapter 3 SNPlex System Protocols 3 30xl Instrument 96 capillary 3 30 Instrument 48 capillary 3 4 384 wells ub 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 0 6 0767050707076707676 0000006 65 65 73 73 81 0 0 0 0 0 0 059 0 07070 p 6 10956565 66 66 74 74 82 0 0707070 0 0 0 6707670 reoeeoccoco9 67 67 75 75 83 0 0 0 0 0 0 0 050 07070 H 5 65 5 12 85 0 8 86 65 68 76 76 84 K Aros L 6 6 2 6 6o 0686 M 0 0 0 0 0 0 0 0 0707070 N 0005156525 66 6069 0 0 6665 666 9 00 9 0 P O 0 0 0 0 0 0 0 0 0 000 01 P 66441508 6 6 q d 65 66 6 6 Qo 9 6 60 6 66 6 b Nod 3 rg 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 A 0 00 0 0 0 0 9 0 0 0 0 B reeecccoccoo00600900000000600 O Blue O Yellow Probe Set A Quadrant 1 O Green Probe Set B Quadrant 2 96 wells two separate runs Injection 1 moe O U gt Ei 6600000 amp o9 90990090 e6e000004 OOOO 0606606660 ro 96600
148. plate record then displays a confirmation message if the import is successful If you set up your text file as recommended the Sample Name Results Group Instrument Protocol Sample Type and SNP Set fields are complete at this point If you have set up the Add Samples options in GeneMapper software Setting Analysis Method Size Standard and Panel Automatically on page 4 7 the Analysis Method Size Standard and Panel fields will be completed automatically when the sample files are imported into GeneMapper software IMPORTANT For GeneMapper software to recognize the SNP set information you must have imported the assay information file into GeneMapper software Importing AIFs on page 4 5 SNPlex Genotyping System 48 plex User Guide 3 31 Chapter 3 SNPlex System Protocols Creating Plate To create the plate record manually Records Manually ll In the tree pane of the Data Collection Software double click GA Instruments gt ga3730 or ga3730x gt Instrument Name gt gt Run Scheduler In the Add Plate field of the Run Scheduler view enter or scan the bar code of a plate that you want to run then press Enter In the Select an Option dialog box click Yes In the New Plate dialog box update the following fields D Barcode Scan or enter the barcode for the plate you want to run e Name Enter a name for the plate Description Enter a description for the plate record
149. ple plate requirements 3 3 Services and Support obtaining xiii sets dye 1 10 3 32 shoulder misidentification 5 16 signal intensity variation across plate 5 24 signal strength checking 5 14 signal strength for troubleshooting 5 4 size calls verifying 5 16 Size Match Editor for checking signal strength 5 14 for size standard peak identification 5 16 size standard setting for analysis 4 7 size standard checking for even peaks 5 17 sizing quality troubleshooting 5 10 SNP control pool contents A 2 using A 5 SNPlex System analysis methods 4 8 SNPlex system prebatch files 3 33 SNPlex System Software Suite 1 2 SNPlex_Rules_3730 about the algorithm B 4 default settings B 5 SNP specific probes 1 3 spatial calibration performing 2 6 spectral calibration performing 2 6 spectral calibration troubleshooting 5 6 Standards kit 3 22 standards calibration 1 10 streptavidin plates preparing 3 18 system files 2 3 Index 3 T Technical Support contacting xiii testrun 2 8 test run evaluating 2 9 test sample plate for mock run 2 8 text conventions xi thermal cycling conditions Exonuclease reaction 3 15 oligo ligation assay with UNG 3 13 PCR 3 17 Purification 3 15 Training obtaining information about xiii troubleshooting allelic ladder 5 18 analyzed data 5 22 cluster plots 5 26 electrophoresis raw data 5 3 ion front 5 7 medial signal intensity 5 24 Negative Hybridization Control NHC 5 25 Positive Hybridiz
150. ple wells pool the boiled gDNA 8 Dilute the gDNA to 18 5 ng uL with 1X TE pH 8 0 Note If using whole genome amplification WGA Applied Biosystems recommends that you dilute the DNA to 37 ng uL with 1X TE pH 8 0 a The concentration of 18 5 ng uL is based on quantification using the TagMan RNase P Quantification Assay If you are using fluorescence or absorbance based assays Applied Biosystems recommends using double the gDNA concentration 37 ng uL Drying Down The SNPlex System assay is equally effective whether you use dried down or wet gDNA gDNA However if your experiment requires multiple plates that use the same gDNA or if you plan to use the same gDNA in several experiments it is convenient to dry down the gDNA in the plates which are then ready for use at any time SNPlex Genotyping System 48 plex User Guide 3 7 Chapter 3 SNPlex System Protocols Dispensing gDNA To dispense gDNA into reaction plates into Reaction Plates 1 Label the reaction plate 96 capillary array 96 well plate 96 capillary arrays 384 well plate 100000000000 gt 00000000000 0 9 616 6 6s 0 e 2606000000000 1990000060 00000000000 0108 86 216 618 0 9 0 100000000000 129600 6 06 6 100000000000 100060000000 606000000000 eOCHODCUUDOCOGOGCLU 60000000000 BO OO DO O O OOOO 606000000000 48 capillary array 96 well plate 1 2 3 4
151. plex User Guide Creating Results Groups and Plate Records To set up results groups for SNPlex System experiments continued 3 Complete the information in the tab as shown in Figure 3 2 and Table 3 1 Note When you create a new results group the Data Collection software displays a single drop down box under each Format section Each time you make a selection as specified in the table below the software adds a drop down box Figure 3 2 shows the Results Group Editor for a sample Results Group used with 3730 instruments running a 48 capillary array Note that for instruments running 96 capillary arrays the Run Sequence Number is unnecessary it Results Group Editor E 3H X General Analysis Destination Naming Sample File Name Format Example Al Sample3_007_ 2002 04 21 lt ext Filename is greater than 26 characters Prefix Marne Delimiter B Format wei Position Sample Mame Capillary Number Date of Run lt none gt Sufi File Extension lt None gt Run Folder Mame Format Example ElAppliedBiosystemsiudcidatacollectioniDataSeqPlate96_QG2 0012 3 Minimum number of characters 63 Prefix Mame Delimiter B Format Plate Mame Plate Quadrant Run sequence Number Run Murnber lt none gt Ok Cancel Figure 3 2 Results Group settings for 3730 instruments running 48 capillary arrays Table 3 1 Suggested minimum sample file and run folder pa
152. pplied Biosystems 3730 3730x DNA Analyzers for SNPlex System Experiments Gel Mer 2 2 Supported COMTIGUIALION a4 siad pe scd ah ab dA ed qe ER AY NERA RR cR Dat 2 2 Required Materials 3 35 EX RR ca a AAA 2 2 Importing SNPlex System Files into the Data Collection Software 2 3 Replacing the PrebatchModule txt File 2 3 Importing the HTSNP36_POP7_V2 Module 2 3 Installing Dye SGE 9 sea oa e dar ss mold oda 2 4 Creating an Instrument Protocol for SNPlex System Experiments 2 4 Preconditioning the Capillary Array llle 2 5 Performing Spatial and Spectral Calibrations 2 6 Performing a Spatial Calibration 2 6 Performing a Spectral Calibration 2 6 Validating Instrument Performance 2 8 Preparing the Test Sample Plate 2 8 Evaluating the SNPlex System Run lille 2 9 Chapter 3 SNPlex System Protocols OVSIVIEW 4 RER sede eee acte ii ts 3 2 Protocols In This CUIR as ised riant Sah eis Ox CREER CERO E nie SOR ORC 3 2 Before YOU BEING EA s ada qe en Ne Re hg 3 9 Designing the Sample Plate Layout 2 3 9 A A UA a vti id ater RU auf see RD 3 3 ASSUMPUONS vica ete edd di de D e s NER ud ded NOT d deut eae o
153. r 19 14 933 236 SNPlex Genotyping System 48 plex User Guide A 3 Appendix A Using the SNPlex System Control Set Table A 1 48 SNPs in the SNPlex System Control Pool continued Celera SNP Minor Allele Frequency Zip Discovery Consortium dbSNP ID System ID ID rs AfA Cauc Chin Japn bus hCV TSC Mb c d e Location M5 1 044 hCV468629 TSC0463040 rs1569125 0 41 0 29 0 22 0 2 Chr 2 236 572 816 M5 1 045 hCV1534177 0 22 0 20 0 27 0 29 Chr 18 75 206 886 M5 1 046 hCV11164916 0 19 0 12 NA NA Chr 7 21 876 339 M5 1 048 hCV2214945 TSC1086371 rs220860 0 25 0 22 0 22 0 22 Chr 11 115 327 978 M5 1 049 hCV8847720 TSC0695101 rs1460239 0 29 0 40 0 19 0 29 Chr 8_105 480 159 M5 1_050 hCV8777053 TSCOO70807 rs954779 0 28 0 18 0 32 0 30 Chr 9 36 409 531 M5 1 051 hCV7443819 TSCO267953 rs963014 0 48 0 40 0 08 0 11 Chr 8 117 999 529 a Indicates the correlation between a SNP and its corresponding ZipChute probe pair b African American c Caucasian d Chinese e Japanese f Indicates chromosome and base position on the chromosome as derived from the SNPbrowser software About the Dried gDNA Plate Each gDNA plate contains 44 unique human DNAs of Caucasian origin Each gDNA is plated at least twice in each quadrant of a 384 well microtiter plate to allow for the asessment of assay reproducibility refer to the SNPlex Geno
154. r required materials Materials Part Item Vendor Number Hybridization oven capable of maintaining a constant MLS temperature of 37 C 1 C Centrifuge equipped to accomodate reaction plates MLS Vortex MLS 96 well aluminum block MLS 384 well aluminum block MLS Multichannel pipettor 250 uL MLS Pipetting reservoirs 25 mL MLS Pipetting reservoirs 100 mL MLS Rotary shaker MLS Standard heat block MLS Sterile wide bore pipette tips VWR 46620 642 1 12 SNPlex Genotyping System 48 plex User Guide Optional Materials Table 1 6 Optional reagents Required Non Kit Materials 1 Part Function Item Vendor Number DNA Qiagen Flexigene Kit Qiagen 51206 Purification choose one Gentra Puregene Kit Gentra D 5000 DNA e TagMan RNase P DNA Applied Biosystems 4343782 Quantitation Quantification Kit m choose one or e TagMan RNase P Detection 4316831 Reagents Kit Note The RNase P kits require one of the TagMan Universal PCR Master Mixes listed below e TagMan Universal PCR Master Applied Biosystems 4304437 Mix r or e TagMan Universal PCR Master 4324018 Mix without UNG PicoGreen dsDNA Quantitation Kit Molecular Probes P 7589 PicoGreen dsDNA Quantitation Kit Molecular Probes P 11496 special packaging PicoGreen dsDNA Quantitation Molecular Probes P 7581 reagent PicoGreen dsDNA Quantitati
155. ral calibration Verify that polymer and buffer Use Dye Set S have not expired Replace old Use DS 40 matrix standard polymer and buffer on instrument and rerun Or if needed If only a few capillaries are affected override spectral calibration from neighboring capillary For more information see section Verify that the array was on troubleshooting resolution preconditioned with Array Conditioning Buffer before use p 2 5 Check Spectral Are pull up or pull down peaks in the raw data p 5 6 For more information see section on troubleshooting spectral calibration Check for lon Front One or two unusually tall or narrow peaks relative to the surrounding peaks that appear near the beginning of the run p 5 7 Verify that most recent version of SNPlex system run module is being used to run samples Replace old polymer and buffer on instrument Verify that polymer and buffer have not expired For more information see section on troubleshooting ion front Proceed to troubleshooting GeneMapper Software Analysis Figure 5 1 Workflow for troubleshooting raw data e To view raw data using GeneMapper software select View gt Raw Data To return to the samples view select View gt Samples SNPlex Genotyping System 48 plex User Guide 5 3 Chapter 5 Troubleshooting Electrophoresis Some problems that you can identify in the raw data Related Problems Signal streng
156. rameters for SNPlex System results groups Parameter Comment Sample File Name In the Format section under Sample File Name Format select Well Position Well Position Sample Name Sample Name Capillary Number Capillary Number Run Folder Name In the Format section under Run Folder Name Format select Plate Name Plate Name Plate Quadrant Plate Quadrant SNPlex Genotyping System 48 plex User Guide 3 27 Chapter 3 SNPlex System Protocols 3 28 About Plate Records Table 3 1 Suggested minimum sample file and run folder parameters for SNPlex System results groups continued Parameter Comment Run Sequence Number Run Sequence Number Note If this parameter is selected the Data Collection software adds a four digit number to the run folder name The number is incremented with every run on the instrument This option is highly recommended for the 3730 instrument when running 48 capillary arrays It is not necessary when running 96 capillary arrays Run Name An alternative to Run Sequence Number If this parameter is selected the Data Collection software appends a text string such as Run InstrumentName Date Time RunSequence Number to the run folder name Run Number Run Number a Preferred over Run Name A plate record is similar to a sample sheet or an injection list that you may have used with some Applied Biosystems instruments Plate records
157. rature for more than 1 hour before use Insufficient PCR product transferred during post PCR step NaOH denaturation step omitted Prepare fresh exonuclease mix and repeat experiment Check protocol Evaluate and optimize pipetting accuracy in assay workflow Repeat experiment with denaturation step SNPlex Genotyping System 48 plex User Guide Troubleshooting Analyzed Data Table 5 1 Troubleshooting cluster plots with low signal and poor cluster information continued PHC NHC i Potential Cause Possible Solution Standard Low OK OK Low ZipChute mix Check concentration of signal concentration ZipChute mix added to hybridization master mix Wrong ZipChute dilution Repeat experiment using buffer used for the correct ZipChute hybridization master mix dilution buffer ZipChute dilution buffer not Repeat experiment using properly diluted ZipChute dilution buffer at correct concentration Bound ZipChute probes Reconfigure the plate stripped off plate washer Set the aspiration tip depth so that 15 to 20 uL of Wash Buffer remains in each well after each aspiration Temperature too high Check temperature in during ZipChute probe hybridization oven and hybridization correct if necessary Low OK Low Incorrect electrophoresis Confirm that correct signal signal protocol injection time electrophoresis protocol too short was used Increase injection time Run allelic ladder plate an
158. re binned correctly Incorrect peak may be appearing in NHC bin NHC is OK Nonspecific hybridization is occuring during the hybridization step or incomplete ZipChute probe removal is occuring during the wash step Verify that these steps are performed according to protocol and that all liquid is removed from wells during the wash step Evaluate cluster plots Refer to section on evaluating cluster plots p 5 25 Figure 5 6 Workflow for troubleshooting GeneMapper software analysis Some problems that can arise from incorrectly setting up GeneMapper software are Poor sizing quality See page 5 10 Problems arising from allelic ladders See page 5 18 Examples of each problem and possible solutions are provided in subsequent sections of this chapter Determining Sizing Quality GeneMapper software sizing quality for SNPlex System results is indicated by symbols or numbers in the SQ sizing quality column of the Samples table SQ values should be 0 98 or greater Lower sizing quality values result in unreliable size calls SNPlex Genotyping System 48 plex User Guide Troubleshooting GeneMapper Software Analysis By default GeneMapper software displays colored symbols in the SQ column However when troubleshooting sizing quality it is easier to use the numerical representation of sizing quality Sizing Quality Representation Example Samples Table Symbols default ELA SE EN DGB_SNPlex
159. rotocol Editor opens Complete the Protocol Editor as shown in the figure below EE een Name SNPlex Protocol Cescnp ion EDEN Select REGULAR Type REGULAR Run Module HTENP36_POPT_ WE 1 un Module for DC V2 0 oct NENEEEEEN HTSNP36 POP7 V2 1 Run Module for DC V3 0 HTSNP36 POP7 V3 Le cores Instrument SNPlex System Experiments Za 3 4 Click OK to save the instrument protocol SNPlex Genotyping System 48 plex User Guide Preconditioning the Capillary Array Preconditioning the Capillary Array Before running SNPlex System chemistry on 3730 3730x analyzers that is before performing spectral or spatial calibrations or SNPlex System protocols you must precondition the capillary array Runs performed using improperly conditioned arrays have poorly resolved peaks To precondition the capillary array l Using the reagents in the SNPlex System Array Conditioning Kit prepare a 500X dilution of Array Conditioning Buffer a Dispense 100 mL of molecular biology grade deionized water into a sterile graduated cylinder b Add 200 uL of Array Conditioning Buffer included in the SNPlex System Array Conditioning Kit PN 4352018 c Cover and invert several times to mix Rinse the plastic array header shipping cover supplied with the new array with deionized water Pour the diluted Array Conditioning Buffer solution into the cover then p
160. rtFiles ga3730 RunModules 4 In E AppliedBiosystems UDC DataCollection bin double click the Import3730RunModules exe file SNPlex Genotyping System 48 plex User Guide 2 3 Chapter 2 Setting Up Applied Biosystems 3730 3730xl DNA Analyzers for SNPlex System Experiments To import the HTSNP36 POP7 V2 module continued 5 Verify that the module has been installed by opening the Module Manager window and observing that HTSNP36_POP7_V2_1 appears in the module list In the Module Manager click Edit then verify that the module has a 45 second prerun at 15 kV Installing To install Dye Set S Dye Set S l Start the Data Collection software 2 Open the Protocol Manager Start a new spectral calibration using Dye Set S e If the protocol is not available install it as explained in step 4 e Ifthe protocol is available exit this procedure In the Protocol Manager click New Click the folder icon then navigate to the S zip file in the Data Collection Files folder in the SNPlex System support CD Click Open Dye Set S should now be available Creating an An instrument protocol contains all the setting necessary to run the instrument Protocol for To create an instrument protocol In the Tree pane of the Data Collection Software click GA Instruments gt ga3730 or ga3730xl gt Protocol Manager In the Instrument Protocols section click New The P
161. s method 1 Start the GeneMapper software 2 Select Tools gt GeneMapper Manager then select the Analysis Methods tab SNPlex Genotyping System 48 plex User Guide Troubleshooting GeneMapper Software Analysis To adjust the blue or green threshold in the SNPlex System analysis method continued 3 Create a new analysis method based on the current method a b C d e Select the current analysis method that you are using to analyze SNPlex System data Click Save As Enter a name for the new method Select a name that easily identifies it as a special ladder analysis method Click OK The new method appears in the list of analysis methods Click Done Select the new analysis method then click Open In the Analysis Method Editor select the Peak Detector tab Change the Peak Amplitude Threshold for green or blue to a value greater than the signal intensity of the misidentified peak Click OK to save the analysis method Click Done to close the GeneMapper Manager then reanalyze the samples using the new analysis method for only the allelic ladder wells IMPORTANT Use the standard analysis method to analyze the rest of the samples IMPORTANT Setting the blue or green thresholds higher than the default value 10 RFU can cause the software to miss sample peaks Use analysis methods with modified threshold settings only for analyzing allelic ladder
162. sers as well as documentation The ligation probes Two sets of hybridization plates 96 well or 384 well A control pool e A dried gDNA plates kit These reagents are described in the following section Reagent Name Part Number SNPlex Genotyping System Core Reagents Kit 48 plex 4362266 SNPlex System Assay Control Kit 4349363 Control DNA SNPlex System SNPlex System Oligonucleotide Ligation Kit 4362268 e Universal Linkers 48 plex SNPlex System e Oligonucleotide Ligation Master Mix SNPlex System e dATP 100X SNPlex System SNPlex System Purification Kit 4349357 e Lambda Exonuclease SNPlex System e Exonuclease Buffer 10X SNPlex System e Exonuclease SNPlex System SNPlex System Amplification Kit 4349358 e Amplification Primers 20X SNPlex System e Amplification Master Mix 2X SNPlex System Hybridization Binding Buffer SNPlex System 4349304 Hybridization Wash Buffer 10X SNPlex System 4349301 ZipChute Dilution Buffer SNPIex System 4349306 SNPlex System ZipChute Kit 48 plex 4349361 e Denaturant SNPlex System e ZipChute Mix 48 plex SNPlex System e Positive Hybridization Controls SNPlex System SNPlex System Standards Kit 48 plex 4349351 e Sample Loading Reagent SNPlex System e Size Standard 48 plex SNPlex System e Allelic Ladder 48 plex SNPlex System SNPlex Genotyping Syst
163. sing the Biomek FX Getting Started Guide FX instrument for automating the OLA portion of the SNPlex System assay e Provides the step by step automation protocols for using the Biomek FX instrument to automate the OLA portions of the SNPlex System assay Document Title eae Contents Availability Number SNPlex Genotyping System 4357460 e Explains how to design SNPlex e Part of the SNPlex 48 plex Assay Design and System ligation probes Genotyping System Ordering Guide e Provides instructions for using the Starter Kit 48 plex Web site to order SNPlex System e Downloadable from the ligation probes SNPlex System Web e Describes the error conditions that site the ent you may encounter when designing on Demand Web site probes and the myScience environment Web site SNPlex Genotyping System 4360856 Describes the SNPlex Genotyping e Part of the SNPlex 48 plex User Guide System 48 plex Genotyping System this document e Explains how to set up the 3730 and Starter Kit 48 plex 3730x instruments for use with the e Downloadable from the SNPlex System SNPlex System Web e Contains the 96 and 384 well site and PO dat protocols for the 3730 3730xl on Demand Web site analyzers e Provides information about analyzing SNPlex System data with GeneMapper Software v3 7 e Discusses the troubleshooting process for SNPlex System experiments SNPlex Genotyping System 4360855 Contains short versions of the 96 and e Part o
164. ss SS 0832909 24006 QOOOQOQOQOQO0 O16 6 3s 6 QOOQOQOQOOO 0000000000000000 0000000000000000 0000000000000000 0000000000000000 0000000000000000 0000000000000000 0000000000000000 0000000000000000s 00000000000000003 0000000000000000 00000000000000005s 00000000000000003 0000000000000000 00000000000000005 00000000000000003 00000000000000003 0000000000000000s3 00000000000000003 4 O O O O O O O O O O O O O O O O T TO c r Ace 48 capillary array 96 well plate a o a D 1 2 3 4 5 6 if 8 9 1 9 10 11 12 13 14 15 16 17 18 19 20 000000000080 50 000000000000 OOO 3 619 6 oe o O 000000000000 O 00 OOO 6 6 3 3 6 o 000000000000 OOO OOO DO OOO o 000000000000 000000000000 10 000000000000 C OOO ODO OOOO NE 000000000000 NTC OO DOO S NO 000000000000 OOOOOOOOOO oQO P O 000000000000 C Control DNA NTC No Template Control L Allelic Ladder For information about proper sample plate layout refer to Designing the Sample Plate Layout on page 3 3 2 Pipette 3 0 uL of OLA reaction mix see step 4 on page 3 10 into each well of the plate IMPORTANT Do not add reaction mix into the allelic ladder wells For information on setting up the allelic ladder wells refer to Preparing Samples for Electrophoresis on page 3 24 3 nto each sample well add 2 0 u
165. ssay IMPORTANT Take care when quantifying gDNA To obtain tight genotype clusters the amounts of the different gDNA samples used for the ligation step must be relatively equal When possible run an aliquot of the quantified gDNA samples on a 0 8 agarose gel to verify equal gDNA concentration TaqMan RNase P Kits Applied Biosystems recommends quantifying the concentration of human gDNA using the TagMan RNase P DNA Quantification Kit or TaqMan RNase P Detection Reagents Kit Both kits require TaqMan Universal PCR Master Mix Using either kit for quantifying DNA is optional The assay provides a functional evaluation of the quality of DNA IMPORTANT If using RNase P kits to quantify DNA quantify the DNA before fragmenting it SNPlex Genotyping System 48 plex User Guide Chemistry Overview There are two copies of the RNase P gene per human cell After RNase P reagents bind to the RNAse P gene the gene is amplified by PCR During amplification the RNase P gene probe is cleaved generating a reporter signal By referencing a standard curve of RNase P gene concentration in human gDNA you can interpolate your starting concentration of gDNA Applied Item Biosystems Part Number TaqMan RNase P DNA Quantification Kit 4343782 e 20X RNase P primer and probe mix e Human DNA standard TaqMan RNase P Detection Reagents Kit 4316831 e 20X RNase P primer and probe mix sufficient to run 200 25 uL reactions
166. stems No rights are granted expressly by implication or by estoppel or under any other patent rights owned or licensable by Applied Biosystems Further information on purchasing licenses to practice the DNA sequencing or fragment analysis methods may be obtained by contacting the Director of Licensing Applied Biosystems 850 Lincoln Centre Drive Foster City California 94404 U S A TRADEMARKS ABI PRISM Applied Biosystems BigDye GeneMapper LIZ ABI PRISM and MicroAmp are registered trademarks and AB Design Applera FAM Celera Discovery System Celera Genomics Hi Di POP 7 ROX SNPlex SNPbrowser TAMRA ZipChute ZipCode and myScience are trademarks of Applera Corporation or its subsidiaries in the U S and or certain other countries GeneAmp AmpErase and TaqMan are registered trademarks of Roche Molecular Systems Inc PicoGreen is a registered trademark of Molecular Probes Inc All other trademarks are the sole property of their respective owners Part Number 4360856 Rev B 6 2005 Contents Preface How to Use This Guide a a xi How to Obtain More Information eee les xii How to Obtain SUDPON 4 0 3o dc ed tx d C CC A ae eb he C rg N xiii Safety Information Safety Conventions Used in This Document xvi Chemical Sale 2425 de Lo Y EA dio A eos des AC a ace e o e s xvii Chemical Waste Safety xviii Biolog
167. store at room temperature in the dark until use 3 8 SNPlex Genotyping System 48 plex User Guide Phosphorylating and Ligating Probes to gDNA OLA Phosphorylating and Ligating Probes to gDNA OLA For a summary of the steps in the OLA procedure refer to Phosphorylating and Ligating Probes to gDNA OLA on page 1 18 Oligonucleotide The components in the SNPlex System Oligonucleotide Ligation Kit PN 4362268 Ligation Kit are listed below The kit contains enough reagent for 5 000 reactions Components Component Storage Temperature C Oligonucleotide Ligation Master Mix 4 to 8 SNPlex System dATP 100X SNPlex System 15 to 25 Universal Linkers 48 plex SNPlex System a The Oligonucleotide Ligation Master Mix is shipped frozen After first use store at 4 to 8 C for up to 1 month Vortex briefly prior to use About SNPlex The pooled SNPlex System Ligation Probe Pools that you order arrive in individually System Ligation labeled tubes Each tube contains a pool of the following three probe types mixed Probes together e Allele specific oligo Al ASO e Allele specific oligo A2 ASO Locus specific oligo LSO The universal linkers are delivered in a separate tube Preparing the To prepare the OLA reactions OLA Reactions 1 Thaw the following reagents at room temperature Universal Linkers 48 plex SNPlex System dATP 100X SNPlex System e SNPlex
168. strument Protocol required required GeneMapper Software Fields Size Standard optional required Analysis Method optional required SNP Set optional required Panel optional required Sample Type optional required When creating plate records note that e You must complete the required 3730 3730xl fields before a run regardless of which GeneMapper software application you are using to analyze the data e If you run the GeneMapper Generic application you can complete the GeneMapper software fields after the electrophoresis run but before analyzing the data Refer to Chapter 4 for information on setting up GeneMapper software Note You cannot analyze the samples in GeneMapper software unless these fields are completed 3 30 SNPlex Genotyping System 48 plex User Guide Creating Results Groups and Plate Records e If you run the GeneMapper lt nstrument Name application you also need to complete the GeneMapper software fields before the electrophoresis run You can preset GeneMapper software to automatically apply the Analysis Method Panel and Size Standard whenever sample files are imported as explained in Importing SNPlex System Data into GeneMapper Software on page 4 6 You can import SNP Sets from assay information files as explained in Importing the AIF on page 4 5 When adding Sample Type and SNP Set information to a plate record enter them in such a way that the data can be readily a
169. t01S_A01_SNPlex_015_20 Size Matches Size Calling Curve Inst018_405_SMPlex_016_20 Inst018_ 405 SNPlex 031 20 Sizing Quality 1 0 Override S Inst018_407_SMPlex_032_20 Ihst018_409_SMPlex_047_20 Ihst018_411_SMPlex_045_20 Inst018_4153_SMPlex_0653_20 Inst018_415_SMPlex_064_20 inst018_417_sMPlex_079_20 Inst018_419_SMPlex_0680_20 Inst018_421_SMPlex_095_20 Inst018_425_SMPlex_096_20 Inati C 1 SMPlex O13 2 1200 1400 1600 1800 4 Ge EE COQ Ok Cancel Apply Figure 5 7 Size standard with good sizing quality Example Poor Sizing Quality Figure 5 8 shows a size standard with poor sizing quality Note the low signal in the figure below tg Size Match Editor X File Edit View Tools X BS AD1 90004949 fea Size Matches size Callinc Curve 203 80004944 fea AOS 90004885 fea Sizing Quality 0 0 Override S 407 390004923 fea E A f5a JL 1200 Ok Cancel Apply Figure 5 8 Size standard with poor sizing quality SNPlex Genotyping System 48 plex User Guide 5 13 Chapter 5 Troubleshooting Example Size Standard Error Figure 5 9 shows a size standard that failed due to no signal which occurs when size standard is omitted from the sample or when the sample is not properly injected Note that all signal strengths are below 12 RFU compared to samples with good sizing quality Figure 5 7 which have signal strengths above 200 RFU 400 600 1200 1600 2000 2400 2000 Figure 5 9 Siz
170. ta based solely on peak height without clustering analysis Clustering This method makes calls based on a minimum confidence value for a sample in a particular cluster Most SNPlex System data is analyzed using this method Modifying the If you select the Filtering method you can specify an Allele Cutoff Value which is Allele Cut off the value below which a peak is recognized as an allele The allele cutoff value is the Value percentage of the larger peak s height required for the smaller peak to be called as an allele IMPORTANT Applied Biosystems does not recommend using this method Heterozygous Homozygous 0 3 A A Allele cutoff value A1 A2 A1 A2 Modifying the If you select the Clustering method for allele calling you can Clustering e Specify how data is clustered Parameters e Set the clustering threshold Modify values of the rules that the algorithm uses to calculate SNP quality Specifying How Data is Clustered e Select Cluster By Run for the software to analyze the samples in one run independently from other runs Most SNPlex System data is clustered according to this method IMPORTANT If you are using the Model method you must select Cluster By Run e Select Cluster By Project for the software to analyze all of the selected samples in a project regardless of whether the samples were run at the same time If you select Cluster By Project make sure that you select the SNP set before analyzing your project
171. tation Denaturant SNPlex System is a possible developmental and birth defect hazard Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves To hybridize the ZipChutes Probes 1 Equilibrate the oven to 37 C 2 Prepare a hybridization master mix by scaling the volumes listed below to the desired number of samples Prepare extra volume to account for losses that may occur during pipetting Component Volume per Reaction uL ZipChute Mix 48 plex SNPlex System 0 05 Denaturant SNPlex System 11 25 ZipChute Dilution Buffer SNPlex System 13 70 Total 25 00 Note Once prepared you can keep the hybridization master mix at room temperature for at least 2 hours without a loss in performance After 2 hours the remaining hybridization master mix may be stored covered in the dark at 4 C for up to 4 days for later use You may prepare a large quantity of hybridization master mix and store it covered in the dark at 4 C for up to 4 days 3 Add 25 uL of the hybridization master mix to each well 4 Cover the plate with one of the recommended plate covers see Table 1 3 on page 1 10 5 Incubate the plates for 60 min at 37 C on a rotary shaker Note During incubation avoid exposure to direct light Note To avoid possible overheating do not place the plate directly on the floor of the oven SNPlex Genotyping System 48 pl
172. tely through the myScience M research environment described in the SNPlex Genotyping System 48 plex Assay Design and Ordering Guide SNP specific probes are not included in the SNPlex System kits About Universal The SNPlex System Oligonucleotide Ligation Kit includes a set of Universal ASO LSO Linkers ASO LSO linkers e Each ASO is ligated to a universal ASO specific linker These linkers contain A universal PCR primer sequence corresponding to the universal forward primer UA sequence A partial cZipCode sequence The ASO linkers anneal to the universal ZipCode sequence of the ASO probes In a 48 plex reaction there are 96 different ASO linkers one for each of the 96 ASO probes each 48 plex utilizes the same set of 96 universal ASO linkers Although the ASO linkers anneal to specific ZipCode sequences they are not SNP specific One additional linker is ligated to the LSO and has a universal sequence that is compatible with all LSOs That is there is only one LSO linker in a 48 plex reaction The sequence includes a partial binding site for a universal reverse primer Each linker contains a spacer that protects a complete ligation product from exonuclease digestion Figure 1 1 shows the interaction between SNP specific probes and universal linkers SNPlex Genotyping System 48 plex User Guide 1 3 Chapter 1 Introduction ASO ASO EME C ASO po ASO 2 TT M LSO gt LSO Linker L
173. tents and applications outside the U S to use only this amount of the product to practice the Oligonucleotide Ligation Assay OLA and Ligation Detection Reaction LDR methods described in said patents solely for the purchaser s own research and development activities when this product is used in conjunction with SNPlex System Oligonucleotide Ligation Kit No other rights are granted expressly by implication or by estoppel or under any other patent rights owned or licensable by Applied Biosystems Further information on purchasing licenses for the OLA and LDR methods and other applications may be obtained by contacting the Director of Licensing at Applied Biosystems 850 Lincoln Centre Drive Foster City CA 94404 U S A SNPLEX SYSTEM AMPLIFICATION KIT 4349358 NOTICE TO PURCHASER LIMITED LABEL LICENSE A license under the non U S counterparts of U S Patents 4 683 202 4 683 195 and 4 965 188 owned by F Hoffmann La Roche Ltd Roche for use in research and development has an up front fee component and a running royalty component The purchase price of the SNPLEX6 SYSTEM AMPLIFICATION KIT includes limited non transferable rights under the running royalty component to use only this amount of the product to practice the Polymerase Chain Reaction PCR and related processes described in said patents solely for the research and development activities of the purchaser when this product is used in conjunction with a thermal cycler whose use is covere
174. tep Type Temperature C Time 1 HOLD 37 90 min 2 HOLD 80 10 min 3 HOLD 4 00 Transfer the reaction plates to the thermal cycler and start the program After thermal cycling is complete spin to collect liquid in the bottom of the wells 10 Add 15 uL of nuclease free water to each well mix then spin down 11 Process the enzyme digested OLA reaction products To use the OLA reaction products e Immediately Proceed to Performing PCR on page 3 16 e Within 21 days Store at 20 C Note For storage seal the plates with one of the recommended plate covers see Table 1 3 on page 1 10 Note For best results use the OLA reaction products immediately SNPlex Genotyping System 48 plex User Guide 3 15 Chapter 3 SNPlex System Protocols Performing PCR For a summary of the steps in the amplification procedure refer to PCR Amplifying Ligated OLA Reaction Products on page 1 21 Amplification Kit The components in the SNPlex System Amplification Kit PN 4349358 are listed Components in the table below The kit contains enough reagent for 5 000 reactions Component Storage Temperature C Amplification Master Mix 2X SNPlex 2 to 8 System Amplification Primers 20X SNPlex System 15 to 25 Required Refer to Required Non Kit Materials on page 1 10 for a complete list of vendors Materials and part numbers Preparing the To prepar
175. th of the allelic ladder not within the recommended 1500 to 5000 RFU range See page 5 4 e Signal stengths of the allelic ladder are uneven See page 5 6 e Poor resolution See page 5 6 e Pull up or pull down peaks spectral calibration problems See page 5 6 A single peak or pair of peaks that is taller and narrower than adjacent peaks 10n front See page 5 7 Examples of each problem and possible solutions are provided in subsequent sections of this chapter Troubleshooting Example Signal Strength mE m Typical signal strengths for ZipChute probes in the allelic ladder are between 1500 and 5000 RFUs as shown in Figure 5 2 Signals that are slightly outside of this range may still be considered normal Also signal strength can vary between instrument types and between instruments of the same type Although signal strengths significantly outside 1500 to 5000 RFU range do not necessarily mean that samples will fail you should consider signal strength when adjusting run conditions for future runs However if signals for the allelic ladder are offscale you must rerun samples Dons Ou rr ET AA MI v NUT k Figure 5 2 Raw data view of an allelic ladder with good signal strength Solution If the signal strength is significantly outside the expected range 1 Confirm that the Allelic Ladder is appropriately diluted see Preparing Samples for Electrophoresis on page 3 24 5 4 SNP
176. tion and wash steps according to the protocol Further ensure that you perform the wash steps thoroughly removing all the liquid from the plate before continuing with the next steps SNPlex Genotyping System 48 plex User Guide 5 25 Chapter 5 Troubleshooting Troubleshooting Cluster plots when used with other troubleshooting tools such as PHCs and NHCs Cluster Plots help you narrow down the causes of a problem Unexpected patterns in cluster plots can originate from components of SNPlex System chemistry DNA quality pipetting and liquid handling robotics and capillary electrophoresis Example Figure 5 16 shows cluster plots from successful SNPlex System assays A B E E Allele 2 A2 Angle in Radians Angle in Radiane Allele 2 A2 z e o Angle in Radians Angle in Radians Allele 2 A2 Allele 2 A2 20 3 o 10 30 40 590 1000 0 0 1000 2000 3020 6000 4000 80008000 8000 0 100 4000 0 90000 14000 10000 logit Intensity log10 Intensity Alilala 4 4844 Alilala 4 4844 Figure 5 16 Cluster plots from successful SNPlex System assays A Polar view B Cartesian plot view The following sections present examples of typical patterns observed in cluster plots The table following each plot describes possible causes when evaluated with the PHC NHC and size standard 5 26 SNPlex Genotyping System 48 plex User Guide Troubleshooting Analyzed Data Example Data Points Have Low Signal I
177. to close the Panel Manager 5 Confirm that the SNPlex System panels and bins were imported properly In the Panel Manager a Select then double click SNPlex 48plex 3730 b Select then double click SNPlex 48plex Panel 3730 c Select an individual marker for example M5 1 005 zs Panel Manager ra Class Microsatellite Example Primer Focus Example De E C3SsNPlex 4B8plex 3730 E C39 SMPlex 48plex Panel 3730 M5 1 004 M5 1 n2 M5 1 03 M5 1 004 M5 1 ODE M5 1 OD7 M5 1 OD8 M5 1 OD8 M5 1 O10 M5 1 011 A 2 9 gt yH 29 lt 2 5 4 manm If the import was successful two bins one gray and one pink are displayed d If you do not see the bins check if the SNPlex System bin set is selected in the bins drop down list e If you do not see the bins reimport the bin set 4 4 SNPlex Genotyping System 48 plex User Guide Importing the AIF Importing the AIF About Assay If your plate records are set up in Data Collection software to include sample names Information Files and SNP sets typically imported from a text file as recommended in Creating for the SNPlex Results Groups and Plate Records on page 3 26 you must import the Assay System Information File AIF into GeneMapper software before you import the SNPlex System sample files fsa Doing so ensures that GeneMapper software reads the sample names and SNP sets from the plate record IMPORTANT
178. typing Dried gDNA Plate Control Pool System CD For all gDNAs a consensus genotype was established using data from repeat SNPlex System assays using the control pool SNPs These genotypes serve as a reference to calculate the accuracy of the system refer to the SNPlexTM Genotyping Dried gDNA Plate Control Pool System CD gDNAs are plated into 384 well microtiter plates appropriate for use with the Applied Biosystems 3730 3730x DNA Analyzer The gDNA plate can be used in conjunction with custom human ligation probe pools Comparing data achieved with the gDNA plate to data achieved with user supplied DNA allows you to assess the quality of your DNAs e The proportion of female to male DNA is approximately 1 1 Users who prefer the 96 well protocol can resuspend the gDNA in one 384 well plate quadrant and transfer the contents to a 96 well plate refer to Using the SNPlex System Dried gDNA Plates file on the CD A 4 SNPlex Genotyping System 48 plex User Guide Using the Control Pool Using the Control Pool To use the SNPlex System Control Pool together with the dried gDNA plate perform the SNPlex System assay as described in Chapter 3 substituting the SNPlex System Control Pool for the SNPlex Ligation Probe Pool See the SNPlex Genotyping Dried gDNA Plate Control Pool System CD for information regarding the dispensing of the allelic ladder Expected Hesults The following results can typically be expected whe
179. uL of undiluted Binding Buffer 0 009 uL of Positive Hybridization Control Note Prepare extra volume to account for losses that may occur during pipetting To bind the PCR product to the hybridization plate l Add 17 5 uL of Binding Buffer containing Positive Hybridization Control to the SNPlex Hybridization Plate Transfer 1 5 uL of each well containing the PCR reaction product into each well of the SNPlex Hybridization Plate and mix Note The notches on the plates do not always line up Make sure you orient the plates with well A 1 at the upper left corner when transfering samples between plates Al Al PCR gt Hybridization Electrophoresis Cover the SNPlex Hybridization Plate with one of the recommended plate covers see Table 1 3 on page 1 10 Incubate for 60 min at room temperature on a rotary shaker set to high Briefly spin the hybridization plates to collect the liquid in the bottom of the wells To isolate the biotinylated strand on the hybridization plates 1 Uncover the SNPlex Hybridization Plates 2 Remove the supernatant from each well 3 Wash each well three times with 100 uL Wash Buffer diluted 1 10 Note Dilute the Wash Buffer 1 10 with deionized water SNPlex Genotyping System 48 plex User Guide 3 19 Chapter 3 SNPlex System Protocols To isolate the biotinylated strand on the hybridization plates continued 4 Bri
180. ue PO Box 6482 Carlsbad California 92008 SNPLEX GENOTYPING SYSTEM LIGATION PROBES 4346978 NOTICE TO PURCHASER LIMITED LICENSE The purchase price of SNPlex Genotyping System Ligation Probes includes a limited non transferable non exclusive license without the right to resell repackage or sublicense under the method claims of U S Patents 4 988 617 and 5 830 711 or counterpart claims in patents and applications outside the U S to use only this amount of the product to practice the Oligonucleotide Ligation Assay OLA and Ligation Detection Reaction LDR methods described in said patents solely for the purchaser s own research and development activities when this product is used in conjunction with SNPIexTM System Oligonucleotide Ligation Kit No other rights are granted expressly by implication or by estoppel or under any other patent rights owned or licensable by Applied Biosystems Further information on purchasing licenses for the OLA and LDR methods and other applications may be obtained by contacting the Director of Licensing at Applied Biosystems 850 Lincoln Centre Drive Foster City CA 94404 U S A SNPLEX SYSTEM CONTROL POOL 4362635 NOTICE TO PURCHASER LIMITED LICENSE The purchase price of SNPlex System Control Pool includes a limited non transferable non exclusive license without the right to resell repackage or sublicense under the method claims of U S Patents 4 988 617 and 5 830 711 or counterpart claims in pa
181. used in conjunction with an authorized DNA sequencing machine or is available from Applied Biosystems No rights are granted expressly by implication or by estoppel or under any other patent rights owned or licensable by Applied Biosystems Further information on purchasing licenses to practice the DNA sequencing or fragment analysis methods may be obtained by contacting the Director of Licensing Applied Biosystems 850 Lincoln Centre Drive Foster City California 94404 U S A SNPLEX SYSTEM MATRIX STANDARD 4349365 SNPlex System Matrix Standard is covered under one or more of U S Patent Nos 5 654 419 5 707 804 5 688 648 6 028 190 5 869 255 6 177 247 6 544 744 5 728 528 U S patent application 10 288 104 and corresponding foreign patents and patent applications licensed from the University of California NOTICE TO PURCHASER DISCLAIMER OF LICENSE This product is optimized for use in the DNA sequencing or fragment analysis methods covered by patents owned or licensable by Applied Biosystems No license under these patents to use the DNA sequencing or fragment analysis methods is conveyed expressly or by implication to the purchaser by the purchase of this product A license to use the DNA sequencing or fragment analysis methods for certain research and development activities accompanies the purchase of certain Applied Biosystems reagents when used in conjunction with an authorized DNA sequencing machine or is available from Applied Biosy
182. which procedure you have selected assemble the OLA reaction as described in e Assembling the OLA Reaction Dried Down gDNA on page 3 11 Assembling the OLA Reaction Wet g DNA on page 3 12 SNPlex Genotyping System 48 plex User Guide Phosphorylating and Ligating Probes to gDNA OLA Assembling the You can set up the reactions at room temperature OLA Reaction Dried Down gDNA To prepare the OLA reaction when using dried down gDNA 1 Retrieve the reaction plates containing the fragmented dried gDNA see Preparing Genomic DNA on page 3 6 If the plates have not been labeled label them as shown in the following figures 96 capillary array 96 well plate 96 capillary arrays 384 well plate OO000000000C as 016 8 OOO OOOO 200000000000000000 OOO DO 6 OOOO 00000000000000000 BOO O OOOO 1900000000900000000 000O0O0O0OO0O0O0OQ 00000000000000090 C AO OOOO 6 6 61678 0000000000000000 NTC sQOOOOOOOOOOQ 100000000000000090 i 00000000000 100000000000000090 48 capillary array 96 well plate 48 capillary array 384 well plate OO0000000000 EE eee 2 0 018 1066 O16 6 CC gt 00000000000000000 OO OOOO IO 00000000000000000 000000000000 de asada 000000000000 C 00000000000000000 C 000000000000 NTC 86000990000009999 NTC 000000000000 i 00000000000000000 QOOOOOOOOOQOOQO 00000000000000090 C Control
183. y on page B 8 for more information e When you have fewer than 40 samples e Samples do not include the no template control NTC and allelic ladder e f x24 samples are included in a run the GeneMapper software will not analyze the data using the model based method Note that to use the Model algorithm probe pools must contain at least 18 SNPs SNPlex Genotyping System 48 plex User Guide Analysis Methods for SNPlex System Assays Terms Used in The following table defines commonly used terms in clustering analysis For more Clustering information about these concepts refer to the GeneMapper Software v3 7 online Analysis help Term Meaning Confidence Value Indicates how confident the software is that a call it has made for a CV particular point is correct Process Reported by the software to aid in finding and fixing problems in Component sample preparation and analysis based Quality Values PQV Quality Flag Based on the value of the PQV For example if SQ 1 the quality flag for SQ will be green pass Sizing Quality SQ If the sizing quality of a SNP falls within the low quality range the software does not process the sample any further Note that SQ values for SNPlex System experiments are higher than that for other applications Genotype Quality Quality flag whose value determines whether the software passes or GQ fails a genotype The calculated value is based on
184. y arrays or eight run folders for 48 capillary arrays The folder structure is generated by correctly setting up a Results Group as described in Creating Results Groups and Plate Records on page 3 26 To import sample files into GeneMapper software 1 Select File gt Add samples to project 2 Find the data that you want to analyze a In the tree pane right side of the workspace click a folder to select it b Click Add to list to add the files contained in the folder The files should appear in the list of files left side of the workspace 3 After adding all relevant files click Add to add the files to the project Analyzing SNPlex System Data GeneMapper software v3 7 provides two methods for analyzing SNPlex System data based on the clustering algorithms used to calculate the SNP quality For more information about analysis methods refer to Appendix B To analyze SNPlex System data 1 Before proceeding with analysis check to see that e Samples have the correct sample type designations Allelic ladder samples are labeled as allelic ladder No template control samples are labeled as negative controls All other samples are labeled sample Analysis Method is set to either SNPlex Model 3730 or SNPlex Rules 3730 for all samples Panelis set to SNPlex 48plex Panel 3730 for all samples e Size Standard is set to SNPlex 48plex v1 for all samples SNP Set is set to the appr

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