ABI Prism® 7900HT Sequence Detection System ... - OpenWetWare

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Managing Sequence Detection System Data 7900HT Instrument Dataflow Data management strategy is a crucial element of successfully integrating the ABI PRISM 7900HT Sequence Detection System into a laboratory workflow. During a single 24-hour period of real-time operation, the 7900HT instrument can produce up to 200 MB of data. To manage the information produced by the 7900HT instrument successfully, it helps to have a basic understanding of how data is collected and processed prior to analysis. The figure below contains a summary of the 7900HT instrument data flow. ABI PRISM 7900HT Sequence Detection System Thermal Cycling and Sequence Detection ♦ SDS files added to and run from the plate queue ♦ SDS file run individually *.sds files Plate document creation Serial Cable Instrument Firmware Data Collection Raw data saved to SDS files *.sds files Data analysis Downstream application specific analysis Computer Automation Controller/ SDS Software Hard drive SDS Software Product Overview 2-15
Working with SDS Software Files Files Used and Created by the SDS Software Average SDS Plate Document File Size 2-16 Product Overview IMPORTANT Never move or delete a SDS software file or folder unless specifically directed to do so by an Applied Biosystems representative or documentation. The SDS software includes many different files and folders. Some of these are created to store run data and calibration data, others are required to run the software. File Type Extension Description Plate Document Files ABI PRISMSDS *.sds A plate document is a virtual representation of a Single Plate consumable (plate) used to contain samples and reagents during an sequence detection run. During the run, the software uses the plate document to coordinate the operation of the instrument (thermal cycling, data collection), to organize and store the data gathered during the PCR, and to store the results of the downstream analysis of the run data. ABI PRISMSDS *.sdt Templates contain can be used to create an unlimited Template File number of plate documents. Templates are optional but useful as a time-saving devices for experiments where samples are run on plates with identical assay configurations. Imported/Exported Files Tab-delimited *.txt The SDS software can export raw or analyzed data in text file tab-delimited (*.txt) format for all or a select group of wells on a plate document. The exported files are compatible with most spreadsheet applications. JPEGa graphic *.jpg The SDS software can export most panes and plots of the files plate document as JPEG graphic files. The JPEG format is compatible with most word processing and spreadsheet applications and can be incorporated directly into HTML documents for viewing by most web browser software. a. Joint Photographic Experts Group The SDS software can produce SDS files of varying sizes depending on the type of runs for which they are created. The table below lists the average sizes of typical files produced by the SDS software. Run Type Average File Size Compressed File Size a Plate-read 150–180 KB 70–90 KB Real-time b 15–25 MB 10–15 MB a. Compressed files sizes shown are estimates based on standard compression using the WinZip® utility. For more information, see “Archiving SDS Files” on page 7-46. b. The maximum file sizes displayed above are nominal for real-time runs (absolute quantification). File size can increase depending on the plate document’s data collection options.
Page 1 and 2: ABI PRISM ® 7900HT Sequence Detect
Page 3 and 4: © Copyright 2001, Applied Biosyste
Page 5 and 6: iv Section:PlateDocumentSetup......
Page 7 and 8: vi A Theory of Operation Fluorescen
Page 9 and 10: Attention Words and Warning Labels
Page 11 and 12: About Waste Profiles A waste profil
Page 13 and 14: Safe Instrument Use Before Operatin
Page 16 and 17: Product Overview 2 In This Chapter
Page 18 and 19: Section: Getting to Know the Hardwa
Page 20 and 21: Interchangeable Thermal Cycler Bloc
Page 22 and 23: Computer Description The computer c
Page 24 and 25: Zymark Twister Microplate Handler D
Page 26 and 27: Instrument Connections Electrical C
Page 28 and 29: Section: Getting to Know the Softwa
Page 32 and 33: Getting Started 3 In This Chapter T
Page 34 and 35: Using the SDS Software Online Help
Page 36 and 37: Turning on the ABI PRISM 7900HT Seq
Page 38 and 39: Using the SDS Software Workspace La
Page 40 and 41: Using the General Toolbar Using the
Page 42 and 43: Basic Software Skills Tutorial Abou
Page 44 and 45: Exercise 3: Opening a Plate Documen
Page 46 and 47: Lesson 2: Viewing and Resizing Pane
Page 48 and 49: Exercise 2: Selecting Multiple Well
Page 50 and 51: Lesson 4: Using the Hand-Held Bar C
Page 52 and 53: Using SDS Plate Documents Using Mul
Page 54 and 55: Run Setup and Basic Operation 4 In
Page 56 and 57: Setup Checklists Experiments/Runs P
Page 58 and 59: Section: Plate Document Setup In Th
Page 60 and 61: Step 2 - Applying Detectors and Mar
Page 62 and 63: Creating an Allelic Discrimination
Page 64 and 65: Step 3 - Configuring the Plate Docu
Page 66 and 67: Step 4 - Programming the Plate Docu
Page 68 and 69: To create a method for the absolute
Page 70 and 71: Step 5 - Saving the Plate Document
Page 72 and 73: Step 7 - Applying Sample and Plate
Page 74 and 75: Section: Running an Individual Plat
Page 76 and 77: Preparing and Running a Single Plat
Page 78 and 79: Operating the 7900HT Instrument Usi
Page 80 and 81:
After the Run Analyzing the Run Dat
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Section: Running Multiple Plates Us
Page 84 and 85:
Adding a Plate Document to the Plat
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To create plate documents from a te
Page 88 and 89:
Adding Plates to the Plate Queue Re
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Loading Plates To load plates onto
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End-Point Analysis 5 In This Chapte
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Section: Allelic Discrimination In
Page 96 and 97:
Algorithmic Manipulation of Raw All
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Before You Begin Using SDS Online H
Page 100 and 101:
Analyzing a Completed Allelic Discr
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Calling Allele Types To call allele
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Scrutinizing the Allele Calls To an
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After the Analysis Changing the Pla
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Real-Time Runs on the 7900HT Instru
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Overview About Absolute Quantificat
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Before You Begin Using SDS Online H
Page 115 and 116:
Analyzing the Run Data Configuring
Page 117 and 118:
Setting the Baseline and Threshold
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Eliminating Outliers For any PCR, e
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Viewing the Standard Curve 6-14 Rea
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6-16 Real-Time Analysis
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Overview About Dissociation Curve A
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Analysis Checklist WhereYouArein th
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Determining T m Values for the Anal
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After the Analysis Changing the Pla
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Recommended Maintenance Schedule Ma
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Replacing the Sample Block When to
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7-6 System Maintenance To remove th
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7-8 System Maintenance To replace t
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7-10 System Maintenance To replace
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Cleaning the Sample Block Wells 7-1
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Prepare a Background Plate Document
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Extracting the Background 7-16 Syst
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Materials Required The following ma
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Extracting Pure Dye Information fro
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Constructing a Custom Pure Dye Plat
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Verifying Instrument Performance Us
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Preparing and Running an RNase P Pl
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Adjusting the Sensitivity of the Pl
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Testing the Adjustment 7-30 System
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Aligning the Plate Handler When to
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7-34 System Maintenance To align th
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Re-checking the Input Stack 1 7-36
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Defining the Positions of the Remai
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Aligning the Fixed-Position Bar Cod
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7-42 System Maintenance To position
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7-44 System Maintenance
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General Computer Maintenance Mainte
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Maintaining the SDS software Admini
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Troubleshooting Table 8-2 Troublesh
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Low Precision or Irreproducibility
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Writing on the Reaction Plates Fluo
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Background Runs Background Troubles
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Pure Dye Runs Pure Dye Troubleshoot
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8-12 Troubleshooting Troubleshootin
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Software and 7900HT Instrument Trou
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8-16 Troubleshooting Troubleshootin
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User Bulletins 9 9 About This Chapt
Page 201 and 202:
Fluorescent-Based Chemistries Funda
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Fluorescence Detection and Data Col
Page 205 and 206:
Normalization of Reporter Signals A
Page 207 and 208:
Determining Initial Template Concen
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Calculating Threshold Cycles A-10 T
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Importing and Exporting Plate Docum
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Configuring the Setup Table File wi
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About the Setup Table File Format S
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Setup Table Elements (continued) Nu
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Exporting Plate Document Data Expor
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Designing TaqMan Assays C In This A
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Design Probes and Primers The follo
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Design Tips for Allelic Discriminat
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Kits, Reagents and Consumables D In
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Consumables and Disposables The ABI
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TaqMan Pre-Developed Assays and Rea
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Contacting Technical Support F Serv
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G-2 Limited Warranty Statement No a
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A(continued) Automation Controller
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I(continued) installing plate adapt
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Q quantifying probes and primers C-
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Headquarters 850 Lincoln Centre Dri
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