User Bulletin. ABI PRISM 7000 Sequence Detection System DRAFT. SUBJECT: Software Upgrade from Version 1.0 to 1.1. Version 1.

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1 User Bulletin ABI PRISM 7000 Sequence Detection System 9/15/03 SUBJECT: Software Upgrade from Version 1.0 to 1.1 The ABI PRISM 7000 Sequence Detection System version 1.1 software is a free upgrade. Also available for separate purchase is an RQ Plate and RQ Study add-on. The minimum system to support version 1.1 is a Pentium III, 1.0 GHz CPU, 256 MB RAM, and a 20 GB hard drive. The supported operating system is the Windows 2000 Professional operating system with Service Pack 3. Version 1.1 Feature Summary This user bulletin describes the new and modified features of the version 1.1 software. The new features include: Auto-C T Automatically finds the appropriate baseline and threshold settings for an Absolute Quantification or Relative Quantification assay. Baseline is performed on a per well basis. Threshold is performed on a per detector basis. You can also perform automatic baseline detection without performing automatic threshold selection. Smoothing Algorithm Reduces noise occasionally seen in the amplification plot data, thus ensuring accurate C T determination. For the version 1.1 software, you can purchase separately an add-on for Relative Quantification. The RQ Plate and RQ Study add-on allows gene expression studies using the comparative C T relative quantification method. You can analyze up to ten RQ plates in a study. You define the calibrator sample, endogenous control, and RQ confidence level. The version 1.1 software also includes changes to: Allelic Discrimination You can now omit a well using Analysis Settings. Plus/Minus The IPC confidence value is correctly reported in the printed results. Amplification Plot You can manually modify log plot maximums to less than ten. Multicomponent View You can adjust the x-axis scale. Report Settings Additional items are available. Grid You can deselect a well in the grid with Control-click. Type of plate document Displayed on documents saved in version 1.1 Printing Includes data previously missing in the report. September 24, :04 am, AB7000v11UserBulletin.fm

2 Auto-C T Auto-C T for Absolute Quantification (and Relative Quantification) allows autobaseline and auto-threshold calculations. You can use auto-baseline and autothreshold, auto-baseline and manual-threshold, or manual-baseline and manualthreshold. Select one of these options in the Analysis Settings dialog box or the Amplification Viewer control panel. After you configure the analysis options, you can analyze the run data. During the analysis, the software mathematically transforms the raw data to establish a comparative relationship between the spectral changes in the passive reference dye and those of the reporter dye. Based on that comparison, the software calculates a cycle threshold (C T ) for each reaction (standard and unknown). The software then generates a standard curve for the run by plotting the standard samples on a graph of C T versus initial copy number. IMPORTANT! If you choose to use the Auto-C T algorithm, verify after analysis that the baseline and threshold were called correctly for each autocalled detector. New SDS documents have the auto-c T auto-baseline and auto-threshold properties. Note: SDS documents created in previous versions do not have auto-baseline or auto-threshold properties. When you open and try to save such documents, the format is converted to include the auto-baseline and auto-threshold properties, which are assigned the values of the current settings. The Auto-C T algorithm: Determines baseline settings. Starts with the Start Cycle and Stop Cycle parameters. Determines baseline on a per-detector, per-well basis. Determines threshold values. Uses units of R n (normalized fluorescence). Determines threshold on a per-detector, per-plate basis. Supports double-stranded DNA binding dye (SYBR Green I). Supports the 5'-nuclease assay for single- and dual-reporter assays. Allows change to the threshold by a horizontal bar in the Amplification Plot or a threshold value text field. Reports C T export start/end baseline cycles with Auto-baseline on. 2 Software Upgrade from Version 1.0 to 1.1

3 Auto-CT Auto-Baseline and Auto-Threshold The SDS software has a proprietary Auto-C T algorithm that can be used to automatically generate baseline and threshold values for individual detectors. The algorithm calculates baseline and threshold parameters for a detector based on the assumption that the data exhibit the typical amplification curve (Figure 1-1). Plateau phase Linear phase Geometric phase R n Background Cycle Baseline Figure 1-1 Typical Amplification Curve Experimental error (such as contamination, pipetting inaccuracy, and so on) can produce amplification curves that deviate significantly from the typical data shown in Figure 1-1. The data from these irregularities can affect the Auto-C T algorithm by causing it to generate incorrect baseline and threshold parameters for the associated detector. For this reason, Applied Biosystems recommends that after analysis of the study data, you review all baseline and threshold parameters determined by the Auto- C T algorithm. Analysis Settings To use the Auto-C T functions, open the Analysis Settings dialog box (Figure 1-2) or Amplification Plot control viewer, then select the Auto-C T options. Figure 1-2 Analysis Settings Dialog Box Software Upgrade from Version 1.0 to 1.1 3

4 Examples Prepare a plate with one or more detectors. Each detector can have a wide range of starting copy numbers and consequently a wide range of C T values. In the example in Figure 1-3, a large dynamic range is being analyzed. Figure 1-3 Amplification Curves with Several Detectors A single baseline region does not work for all samples from the selected detector. Auto-baseline improves the ability to select a single threshold, as shown in Figure 1-4. Figure 1-4 Amplification Curves with Auto-Baseline Next, select a single threshold. The auto-threshold can optionally be applied to provide the final solution or begin the selection, and allows you to fine-tune the threshold. Smoothing Algorithm Data processing using the smoothing algorithm reduces noise occasionally seen in the amplification plot data, thus ensuring accurate Ct determination. 4 Software Upgrade from Version 1.0 to 1.1

5 RQ Plate and RQ Study Add-On RQ Plate and RQ Study Add-On Relative Quantification Plate Relative Quantification Study The Relative Quantification (RQ) assay calculates relative quantification values from C T values generated during sequence-detection PCR. The data-collection part of the assay is a single-plate document called the Relative Quantification Plate. The dataanalysis part of the assay is a multiple-plate document called the Relative Quantification Study. Note: The data from an RQ Plate can be analyzed only within an RQ Study. The RQ Plate assay is a data-collection assay used to thermal-cycle the plate and collect real-time PCR plate data. Amplification data from PCR runs is stored with sample setup information on the plate. Amplification of an endogenous control must be performed to standardize the amount of sample RNA or DNA added to a reaction. For the quantification of gene expression, researchers have used ß-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal RNA (rrna), or other RNAs as the endogenous control. An RQ Study document allows multiple analyses of RQ Plates. You can analyze up to ten RQ plates in a study. You define the calibrator sample, endogenous control, and RQ confidence level. RQ Study documents neither control the instrument nor do they provide tools for setting up plates. The RQ application within the SDS software supports only the comparative method. You can choose to amplify and detect the target amplicon and the endogenous control amplicon in different tubes using singleplex PCR, with one primer pair in each tube. In addition to the singleplex reaction, the availability of distinguishable reporter dyes makes it possible to amplify and detect the target amplicon and the endogenous control (reference) amplicon in the same tube using multiplex PCR, which uses more than one primer pair in the same tube. You can use multiplex PCR in relative quantification where one primer pair amplifies the target and another primer pair amplifies the reference in the same tube. Software Upgrade from Version 1.0 to 1.1 5

6 RQ Study Features The RQ Study software has: Auto-C T system Can automatically baseline R n vs. Cycle data on a per-well basis and threshold R n vs. Cycle data on a per-detector basis (across the study). Gene Expression Bar Graph Allows you to set the bar widths. Bars correspond to samples or detectors. Bar graphs are two-dimensional (2D) by default but can be viewed as 3D. Detector Grid Lists detectors found in the study. Shows all C T -related analysis settings per detector. Sample Summary Table Lists samples associated with detectors highlighted in the Detector Grid. Shows RQ-related results for these samples. Allows omitting sample replicates. Well Information Table Lists wells associated with detectors highlighted in the Detector Grid. Shows well-related setup and C T setting and results, and shows which wells are undetermined and filtered out due to baseline noise. Allows omitting individual wells. Plate Grid Lists plates added to the study, with their last modification dates. Control Type (multiplex vs. non-multiplex) Based on the experimental setup on the RQ plate. If multiplex and non-multiplex wells share the same endogenous control, you can select which control type to analyze. Export Allows you to export Sample Summary, Well Information, or Both results, as well as sample setup, calibration, spectra, and so on. Omitted wells are flagged as Omitted. Minimum/Maximum confidence Allows you to change the confidence level. Note: When creating an RQ Study with both multiplex and non-multiplex endogenous controls (Mixed RQ Study), plates with the multiplex controls need to be added before plates with non-multiplex controls. (Otherwise, multiplex plates added after non-multiplex plates may or may not be included in results when multiplex analysis is done.) The software does not verify that multiplex plates are added prior to non-multiplex plates. 6 Software Upgrade from Version 1.0 to 1.1

7 RQ Plate and RQ Study Add-On Software Upgrade from Version 1.0 to 1.1 7

8 Copyright 2003, 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. TRADEMARKS: ABI PRISM and Applied Biosystems are registered trademarks of Applera Corporation or its subsidiaries in the U.S. and/or certain other countries. AB (Design) and Applera are trademarks of Applera Corporation or its subsidiaries in the U.S. and/or certain other countries. AmpErase, AmpliTaq, AmpliTaq Gold, GeneAmp, and TaqMan are registered trademarks of Roche Molecular Systems, Inc. SYBR Green is a registered trademark of Molecular Probes, Inc. Microsoft and Windows are registered trademarks of Microsoft Corporation. All other trademarks are the sole property of their respective owners. Headquarters 850 Lincoln Centre Drive Foster City, CA USA Phone: Toll Free (In North America): Fax: Worldwide Sales and Support Applied Biosystems vast distribution and service network, composed of highly trained support and applications personnel, reaches 150 countries on six continents. For sales office locations and technical support, please call our local office or refer to our Web site at Applera Corporation 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. Printed in USA, 09/2003 Part Number Rev. A, Stock Number 117UB09-01