Precision Melt Analysis™ Software Instruction Manual Catalog #184-5025
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Bio-Rad Resources Bio-Rad provides many resources for scientists, including rich technical resources on a wide variety of methods and applications related to PCR, real-time PCR, gene expression and HRM analysis. Table 1 lists Bio-Rad resources. Table 1. Bio-Rad resources Resource How to Contact Local Bio-Rad Laboratories representatives Find local information and contacts on the Bio-Rad website by selecting your country on the home page (www.bio-rad.com).
Precision Melt Analysis Software Instruction Manual Table of Contents Bio-Rad Resources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ii Writing Conventions Used in this Manual . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ii Chapter 1. HRM Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Introduction to HRM Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Table of Contents Chapter 5. Analyzing Melt Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 Processing Melt Data. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Precision Melt Tab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Precision Melt Data Tab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Melt Curve Tab . . . . . . . . . . . . . . . . .
Precision Melt Analysis Software Instruction Manual 1 HRM Analysis Read this chapter for information about high resolution melt (HRM) analysis.
HRM Analysis SNP Genotyping Representative of the smallest genetic change, the detection and genotyping of SNPs underlines the sensitivity of HRM analysis. Unknown mutations are often a single nucleotide change, but they may also comprise multiple base changes, insertions and/or deletions. In general, the more base changes in the DNA, the easier they are to detect by HRM. SNPs have been divided into four classes as summarized in Table 3, the most difficult to genotype are the class 4 (A>T conversions).
Precision Melt Analysis Software Instruction Manual 3. Use sufficient pre-amplification template Analyzing real-time PCR amplification data can be extremely useful when troubleshooting HRM analyses. Samples should have a quantification threshold (Cq) less than 30 cycles. Products that amplify late due to too little starting template amount or template degradation produce variable HRM results. 4. Normalize template concentration The amount of template added to the reaction should be consistent.
HRM Analysis • Wittwer CT, Reed GH, Gundry CN, Vandersteen JG, Pryor RJ (2003). High resolution genotyping by amplicon melting analysis using LCGreen. Clin Chem. 49(6):853-60. • Liew M, Pryor R, Palais R, Meadows C, Erali M, Lyon E, Wittwer C (2004). Genotyping of single-nucleotide polymorphisms by high-resolution melting of small amplicons. Clin Chem. 50(7):1156-64. • Vossen RH, Aten E, Roos A, den Dunnen JT (2009). High-resolution melting analysis (HRMA): more than just sequence variant screening.
Precision Melt Analysis Software Instruction Manual • Montgomery J, Wittwer CT, Kent JO, Zhou L (2007). Scanning the cystic fibrosis transmembrane conductance regulator gene using high-resolution DNA melting analysis. Clin Chem. 53(11):1891-8. • Taylor CF (2009). Mutation scanning using high-resolution melting. Biochem Soc Trans, 37:433-437. • Erali M and Wittwer CT (2010). High resolution melting analysis for gene scanning. Methods, 50:250-261.
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Precision Melt Analysis Software Instruction Manual 2 Precision Melt Analysis™ Software Read this chapter for information about installing Precision Melt Analysis software.
Precision Melt Analysis™ Software A melt calibration kit (catalog number 184-5020) will also be shipped with the following components: • Melt Calibration DNA standard • Melt Calibration Primers • Precision melt supermix • Melt Calibration instructions for Precision Melt Analysis software Software Installation Precision Melt Analysis software is run on a PC computer with either the Windows XP or Windows 7 operating system. Table 4 lists the computer system requirements for Precision Melt Analysis software.
Precision Melt Analysis Software Instruction Manual 3. Click Next on the software installation screen (Figure 1). Figure 1. Software installation screen. 4. To install the software, you must accept the terms in the license agreement. Click the radio button to accept the terms. Click Next (Figure 2). Figure 2. End User License Agreement. 5. Click Next to install the software to the specified, default destination folder (Figure 3). Figure 3. Installation folder designation.
Precision Melt Analysis™ Software 6. The wizard is now ready to begin installation. Click Install to install the software program in the specified destination folder (Figure 4). Figure 4. Software installation. 7. When the software installation is complete, click Finish to exit the installation wizard. 8. When completed, the Bio-Rad Precision Melt Analysis software icon appears on the desktop of the computer (Figure 5). Figure 5. Precision Melt Analysis software desktop icon.
Precision Melt Analysis Software Instruction Manual Preparing the Melt Calibration Plate Bio-Rad Laboratories provides the following materials in the Melt Calibration kit (184-5020) to create a melt calibration plate: • 10016289. Melt Calibration DNA Standard • 10016273. Melt Calibration primers • 1725110.
Precision Melt Analysis™ Software 5. Seal the reaction plate with Microseal ‘B’ adhesive film. Centrifuge the melt calibration plate to move all the reaction components to the bottom of the well. Performing the Melt Calibration Experiment The melt calibration experiment is run on a CFX96, CFX Connect, or CFX384 real-time PCR system using the prepared melt calibration plate. To run the melt calibration to generate a melt calibration data file 1. Turn on the real-time PCR system. 2.
Precision Melt Analysis Software Instruction Manual 13.Click the Start Run button to begin running the experiment. 14.At the prompt, save the melt calibration data file as “Melt Calibration_today’s date”. 15.When the melt calibration run is complete, the data file is automatically displayed by CFX Manager software. Check the data file to ensure all wells display a tight amplification and a single melt peak (Figure 6) Figure 6. Melt calibration data. Importing the Melt Calibration File 1.
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Precision Melt Analysis Software Instruction Manual 3 Introduction to Precision Melt Analysis™ Software Read this chapter for information about getting started with Precision Melt Analysis software. • Main software window (below) • Startup Wizard (page 18) • Analysis Options Manager (page 18) • Software files (page 20) • Software help tools (page 21) • Tips and tricks (page 21) Main Software Window Precision Melt Analysis software processes melt files (.
Introduction to Precision Melt Analysis™ Software • Startup Wizard window. Access common software commands (page 18) Figure 9. Main software window. Menu Bar The menu bar of the Precision Melt Analysis software provides access to the functions and commands listed in Table 7. Table 7. Menu bar items in the main software window Menu Item Command Function File New Melt File Create a new melt file (.melt) from a CFX Manager™ data file (.pcrd) New Melt Study File Create a new melt study file (.
Precision Melt Analysis Software Instruction Manual Table 7. Menu bar items in the main software window (continued) Menu Item Command Function Help Contents Open the software Help to find information about running the software and instruments Index View the index in the software Help Search Search the software Help About Open a window to see the software version Toolbar Buttons Click a button in the toolbar of the main software window (Table 8) for quick access to common software commands.
Introduction to Precision Melt Analysis™ Software Startup Wizard The Startup Wizard automatically appears when Precision Melt software is first opened (Figure 10). If it is not shown, click Startup Wizard on the main software toolbar. Figure 10. Startup Wizard window. Options in the Startup Wizard include the following: • Create a new melt file. Create a new melt file (.melt) by importing a data file (.pcrd) generated by CFX Manager software • Open a melt file.
Precision Melt Analysis Software Instruction Manual • Select Tools > Analysis Options Manager in the main software window menu bar Figure 11. Analysis Options Manager Window. Melt Analysis Settings Profiles Use the Analysis Options Manager to choose the settings for analyzing a melt file in the Data Analysis window. Customized analysis settings can be saved and applied to different melt files. The name of the current analysis settings profile is displayed in the Name pull-down menu.
Introduction to Precision Melt Analysis™ Software Analysis Options Use the Analysis Options Manager window to choose the settings for analyzing a melt file. • Auto detect the melt region. Select the checkbox to enable the software to automatically define the pre-melt and post-melt temperature ranges. To manually define the pre-melt and post-melt ranges, deselect the checkbox and enter temperature values by typing a number in the text box or using the up-down arrows. • Normalized view.
Precision Melt Analysis Software Instruction Manual Software Files Precision Melt Analysis software stores information about experiments in specific files (Table 9). Table 9. Precision Melt Analysis software file types. File Type Extension How to View and Edit File Data .pcrd View and analyze in Data Analysis window Melt .melt View and analyze in Data Analysis window Calibration .mcal Cannot be viewed Melt Study .
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Precision Melt Analysis Software Instruction Manual 4 Data Analysis Overview Read this chapter for information about data analysis in Precision Melt Analysis™ software.
Data Analysis Overview . Figure 12. Layout for the Precision Melt tab. Data Analysis Menu Bar The menu bar in the Data Analysis window provides the menu items listed in Table 10. Table 10.
Precision Melt Analysis Software Instruction Manual Data Analysis Toolbar The toolbar in the Data Analysis window provides quick access to important data analysis functions (Table 11). Table 11.
Data Analysis Overview • Run Information tab. Shows information about the experiment, including the protocol, optional notes, optional ID, and run log (Figure 14). Enter and edit the data ID for the run by typing in the ID box. View the Other section to see events, such as error messages, that might have occurred during the run Figure 14. Layout of the Run Information tab in the Data Analysis window. TIP: Right-click any chart, spreadsheet, or well selector for more options.
Precision Melt Analysis Software Instruction Manual • Unselected, loaded wells (light gray). The data from unselected wells do not appear in the charts and spreadsheets in the Data Analysis window • Empty wells (dark gray). These wells were not loaded in the Plate Editor window Figure 15. Different colored wells appear in a well selector. Well Selector Right-Click Menu Items Right-click any well selector view to select the items listed in Table 12. Table 12.
Data Analysis Overview Common Right-Click Menu Items for Charts Right-click menu items are available on all charts. Some of the available items are present for all charts, and these items can be used to change how the data are displayed or to easily export the data from a chart (Table 13). Table 13. Right-click menu items for charts Item Function Copy Copy the chart into the clipboard Save Image As... Save the chart image in the selected image file type.
Precision Melt Analysis Software Instruction Manual Table 14. Right-click menu items for spreadsheets (continued) Item Function Copy as Image Copy the spreadsheet view as an image file and paste it into a file that accepts an image file such as text, image, or spreadsheet files Print... Print the current view Print Selection... Print the current selection Export to Excel... Export the data to an Excel spreadsheet Export to Text...
Data Analysis Overview Well Groups Manager Set up well groups using the Well Groups Manager to independently analyze sets of wells within the same plate in the Data Analysis window. For example, set up well groups to analyze multiple experiments that were run in one plate. You can also analyze the same wells with different analysis settings. To open the Well Groups Manager window, open the Plate Editor and click Well Groups in the toolbar (Figure 17). Figure 17. Well Groups Manager window.
Precision Melt Analysis Software Instruction Manual Step Number Selection CFX systems can acquire fluorescence data at multiple protocol steps; the software maintains the data acquired at each step independently. Precision Melt Analysis software displays the Step Number selector below the Difference Curve chart on the Precision Melt tab whenever a protocol contains more than one melt curve data collection step.
Data Analysis Overview 3. Click Exclude Wells in Analysis (Figure 20) to exclude the selected wells. This checkbox is at the bottom of the Plate Editor controls on the right side of the window. Figure 20. Exclude Wells in Analysis checkbox. In Figure 21, multiple wells were excluded from data analysis in the Plate Editor. The excluded wells are marked with an asterisk (*) and appear grayed out in color. Figure 21. Excluded well (marked with *) in the Plate Editor.
Precision Melt Analysis Software Instruction Manual 5 Analyzing Melt Data Read this chapter for information about analyzing melt data in Precision Melt Analysis™ software. • Processing melt data (below) • Precision Melt tab (page 33) • Precision Melt Data tab (page 42) • Melt Curve tab (page 46) • Melt Curve Data tab (page 48) Processing Melt Data Precision Melt Analysis software plots the relative fluorescence unit (RFU) data collected during a melt curve as a function to temperature.
Analyzing Melt Data Precision Melt Tab Use the data in the Precision Melt tab to set data analysis conditions, including normalization and, if required, temperature shift. The Precision Melt tab shows data in four views (Figure 22): • Melt Curve chart. Shows the RFUs for each well plotted against temperature. Each trace represents data from a single fluorophore in one well • Difference Curve chart. Shows the difference RFU plotted on the y-axis against temperature on the x-axis • Well selector.
Precision Melt Analysis Software Instruction Manual • Normalized view. Select Normalized view at the bottom of the chart to view normalized melting curves (Figure 23). Changing the view of the data in the Melt Curve chart does not change the data clustering. Figure 23. Normalized view option selected in Melt Curve chart. • Adjusting pre-melt and post-melt regions. Two pairs of adjustable vertical sliders correspond to the pre-melt region (green) and the post-melt region (red) as shown in (Figure 24).
Analyzing Melt Data view is selected. The default value for the temperature shift bar height is specified in the Analysis Options Manager window. Figure 25. Temperature Shifted view option selected in Melt Curve chart. TIP: To magnify any area of the Melt Curve chart, click and drag the mouse across an area of the chart. To return the chart to a full view, right-click and select Set Scale to Default from the menu.
Precision Melt Analysis Software Instruction Manual Analysis Options Window Select Analysis Options from the toolbar of the Data Analysis window. In the Analysis Options window, refine clustering results by increasing or decreasing the stringency used to classify well data into different clusters (Figure 27). Figure 27. Analysis Options manager window. Choose the settings for analyzing melt file data in the Data Analysis window.
Analyzing Melt Data MELT CURVE SHAPE SENSITIVITY Clustering shape sensitivity determines the stringency used to classify melt curves into different clusters. To refine clustering results, increase or decrease the sensitivity of clustering based on the shape of the melt curve. Enter a numerical value or move the slider bar to the left or to the right. Entering a lower percentage value or sliding the bar to the left reduces stringency and results in less heterozygote clusters.
Precision Melt Analysis Software Instruction Manual Manual Assignment of Clusters Right-click on the fluorescence trace of a well in the Melt Curve or Difference Curve chart to override the cluster assignment or change the cluster name (Figure 28). Figure 28. Changing the cluster assignment for a sample. NOTE: Multiple wells can be called at once by holding down the right mouse button and dragging over the wells.
Analyzing Melt Data Changing Cluster Parameters Right-click on the fluorescence trace of a well in the Melt Curve or Difference Curve chart to change cluster parameters (Figure 30). Figure 30. Changing cluster parameters. Changes made to the cluster are applied to all samples in the cluster. Select from the options listed in the Selected Cluster menu: • Select to assign a name to the cluster.
Precision Melt Analysis Software Instruction Manual Confidence Level Precision Melt Analysis software determines a probability distribution for each cluster based on the standard deviation of the melt curves within the same cluster. Each sample is mapped onto each cluster's probability distribution, based on that sample's similarity to the mean melt curve across each sample in the cluster. The confidence value is an indication of the relative probability the sample has of being in a cluster.
Analyzing Melt Data Precision Melt Data Tab The Precision Melt Data tab shows spreadsheets that describe the melt data collected in each well. Select one of the six options to show the data in different formats. • Results. Displays a spreadsheet view of the data • Charts. Displays multiple data charts in a single sheet • Plate view. Displays a view of the data in each well as a plate map • Raw RFU. Displays the RFU value in each well for each temperature • Normalized RFU.
Precision Melt Analysis Software Instruction Manual Charts View Spreadsheet Select the Charts spreadsheet (Figure 33) to view multiple data charts for all wells. Figure 33. Precision Melt Data tab with Charts spreadsheet selected. Select a fluorescence curve in a chart to display the information listed in Table 17. Table 17. Content displayed for a highlighted fluorescence curve.
Analyzing Melt Data Plate View Spreadsheet Select Plate View spreadsheet to see a plate map of the data. Figure 34 shows the Plate view spreadsheet as plate map. Figure 34. Plate spreadsheet in Precision Melt Data tab. The Plate View spreadsheet includes the type of information shown in Table 18, including the selected fluorophore in the plate map. Table 18.
Precision Melt Analysis Software Instruction Manual The raw RFU spreadsheet includes the type of information shown in Table 19. Table 19. Raw RFU spreadsheet contents Information Description Well number (A2, A3, A4, A5, A6...) Well data, listed by position in the plate for all the loaded wells Temperature Temperature data listed in ascending order for all the loaded wells Normalized RFU Spreadsheet Select the Normalized RFU spreadsheet to view normalized fluorescence readings for each well.
Analyzing Melt Data Difference RFU Spreadsheet Select the Difference RFU spreadsheet to view the difference fluorescence readings for each well. Negative wells do not appear in this spreadsheet because they are excluded from difference processing (Figure 37). Figure 37. Precision Melt data tab showing the Difference RFU data. The Difference RFU spreadsheet includes the type of information shown in Table 21. Table 21.
Precision Melt Analysis Software Instruction Manual Figure 38. Layout of the Melt Curve tab in the Data Analysis window. Adjusting Melt Curve Data Adjust the melt curve data by any of these methods: • Click and drag the threshold bars in the Melt Peak chart to include or exclude peaks in data analysis • To change the color of the traces in Melt Curve and Melt Peak charts, right-click on a chart and select Trace Styles from the menu.
Analyzing Melt Data Melt Curve Data Tab The Melt Curve Data tab shows the data from the Melt Curve tab in multiple spreadsheets that include all the melt peaks for each trace. Select one of these four options to show the melt curve data in different spreadsheets: • Melt Peaks. List all the data, including all the melt peaks, for each trace • Plate. List a view of the data and contents of each well in the plate • RFU. List the RFU quantities at each temperature for each well • -d(RFU)/dT.
Precision Melt Analysis Software Instruction Manual Plate Spreadsheet Select the Plate spreadsheet (Figure 40) to view melt curve data in a plate format. Figure 40. Plate spreadsheet in Melt Curve Data tab. NOTE: To adjust the peak that the software calls, adjust the threshold line in the Melt Peak chart on the Melt Curve tab. The Plate spreadsheet includes the types of information shown in Table 24. Table 24.
Analyzing Melt Data Table 25 lists the type of information shown in the RFU spreadsheet. Table 25. RFU spreadsheet content Information Description Well number (A1, A2, A3, A4, Well position in the plate for the loaded wells A5...) Temperature Melting temperature of the amplified target. Plotted as one well per row, and multiple wells for multiple products in the same well -d(RFU)/dT Spreadsheet Select the -d(RFU)/dT spreadsheet to view the type of data shown in Figure 42. Figure 42.
Precision Melt Analysis Software Instruction Manual • Preview (right side of window). View a preview of the current report Figure 43. Example of a Report window for a melt file. TIP: The layout of the report can define the type of information that appears in any report if you save the report as a template. Select Template > Save or Save As to save the layout of the current report as a template. Creating a Data Analysis Report To create a report in the Data Analysis window, follow these steps: 1.
Analyzing Melt Data 7. (Optional) Create a report template with the information you want. To save the current report settings in a template, select Template > Save or Save As. Then load the report template the next time you want to make a new report. Report Options List A report can include any of the options in each category described in Table 26. Use checkbox selection in the Report Options list to determine the information that appears in the report. Table 26.
Precision Melt Analysis Software Instruction Manual 6 Melt Study Analysis Read this chapter for information about performing melt study analysis in Precision Melt Analysis™ software. • Melt Study window (below) • Study Setup tab (page 54) • Study Analysis tab (page 55) • Melt Study report window (page 55) Melt Study Window Precision Melt Analysis software can compare melt curve data from different runs of the same instrument using a single melt study.
Melt Study Analysis Figure 44. Melt Study window. Study Setup Tab The Study Setup tab (Figure 44) shows a list of all the experiments in the Melt Study. • Adding experiments. Click Add Melt Files to select a file from a browser window. To quickly add experiments to a Melt Study, drag the melt files (.melt extension) to the Melt Study window NOTE: When adding a melt file with multiple well groups, select the well group(s) to add to the study in the setup tab. • Removing experiments.
Precision Melt Analysis software Instruction Manual Study Analysis Tab The Study Analysis tab shows the data from all experiments that are added to the Melt Study. The Study Analysis tab includes the same features as the Precision Melt Analysis tab. For example, highlighting a sample in the Melt Study chart highlights the corresponding cell in the spreadsheet below the chart (Figure 45). Figure 45. Study Analysis tab in Melt Study window.
Melt Study Analysis Figure 46. Example of a Report window for a Melt Study file. TIP: The layout of the report can define the type of information that appears in any report if you save the report as a template. Select Template > Save or Save As to save the layout of the current report as a template. Creating a Melt Study Report To create a report, follow these steps: 1. Click Report in the Study Analysis toolbar to open the Report window. 2. Change the options you want to include in the report.
Precision Melt Analysis software Instruction Manual Report Options List A Melt Study report can include the options in each category described in Table 29. Use checkbox selection in the Report Options list to determine the information that appears in the report. Table 29.
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Precision Melt Analysis Software Instruction Manual Index Menu bar, 24 Plate content, 23 Toolbar, 25 Well contents, 23 Well selector, 26 Data file Report, 50 Data report Creating, 51, 56 Detected Instruments, A Adjusting Data analysis charts, Melt Curve Data, 47 27 B Bio-Rad Laboratories Resources, ii E C CFX Manager Files, 21 Chart Data analysis, 27 Clearing wells in Plate Editor, Creating Data report, 51, 56 D -d(RFU)/dT Spreadsheet, 50 Data Files, 21 Data analysis About, 23 Adjusting, 23 Charts, a
Index Gene Study, 55 RFU Melt Curve tab, 46 L Layout Quantitation tab, 28, 34 S M Main software window, 15 Melt Curve Adjusting data, 47 Data analysis, 46 Opening tab, 46 RFU data, 46 Spreadsheet, 47 Melt Curve Data, 49 Amplification Data (RFU), Plate, 49 Tab, 48 Melt Peaks Spreadsheet, 48 Menu bar Data analysis, 24 Main software, 15, 16 O Opening Melt Curve tab, 46 49 Safety Warnings, ii Software Glossary, 21 Main window, 15 Tips and tricks, 21 Spreadsheet Amplification Data for Melt Curve, -d(RFU
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Bio-Rad Laboratories, Inc. Life Science Group Bulletin 10014811 Rev B Web site www.bio-rad.
