LEGENDplex Data Analysis Software

LEGENDplex Data Analysis Software Version 7.0 User Guide Copyright 2013-2014 VigeneTech. All rights reserved.

Contents Introduction... 1 Lesson 1 - The Workspace... 2 Lesson 2 Quantitative Wizard... 3 Lesson 3 Qualitative Wizard... 12 Lesson 4 Selected Functions... 17 Load the Shared Files... 17 Set Standard Curve Manually... 20 Data Sorting... 20 Replicates... 20 Define Sample Dilution... 21 Edit Sample ID... 21 Flag and Unflag... 22 Edit Standard Curve... 22 Set Control Type... 24 Lesson 5 - Gating... 25 Gating Protocol... 26 Auto Gating... 26 Manual Gating Tools... 27 Lesson 6 View... 28 3D Result View... 28 Bar Chart View... 28 Standard Curve View... 29 Clustering view or Heat Map... 31 Lesson 7 Report... 32 Detail Report... 32 Summary Report... 32 Contacting VigeneTech... 33

Introduction LEGENDplex Data Analysis Software is a quantitative analysis package for FCS data generated by a flow cytometer. It is designed to provide a user-friendly interface, accurate analytical results, and automated data report. LEGENDplex offers: Easy-to-use ribbon style user interface Simple wizard for repetitive tasks required to analyze flow data Fully automated analysis including data reduction and concentration calculation Robust curve fitting and accurate detection limit determination Completely integrated visualization between original data, curve mapping and results Standardized reports with graphic presentation Support both quantitative and qualitative analysis Normalization options for control analyte or control sample Summary and detailed reports 1

Lesson 1 - The Workspace LEGENDplex is similar to Microsoft Office products, which places a greater emphasis on visual orientation to software features to facilitate ease of use. The hierarchy is that Ribbons contain Tabs, Tabs contain Groups, and Groups contain Tasks. Click to hide/display the tabs details. Figure 1. LEGENDplex Ribbon Ribbons Ribbons contain both tabs and groups, along with access buttons for different functions. Tabs Tabs are a set of groups, organized by similar function. For example, the Analysis tab contains the Wizard, Preference, Edit, Analysis, Protocol and Gating groups. These groups are all related because they are the commonly used groups of tasks during the analysis process. 2

Groups LEGENDplex Data Analysis Software User Guide Groups are a set of similar features or program tasks similar to menus, but with images, and tool tips. For instance, the View group contains 3D, Bar Chart, Standard Curve and Sample Detail. These are all visual presentations of results but in different forms. Lesson 2 Quantitative Wizard 1. Click to start a new quick wizard session. 2. Click to open the Add Files dialog box. 3

3. Select the files to be analyzed and drag them to the FCS Files list. You can also drag the file folder to the FCS Files list; LEGENDplex TM will load all the.fcs or.lmd files in this file folder. 4

4. Click Next and adjust the parameters on the Settings panel according to the assay. In the example shown above, we set Highest Conc. to 4000, Dilution Factor to 4, and Decimal Places to 0. The AABBCC mode will replicate samples in consecutive manner when the number of replicates is defined (e.g. sample 1, sample 1; sample 2, sample 2;..). The ABCABC mode will replicate samples in repeating groups with defined group size (e.g. sample 1, 2, 3, 4 ; sample 1, 2, 3, 4 ). It is recommended that the Auto Gate be set at OFF position. Although a single highest standard concentration is set here, you will have options in Wizard Step 8 to assign different concentrations to different analytes if they are different. 5

5. Click Next. Sort the loaded files to the order you need by clicking the column titles File Name, or Data modified in the file list, the program will sort the files in ascending/descending order. In your experiment, if you follow the LEGENDplex kit Plate Map or Rack Map and acquire data for standards and samples in the same sequential order as that in the Plate Map, you can do file sorting by clicking on Date modified to put files in correct order, select the consecutive files and then click on standard curve parameters according to the default Settings. to apply the Note: You should click to apply the curve options whenever a parameter of Standard Curve settings has been altered. If the FCS files for the standard curve are inconsecutive in the list, you can press and hold down the Ctrl key and select the files in the correct order of the standard curve, then right click Apply Curve Options. Alternatively, you may drag the small round buttons labeled as C0, C1, C2 on the top of the file list individually to appropriate files corresponding to the standard points (such as C0 for the 0 Standard, C1 to the lowest standard concentration, C2 for the second lowest concentration of the standard curve, etc.). To define sample 6

replications and dilution factors, you may highlight selected samples, then right click your mouse and go to Edit Samples functions. 6. Click Next, the Bead Gating window will pop up automatically. Adjust # of Bead Size, Report Signal, # of Beads for Size 1, and # of Beads for Size 2 and select the axis for the dot plots. Click the arrow to unfold the detail of Bead Size and define the Bead ID and Analyte, if so desired. When entering Bead ID information, always enter the Bead IDs in increasing alphabetical order (e.g. A4, A5, A6, the B2, B3, 7

B4 ). LEGENDplex Data Analysis Software User Guide Type here Bead ID and analyte identity, as provided in the instruction manual of the kit. You will need to select the X-axis and Y-axis labels (channels) of the bead dot plots. The channels vary depending of the flow cytometer used to collect the data. The default settings are for the BD FACSCalibur TM instrument. Then click on the top of the Bead Gating window, LEGENDplex will find bead areas automatically. For LEGENDplex beads-based assays, the auto gating works well if you follow the kit protocols and the flow cytometer used for sample reading is properly set up and compensated (if needed). If for some reason autogating failed to properly separate the bead populations, please use following options: a. Decrease Gate Setting (e.g. from 100 to 50), or b. Use manual gating: You may need to click on and then click on the bead population to remove the gate set by auto gating. Click and then draw a gate around the bead size population(s). The new gating will automatically apply. If you find that the Band Panel (lower panel) is not acceptable, you may also use 8

to erase the rectangular gate for a particular population and re-draw a new gate using. After manual gating if you click on gating., it will remove all manual c. Use a different sample to do gating. This is usually the most practical method. You can click to save current gate settings to a gating protocol. 7. Click to finish gating. Once completed, click. 9

Standard curves and data calculation options can be edited using the two buttons labeled as Curve and Options. Variations in standard concentrations for different analytes can be modified in the Curve and Options. Curve fitting method can also be changed in Options. If you forget to define files for standard curves in Step 5, the Curve and Option button may not work. You will need to click on the Previous button to go back to the previous page to define the files used for standard curves. 8. Click. A Process window will pop up to show you the analysis process. 10

The result will be displayed as shown below. 9. Click View on the Ribbon menu and select a view type to see different results. You may use this function to view selected sample concentrations in a 3D or 2D graph format. The image can be saved or copied by right clicking your mouse. 11

10. Click Report on the Ribbon menu and select a report type to generate a report. The report can be printed directly or exported to Excel, Word or PDF files. Lesson 3 Qualitative Wizard 1. Click to start a new quick wizard session. 2. Click to open the Add FCS Files dialog box. 12

3. Select the.fcs files or.lmd files (a special form of.fcs files) and drag them to the FCS Files list. You can also drag the file folder to the FCS Files list; LEGENDplex TM will load all the.fcs files in this file folder. Once the.fcs files are loaded, the workspace will be displayed as below: 13

4. Click Next; select Qualitative as Type in Settings. 14

5. Click on the bottom left of the window to do gating procedure. 6. Click Next to go to the next step. 7. Do the followings if necessary. Edit Sample ID Edit Options 15

8. Click. A Process window will pop up to tell you the analysis process. 16

9. The result will be displayed as below. The results include Normalized MFI, Median CV, Median, and Count. Click tabs to switch between the views. Lesson 4 Selected Functions More detailed functions of the software are described below. Load Shared Files LEGENDplex allows the users to load files on a network. However, a network drive may need to be mapped first. To map a network drive, follow the instructions below. 1. Open Computer by pressing Win+R or double click the Computer icon on your Desktop. 17

2. Select Map network drive marked in the above picture to open Map Network Drive dialog box. 3. In the Drive list, click a drive letter. You can choose any available letter. 18

4. In the Folder box, type the path of the folder or computer, or click Browse to find the folder or computer. 5. Click Finish. Your computer is now connected, or mapped, to the network drive. Next time, you can load the shared files from a network drive when loading FCS files. 19

Set Standard Curve Manually LEGENDplex Data Analysis Software User Guide If the FCS files for the standard curve are in the middle of the file list, you may drag the small round buttons labeled as C0, C1, C2.. on top of the file list individually to appropriate files corresponding to the standard points (such as C0 for the 0 Standard, C1 for the lowest standard concentration, and C2 for the second lowest concentration of the standard curve, etc.). You can also select C from the drop down list of Type column in the FCS Files list and manually type a concentration in the Concentration column. The designates a sample and to ( please change to C0 to C7) indicates calibrators (i.e. Standards). Move the mouse over the icons, a tooltip will display the theoretical standard concentration according to Quantitative Settings. Alternatively, the files corresponding to Standards can be highlighted and right clicked to change file types to C. The standard concentrations can be typed in the Concentration column. Data Sorting In the FCS Files list, the column title such as File Name, and Data modified can be used to sort the files in ascending/descending order. Common use of the sorting function is to arrange the order of files for standard curves to match with experimental layout so that data can be calculated correctly. Replicates Sample replicates can be defined in two ways: 1. Double click the Name column of the FCS files for the samples in the FCS Files list and type in the same name for sample replicates, or 20

2. Highlight samples, right click in the FCS Files list and select Edit Name to open the Define Names dialog box. Define the Replicate Mode and Replicate Number. Define Sample Dilution Select sample files, right click in the FCS Files list and select Edit Name to open the Define Names dialog box. Input the Dilution value. Or you can input the dilution value directly in the Dilution Fold column of the FCS Files list for each sample. Edit Sample ID Select a file or multiple files in the FCS Files list, then right click in the list and select Edit Names to open the Define Names dialog box. Prefix --- Enter a Prefix of sample well name series (The default is Sample). Start Number --- Enter a starting number of sample name series. Replicate Mode --- Select a Replicate Mode: AABBCC or ABCABC. The AABBCC mode will replicate samples in consecutive manner when number of replicates is defined. The ABCABC mode will replicated samples in repeating groups with defined size. 21

Replicate number --- Enter number of replicates for the selected samples. Dilution Factor --- Enter a Dilution Factor if the sample is diluted. The software will report sample concentrations adjusted for dilution factors in the final report. Flag and Unflag Flag data: Select one or a group of data, right click the data and select Flag from the popup menu, or Click on the Analysis tab. Unflag Data Select one or a group of flagged data, right click the data and select UnFlag from the popup menu, or Click on the Analysis tab to cancel the flag. Edit Standard Curve Standard curve can be further edited prior to analysis. Step1: Open Edit Standard Curve dialog box. Click on the left bottom at Wizard Step 7 (Quantitative Analysis) to open the Edit Standard Curve dialog box. 22

Step2: Step3: Verify or redefine the standard curve in the left panel (Standard Curve). Select the check boxes in front of the IDs. If the check box is blue, it means that it is the replicate of the well above. Enter a value for the highest concentration, dilution factor for the standard curve and the number of decimal digits for the calibrated sample concentrations. Check Background (C0) check box to indicate that the standard curve files include background wells. Uncheck it to indicate the standard curve files do not include background files. Check Direction: Increasing to define standards curve files that have a serial dilutions from low to high (upward curve shape); or uncheck Direction Increasing for standard concentrations from high to low (downward curve 23

LEGENDplex Data Analysis Software User Guide shape). Increasing is used most often with typical sandwich assays involving S shaped curves. Decreasing is often used for competition assay curves. If all analytes have same starting concentrations, select Apply to All Analytes check box (default). Click button to continue. If not all analytes have the same starting concentrations, uncheck the box for Apply to All Analytes, select the analytes which share the same concentrations, enter the concentration in the Highest Concentration box, and then click for these selected analytes. Repeat the same operations for other targets which have different highest concentrations. When all analytes are defined, click OK button. Set Control Type In the Qualitative wizard 5, click to open the Bead Gating window. Click the drop down list of Control Type to select a control type: Analyte, Negative Control or Positive Control. 24

Reanalyze LEGENDplex Data Analysis Software User Guide Lesson 5 - Gating Gating is the process of separating bead populations. Bead size populations can be further gated to generate subpopulations. LEGENDplex Data Analysis Software offers automatic tools and advanced manual gating tools to facilitate this process. First, open the Bead Gating window. Select any sample and click or the same icon on the Analysis ribbon tab at Wizard step 6. on the left bottom Gating Toolbar provides a series of tool to finish the gating process. Gate Setting is where to define the gating parameters. The Size Panel and Band Panel display the gating results. In Qualitative wizard, the only difference is the part in Gate Settings as shown below: 25

Gating Protocol Gating protocol is a.gating.xml file which contains gate setting and gating results. You can load a saved gating protocol file by clicking to browse. Also you can click gating protocol. to save current gating setting and apply gating results to a Auto Gating Adjust the value of # of Bead Size, #Bead of Size 1, Size 2 and change the x and y axis parameters of the Size Panel and the Band Panel to match the experiment. To define the size, choose SSC-H in the y axis and FSC-H in the x axis of the Size (upper) panel. To define the subpopulations of beads, choose FL3 or FL4 in the y axis and FL2 in the x axis of the Band (lower) panel. The selection may vary with data acquisition software on flow cytometers and user should select appropriate parameters for proper display of data. 26

Then click on the top of the Bead Gating window, LEGENDplex will find bead clusters automatically. Manual Gating Tools If there is severe background noise which may affect the gating process or the gating results don t match your expectation, LEGENDplex provides a set of tools to perform gating manually. Add a polygon cluster in Size Panel or a rectangle in Band Panel. Delete a cluster in Size Panel or Band Panel. 27

Lesson 6 View LEGENDplex Data Analysis Software User Guide 3D Result View To display results in 3D view, highlight selected Concentration or Median data and click on the View ribbon tab, the 3D View Window will be displayed as below. The 3D view can be rotated or zoomed with your mouse. The 3D image can be saved or copied to other applications by right clicking your mouse and select the Save or Copy function. Bar Chart View To display results in bar chart view, highlight selected Concentration and Median data and click on the View ribbon tab, the Bar Chart View Window will be displayed as below. The bar chart can be saved or copied by right clicking your mouse and select the appropriate functions. 28

When the mouse moves over each bar, a tooltip will display the specific value. If too many data points are highlighted, the bar chart may not have enough room to display properly. You can enlarge the display window to properly display the chart. Standard Curve View In the Concentration tab, click on View ribbon tab, the standard curve for each analyte will be displayed individually. The curve-fitting parameters including curve coefficients, assay minimum and maximum detectable concentrations (DC) are displayed in an area just above each standard curve. 29

Sample Detail View Highlight one data and click as shown below. on the View ribbon tab to display single sample data 30

Select the analyte (e.g. d) and sample(s) (e.g. F026) on the top of the window to display the corresponding sample detail view. Sample will be mapped in the standard curve. All sample concentrations can be mapped to a standard curve. Clustering view or Heat Map Highlight one or more data, click displayed: on View ribbon tab. The clustering view will be 31

Lesson 7 Report LEGENDplex offers 2 report types: Detailed Report and Summary Report. Select a report type from the drop down list of can be exported in Excel, PDF, or Word format. on the Main Toolbar. The reports Detail Report The Detailed Report includes sample concentration details, standard curve details, file information, standard curve montage and signature. Summary Report The Summary Report includes sample concentrations, sample MFI, file information, standard curve montage and signature. 32

Contacting VigeneTech For technical support and product information of LEGENDplex TM Data Analysis Software, please contact VigeneTech customer support and technical support. Web site E-mail www.vigenetech.com supports@vigenetech.com Telephone 1-978-371-5959 Fax 1-978-371-9559 33