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1. Bio Plex Data Pro Sample Project File Edit Data View Help f amp cum Cb 7 E Condition Gender Data Set y Sample Type Description Well Locations Sample Label y Obs Conc 1 Normal Male CDG kiti Unknown Normall D3 D4 xi 74 51 p Normal Male CDG kiti Unknown Normal2 E3 E4 x2 69 67 Normal Male CDG kiti Unknown Normal3 F3 F4 x3 41 67 Normal Male CDG kiti Unknown Normal4 G3 G4 xa 113 02 Normal Male CDG kiti Unknown Normal5 H3 H4 x5 61 45 Normal Male CDG kiti Unknown Normal6 A5 A6 x6 97 93 Normal Male CDG kiti Unknown Normal7 B5 B6 X7 3945 Normal Male CDG kiti Unknown Normal C5 C6 X8 85 68 Normal Male CDG kiti Unknown Normal DS D6 xs 89 07 Normal Male CDG kiti Unknown Normal10 E5 E6 86 66 Normal Male CDG kiti Unknown Normalii F5 F6 97 93 Normal Female CDG kiti Unknown Normal12 G5 G6 94 72 Normal Female CDG kiti Unknown Normal13 H5 H6 90 97 Normal Female CDG kiti Unknown Normal14 A7 A8 77 13 Perform Analysis Normal Female CDG kit1 Unknown Normal15 B7 B8 67 18 Cete Set Normal Female CDG kiti Unknown Normali6 C7 C8 96 34 Normal Female CDG kiti Unknown Normal17 D7 D8 70 22 Normal Female CDG kiti Unknown Normal18 E7 E8 66 90 Normal Female CDG kiti Unknown Normali9 F7 F8 98 16 Normal Female CDG kiti Unknown Normal20 G7 G8 85 68 Normal Female CDG kiti Unknown Normal21 H7 H8 97 48 Normal Female CDG kiti Unknown Normal22 A9 A10 87 87 Normal Female CDG kiti Unknown Normal23 B9 B10 57 57 Normal Female CDG k
2. Bio Plex Data Pro Software User Guide Merging Data Viewing Merged Data in the Results Table In the Results table the Merge column displays the number of rows that have been merged for each group The lock in the first column indicates that the data in these rows cannot be edited ft oe amp b BY E amp ERE BR ve O reme JOE GEBEN EE Cemdiian ma Matric e Gender 1 DataSet xj 1 Detention 13 Horm l Plaats Menea Normal vs Dibe se t Normal Lung Cancer Plasma Merged Normal ws Disease plasma 1 lang cancer Cees Cecer Plasma Merged Nee mal vs Diipease plapma l Un 44 EMI Breast Carei Placa Female Nerma l wi reiia plabas 1 Ahern bread caer File Edit Data View Merge Column gt 14 XM4 13 T3 XMS 8 38 XM2 8 48 XM1 8 Locked Rows Manage Data Sets TT In a merged table Observed Concentration Obs Conc and Concentration of the coefficient of variation Conc CV columns report average values for merged rows 42 Merging Data The multiple analyte view shows all the analytes and observed concentration merged values for each analyte Viewing Merged Data in the Sample Table Merged data in the Sample table are displayed as individual rows The rows are sorted so that all merged rows are grouped next to each other You can edit the merged data in the Sample table for instance if you mislabeled a sample The data will be remerged dynamically based on your edi
3. You control the data that are imported in one of the following ways e Add data files using the Import Bio Plex Results File command e From Bio Plex Manager import your results directly into Bio Plex Data Pro All files that appear in the Data Set Files list are imported into the selected project You can combine data from multiple experiments into a project You can import up to ten data sets RBX files into a single Bio Plex Data Pro project Each RBX file represents one data set You can import data set files into multiple projects by selecting another project The procedure for importing data in Bio Plex Data Pro differs slightly from importing through Bio Plex Manager See Importing Data into Bio Plex Data Pro Software on page 16 and see Importing Data Directly from Bio Plex Manager Software on page 17 for more information 15 Bio Plex Data Pro Software User Guide Importing Data Sets 16 Importing Data into Bio Plex Data Pro Software The following procedure describes how to import data into Bio Plex Data Pro To import data sets into a project 1 From Bio Plex Data Pro click Import Bio Plex Results File in the toolbar The Import Bio Plex Results File dialog box appears 2 Select the desired project from the Import Into drop down list or create a project by clicking New Project 3 Click Browse to navigate to and select the data sets you want to import 4 Click Open The Data File field displays the file name
4. A Error Messages As you view your results in the Perform Analysis Create Charts view using different measurements you may see errors The following table describes the conditions in Bio Plex Manager that may cause the error Bio Plex Data Pro Measurement Error Message Error Condition Fl Acquisition Error No data present or not run Not Run No Bio Plex Manager data for that analyte Obs Conc Acquisition Error No data present or not run Cannot Calculate No data present or not run for other reasons Not Run No Bio Plex Manager data for that analyte CN oom ooe Conc in Acquisition Error No data present or not run Range Cannot Calculate No data present or not run for other reasons Not Run No Bio Plex Manager data for that analyte OOR gt OOR gt 61 Bio Plex Data Pro Software User Guide Bio Plex Data Pro Error Message POR o Norm Ratio Acquisition Error No data present or not run Not Run No Bio Plex Manager data for that analyte Ratio Acquisition Error No data present or not run Not Run No Bio Plex Manager data for that analyte a Not run means the sample is not run for any analytes and any replicates All measurements are error not set Measurement Error Condition 62 Bio Plex Data Pro Software User Guide Glossary Analyte The substance being measured identified quantitated or otherwise analyzed in an experiment In the Bio Plex system analytes are molecules that ar
5. Manager Software The Export Results to Bio Plex Data Pro feature in Bio Plex Manager allows you to import data directly into Bio Plex Data Pro You initiate the data export from Bio Plex Manager Then the Import Bio Plex Results File dialog box in Bio Plex Data Pro displays automatically If the Bio Plex Data Pro program is not already running it launches automatically Tono e ele Import Into Diabetes v New Project Data Set Files 0000000000000 P Status Data File Browse 4 Ready Diabetes 12 Plex Plate 1 1 35 rbx from Bio Plex Manager Import 17 Bio Plex Data Pro Software User Guide Editing a Data Set The Data File field displays the file name and indicates the source as Bio Plex Manager You may import this data set just as you would any RBX file You can add more data sets to this list by using the Browse button You may also return to Bio Plex Manager to export another data set For more information on this feature see the Bio Plex Manager Software 6 1 User Manual Editing a Data Set You can edit the name and description of a data set To edit a data set 1 Open the project whose properties you want to edit 2 Inthe Data Sets table right click the data set you want to edit and then click Edit Data Set in the context menu The Edit Dataset dialog box appears Edit Dataset Name Diabetes 12 Plex Plate 1 1 35 Description Human Diabetes 12 plex lot 501
6. and you may not be working with all of them at the same time You can organize your projects by removing projects that you are not currently working on from the list of active projects and moving them to the Inactive Projects list You can perform any of the commands on an inactive project that you perform on an active project you Can open and edit the project view the data and perform statistical analysis on the data and so on You can return inactive projects to the Active Projects list at any time To move a project to the Inactive Projects list e From the Active Projects tab in the Open Projects dialog box select the project you want to move to the Inactive Projects list and click Deactivate To return a project to the Active Projects list e From the Inactive Projects tab in the Open Projects dialog box select the project you want to move to the Active Projects list and click Activate Bio Plex Data Pro Software User Guide 3 Working With Data Sets Bio Plex Manager software results files are imported into Bio Plex Data Pro software for analysis You can view the data set in the Manage Data Sets view You can also edit the properties of the data set and delete data sets if you no longer need them for your analysis Importing Data Sets Data are imported into projects from the Import Bio Plex Results File dialog box You can select an existing project or you can create a new project into which the files are imported
7. 4 7 HuPDGF BB 0 00023 one way ANOVA 2 70 3380 85 3006 64 88 93 37 37 0 4 8 Hu PECAM1 0 714 one way ANOVA 0303 699 84 264 99 37 86 37 37 0 9 Hu Prolactin 0 244 one way ANOVA 143 967 24 131137 135 58 37 37 0 4 10 Hu SCF 0 00992 one way ANOVA 0640 42213 138 26 3275 37 37 0 11 Hu sEGFRi 0 145 one way ANOVA 0 541 3090 53 1275 83 41 28 37 37 0 12 Hu sHER2 neu 0 070 one way ANOVA 0 517 5007 05 1469 38 29 35 37 37 0 4 13 Hu sil 6Ra 0 00068 one way ANOVA 0 740 752782 2504 95 3328 37 37 0 4 Interpreting P Value Statistics 52 Bio Plex Data Pro software automatically calculates P values You can quickly sort the analytes by their P values and examine the analytes with the lowest P values in order to visually see the differences between the samples groups Bio Plex Data Pro uses P values to discriminate between sample groups Interpreting P Value Statistics The smaller the P value the greater the probability that two or more groups of samples have significantly different analyte measurements Typically a P value of less than 0 05 5 is considered significant Note that when a project contains many analytes some of them may have a low P value by chance even if the samples from different groups do not truly differ Therefore a low P value is not itself an indicator of a good separation between sample groups Analytes with low P values may have a large overlap of concentrations between the sample groups However a large P value
8. Project in the toolbar 2 Type a name for the project in the Name box Bio Plex Data Pro Software User Guide Creating a Project 10 Type the name of the project owner in the Owner box Optional Type the name of the team Optional Type the name of the supervisor Optional Type a description for the project up to 255 characters Click OK The Import Bio Plex Results File dialog box appears Import Bio Plex Results Fi Sox Import Into Sample Project 1 E New Project DU oS qe o A Data Set Files E Status Data File Browse Note The name of the project appears in the Import Into list 8 Click Browse and navigate to the data sets you want to import 9 Select the data sets you want to import and click Open 10 In the Import Bio Plex Results File dialog box click Import The data sets are copied into the database When the Status displays Done the data sets have been successfully copied 11 Click Close The project opens in the Manage Data Sets view and displays the Data Sets table Data Sets Table The Data Sets table displays the details for the data sets imported from RBX files at the plate level It is most useful immediately after data import when you can use it to verify that the imported data are what you want to analyze Opening a Project Fie Edna Osta View Help amp dw o BV E Qs Name e Description Imported Date 1975 ATA E 8 17 2011 5 3 Edit Samples Add Colume
9. Sample ID you can edit this field Data Set one source data set if the sample belongs to multiple data sets the sample is listed multiple times Sample Type Unknown Control Standard or Blank Description displays information that was entered in the sample description in the originating Bio Plex Manager results file You can edit this field Well Locations the well on the plate where the experiment was run for example A1 B1 or C1 C2 C3 Sample Label name of the sample for example S1 Sn X1 Xn and C1 Cn Samples with the same data set and sample label are merged Obs Conc observed concentration custom column Conc CV concentration of the coefficient of variation custom column View Results for All Analytes You can view the results for all analytes by clicking Multi Analyte Table in the toolbar Columns are displayed with headings and subheadings You can sort data in the columns but you cannot change the order of the columns See Sorting Data on page 29 You can customize the table by showing and hiding columns See Hiding a Column on page 28 Filters applied elsewhere in the application are preserved here but you cannot apply a filter in the Multi Analyte table This table is useful for copying data to spreadsheet software for further analysis 39 Bio Plex Data Pro Software User Guide Merging Data Pie Edi Data Worw Hep A EI NE Semple Properbes Perfaeem
10. a 14 Chapter 3 Working With Data Sets 15 Iniporung Data SEIS id E es pradidt deor duode Beh qe od edel dci de eee ied red 15 Importing Data into Bio Plex Data Pro Software 16 Importing Data Directly from Bio Plex Manager Software 17 Editing a Data Seb c mace Xo ue dd oss abita we lo pee odes 18 Deleting a Dalia Sets tay ss cout dwt te ESL E tt ood faros ipu Es 18 Chapter 4 Organizing the Data 21 Selecting Samliples 22 22 4 9 21 0 0 9 ded Doe M ee dO oer di Hoe e M Re dd i d 21 Viewing and Editing Samples llle 22 ime Sample Tables sew a SE CERTO EM E oa SC CB Po aeu e 22 About the Sample and Results Tables o oooooooomoo 24 Fieno Dalan CC LLL 24 Filtering Data in the Sample and Results Tables 24 Filtering Data in the Perform Analysis Create Charts View 26 Bio Plex Data Pro Software User Guide Table of Contents REMOVING FINES r porosa rada chewed oe etn Ea cea do 21 Adding a COM Nis ca a ae dd 27 Hicling a olco din UE RC hee a pend UR eee eee ERR E d CR 28 DOING MO Ata cr a de bette Coetu ede uci dedere Sunk eet oai gota E 29 Defining a Sort in the Sample or Results Tables 30 Clearing Data Sorting llle 32 Basic Sorting in Bio Plex Data Pro Software 34 Copying Data in Tables senera vie oe v a RC ea d e mo RA o d 34 Copying d Data BOW erica dara b
11. are excluded from the analysis To select the samples to be used in your analysis 1 Click Select Sample Type for analysis in the toolbar 2 Inthe Filter Samples dialog box clear the checkboxes for the samples you want to exclude from your analysis 3 Click OK 21 Bio Plex Data Pro Software User Guide Viewing and Editing Samples Viewing and Editing Samples The Edit Samples Add Columns view displays a tabular list Sample table of all unique samples in a project in the raw form imported from the RBX files In this view you add columns to the Sample table which allows you to define your biological replicate groups and assign other important information such as demographic data strain or gender associated with each sample Editing the data adds descriptive information that enables you to make meaningful comparisons when you analyze the data This descriptive information is a prerequisite for data visualization and statistical calculations Annotation assists you in identifying data groupings You can edit the data on a plate by plate basis using the data set selector You can copy paste or edit data into table columns from a spreadsheet You can also change the order of the columns Columns and order selections persist until you change them Once you add columns you can add data from a spreadsheet to each cell one row or one column at a time or all at once The Sample Table 22 When you import data into Bio
12. graphs to a Portable Network Graphics png JPEG bitmap or TIFF format In addition to Edit menu commands you can use the following commands to copy paste and delete data in tables CTRL A Select All selects the entire table in tables that allow multiple selections Bio Plex Data Pro Software User Guide The Workspace Copy Tableto Right click the table and select Copy Table to Clipboard Clipboard on the context menu to copy the entire table to the clipboard including the column titles CTRL C Copies the contents of selected cells to the clipboard Delete Deletes the content of selected cells if they are not read only CTRL X Copies contents of selected cells to the clipboard and deletes content of selected cells that are not read only CTRL V Pastes clipboard contents into the table Read only cells are skipped When clipboard content is a single value its content is copied to each selected cell in the table Data Menu Commands The following commands can be accessed from the Data menu Select Sample Type for analysis opens a dialog box in which you select sample types to use in your analysis These sample types are the standards controls blanks and unknowns imported from Bio Plex Manager Sample Types Y Name O Blank Unknown 4 Control 6 Standard mum Cancel Clear All Analysis Filters clears all filters in the project Reset Sample Lists to Default Sort c
13. into the table Read only cells are skipped When clipboard content is a single value its content is copied to each selected cell in the table The Workspace e Customize Sample Table opens a dialog box in which you can select or clear columns or add new columns to the sample table Name Datatype Unit Condition Text Matrix Text Gender Text Sample ID Text Clear Am Data Set Text AAA New Column Sample Type Text Description Text Well Locations Text Sample Label Text Cancel e Customize Result Table opens a dialog box in which you can hide or display columns add new columns and reorder columns 8 Customize Result Table t ou X J Name Datatype Unit s E m Select All Sample ID Text I I Data Set Text Clear All Sample Type Text New Column Description Text Well Locations Text Sample Label Text Condition Text Gender Text Matrix Text FI Numeric FI CV Numeric FI Std Dev Numeric Obs Conc Numeric ISB ES 99 Y Y Y Y A Y S S S Conc in Ranne Numeric Cancel e Copy Table to Clipboard copies the entire table to the clipboard including the column titles e Copy Table to Clipboard Full Precision copies the data with full precision to the clipboard e Copy Plot to Clipboard copies selected scatter plot or box and whisker plot to the clipboard e Export Plot exports plots or
14. is generally an indicator that significant differences do not exist Statistical tests require that two or more groups be identified and they require that each group have more than one sample If the Statistical Test options are disabled then your groups do not meet these requirements Select a different choice from the Compare by drop down list or edit sample attributes to identify more than one group with the required minimum number of samples If samples are grouped by the Compare by column the P value column displays the P value and the P value Method column displays the method used to calculate the P value Bio Plex Data Pro uses different algorithms to calculate the P values depending on the number of sample groups per analyte and the user s preference for one test or another All P values are two tailed probabilities Bio Plex Data Pro does not make any assumptions about the direction of difference between two or more groups The following describes which statistical method is used to calculate the P value For data in a normal distribution select t test One Way ANOVA The following logic is used to determine which test to apply e lf there are two groups a t test is applied e If there are three or more groups then the One Way ANOVA test is applied For data that are not in a normal distribution or if you are uncertain select Mann Whitney Kruskal Wallis The following logic is used to determine which test to a
15. o o n a 0 D o d uU a So ROR j FGF basic 2 H Follistatin H Hi Hi Hovering over a cell in the heat map displays a description of that cell s contents Heat Map Result View Bio Plex Data Pro Sample File Edit Data View Chart Options Help amp c ww CB o7 BY E Sample Label Field Sample Label Z Manage Data Sets Group by O Ascending Data Set H Hi Descending H E Show in sample label Edit Samples Then by E ML di ci Se ODS Sample Label v ica Show in sample label oes xXx Un de X8 X7 Normal vs Disease plasma 1 Normal Condition Normal Gender Female Matrix Plasma Analyte Hu PDGF BB Obs Conc 825 12 Log2 Obs Conc 9 69 Normalized Intensity 34 00 Fe y own Then by lt none gt su it Perform Analysis Create Charts XXXXXXXXXXXXXXXXXXXXXXXXXX bi QI GI Gi DI OI 0 Gi RS S NS S S S SR AN m m s s s a a 4 h b 6 M OQ ID O O Ui P o6 M 9 OU 4m 0 b 0 M Commands to the left of the heat map control the order of the samples and the labels used in the heat map The Chart Options menu allows you to change the manner in which the intensities are calculated and displayed CHART OPTIONS MENU COMMANDS From the Chart Options menu you can access the following commands Frame Cells a toggle that turns on or off contrasting lines framing each cell Sample Label Field opens a drop do
16. of Fold Change Control Group Compare Group 1 1 COS E 2 A A 4 1o pa Obs JO H 46 ele A m m Condition Samples Valid Samples P Value P Value Method Fold Change Mean Std Dev CV Standard Error Min Qi M 1 Normal 13 13 3093 81 2980 32 96 33 826 59 179 50 581 63 231 12 Breast Cancer 8 8 0 082 Mann Whitney 0 709 5055 69 2958 24 58 51 1045 89 1316 75 2200 50 44 3 Colon Cancer 8 8 0 562 Mann Whitney 0 190 3529 25 2650 83 75 11 937 21 696 00 1362 06 24 4 Lung Cancer 8 8 0070 Mann Whitney 0 684 4969 59 2213 98 44 55 782 76 1632 75 3412 88 47 Analyte Charts and Tables Export to Statistics Software This table displays each group as a column and includes only the sample values Unlike the Results by Sample table it includes only samples with valid values This table makes it possible to copy data to third party software applications that support this format for sample groups Qi JO ii 8 gh p Normal Breast Cancer Colon Cancer Lung Cancer 1 1088 50 6914 25 6539 00 3765 75 2 333 00 8459 00 1071 25 1632 75 3 2364 75 484325 696 00 4893 50 4 3978 00 1316 75 4639 50 7999 25 5 6588 00 9326 75 2370 00 3295 25 6 51475 4106 50 8102 00 5672 50 7 1472 50 1661 50 2581 75 7977 00 g 3313 00 3817 50 2234 50 4520 75 9 548 50 10 2923 75 11 179 50 12 7657 50 13 9157 75 Results by Sample This table displays all the samples for the current analyte in rows If a Compare by column is selected the table in
17. your results in biologically relevant groupings Proper annotation of your samples is a prerequisite for using this view At a minimum the Compare by drop down list should include column headings that contain the biological replicate groups you want to compare and display on the x axis of your graphs Subpopulations of samples can be analyzed by excluding samples from the view with the sample Filter drop down list Changing the column in the Compare by or Filter by drop down lists dynamically updates all views The column selected in the Control by drop down list is the biological replicate group that represents the control for your experiment By choosing a control group several calculations are automatically generated including a pair wise P value calculation either a t test or Mann Whitney depending on which test is selected and a log scale fold change between the control and each of the experimental groups Statistical analysis is useful for identifying analytes that display differences between the groups under observation Selecting the Perform Analysis Create Charts view displays a table of statistical information for all analytes This view enables visualization and discovery of statistically significant differences P value and fold change among sample groups within each analyte in the project 49 Bio Plex Data Pro Software User Guide Analysis Workflow Analysis Workflow The statistical analysis wor
18. 170 South Africa 27 861 246 723 Spain 34 91 590 5200 Sweden 08 555 12700 Switzerland 061 717 95 55 Taiwan 886 2 2578 7189 Thailand 66 2 6518311 United Kingdom 020 8328 2000 11 1373 0911 Sig 0211
19. 39 Edit menu 4 Edit Samples Add Columns view 22 Editing merged data 42 43 project properties 13 Editing commands 5 Export to Statistics Software 54 Exporting graphs 36 plots 36 F FI fluorescent intensity 38 File Menu 4 Filtering icons 33 66 Filtering data 24 26 Frame Cells command 45 Full precision data values viewing 35 G Graphs copying 35 exporting 36 Heat map 38 44 47 copying 44 definition of 44 labeling the 46 Importing data 15 18 from Bio Plex Manager 17 18 status of 16 In Range 38 Inactive project definition of 14 Intensity Tyoe command 45 K Kruskall Wallis test 53 Mann Whitney test 53 Merge column 42 Merging data 40 43 Muilti Analyte table filtering data in 24 Multi Analyte table 39 O Obs Conc 38 Obs Conc column 39 Observed Concentration 42 One way ANOVA test 53 P Perform Analysis Create Charts view filtering data in 26 Plot box and whiskers 55 scatter 55 Plots copying 35 exporting 36 Project creating 9 10 editing 13 name changing 13 opening a 11 12 organizing 14 Projects inactive 14 Projects active 14 P value 53 Ratio 39 RBX file 15 Reset to Default Sort 33 Results by Sample 54 Results table filtering data in 24 25 hiding columns 29 merged data in 42 synchronized with Sample table S Sample annotation 49 Sample ID column 39 Sample Label column 39 Sample Label Field command 45 Sample table 22 filtering data in 24 25 hiding c
20. 7697 control 5017698 chi flu study run march 2010 Cancel 3 Edit the name and description and click OK Deleting a Data Set The following procedure describes how to delete a data set from your project To delete a data set 1 In the Data Sets table select the data sets you want to delete 2 Right click and select Delete Data Set in the context menu 18 Deleting a Data Set A message asks you to confirm that you want to delete the data set 3 Click Yes The data sets are deleted from the project 19 Bio Plex Data Pro Software User Guide Deleting a Data Set 20 Bio Plex Data Pro Software User Guide 4 Organizing the Data After data sets are imported into Bio Plex Data Pro software you can manage the data that is used in your analysis You can add data to your samples with data from your experiments You can filter your data and include or exclude samples Finally data can be copied or exported to other applications for analysis Selecting Samples When you import Bio Plex Manager software data sets into Bio Plex Data Pro all of the samples in your experiment are imported Typically you will want to exclude some samples for example Blank and Standard samples Use Select Sample Type for analysis in the toolbar to specify which samples to include This is a global setting that applies to all views of the project in Bio Plex Data Pro Note By default the Blank and Standard samples
21. Anabysis Creabe Chants Data Set Sample Type Normal vs Disease plasma 1 Unknown Moral vk Drsear plasma 1 Unknown 1 Normal v nosebe platma l Unknown 4 Mormal vt Diseate plasma l Linknowrna Normal vs Disease plasma 1 Linknewn amp Normal vs Disease plasma 1 Unknown Moral v Drseacr plasma 1 Unknown Normal vc Dieses platma l Unknown a Mormal vt Diseate plasma l Linknowrna I Nommalws Disease plasma l Linkncwn Mormal vt Diseape plasma l Linknowrna Formal vs Disease plasma 1 Lnknewn 17 Normalws Disease plasma l Unknown id Moral v Dres plasma 1 Unknown Normal v Dices jlacma l Unknown 160 ere wi Disease plasma l Unknown Homma vs Disease plasma 1 Unknewn 1a Nerenalvs Disease plasma l Unknown 15 Moral vk Dres plasma 1 Unknown m Normal v Dipende platma l Unknown Mormal vt Diseate plasma l Linknowna Normal vs Disease plasma 1 Lnknewn 3 4 Momma vs Disease plasma 1 Unkn awn Merging Data Samples may be run several times for one of the following reasons 40 e Dilution requirements may be very different for different assays Descripbon Mermal Normal Porra Nesrrsal Iewrrsal Mermal Normal Normal MNewrrsal Ha Ma Mewrrsal Miss CoSon Cancer Coos cancer eee Seer coson cancer ean Sater cole gingii hung cancer lung cancer Fl Condibon Nara Horma Blareal dorm l oral Nora Normal hiyaan Marm l Nora Hor mal eru Aenea Doon Cancer Colon Cancer Colon Lancer Colon Cancer Col
22. Bio Plex Data Pro Software User Guide Version 1 0 Bio Plex Data Pro Software User Guide Bio RAD TECHNICAL SUPPORT DEPARTMENT The Bio Rad Technical Support Department in the U S is open Monday through Friday 5 00 a m to 5 00 p m Pacific Standard Time Worldwide technical support is available on the Web at www consult bio rad com Phone 1 800 424 6723 option 2 Fax 1 510 741 5802 Email LSG TechServ US Bio Rad com U S LSG TechServ Intl Bio Rad com International Web www consult bio rad com NOTICE No part of this publication may be reproduced or transmitted in any form or by any means electronic or mechanical including photocopy recording or any information storage or retrieval system without permission in writing from Bio Rad Bio Rad reserves the right to modify its products and services at any time This user guide is subject to change without notice Although prepared to ensure accuracy Bio Rad assumes no liability for errors or omissions or for any damage resulting from the application or use of this information The following are trademarks of Bio Rad Laboratories Bio Rad Bio Plex Bio Plex Manager and Bio Plex Data Pro Luminex xMAP and xPONENT are trademarks of Luminex Corporation Windows and Excel are trademarks of Microsoft Corporation Pentium is a trademark of Intel Corporation No rights or licenses under any of Luminex Corporation s patents are granted by or shall be implied from t
23. Disease E Disease E Disease E Disease E Disease E Disease E Disease E Disease E Disease p 7 k 43 Bio Plex Data Pro Software User Guide Heat Map Result View Heat Map Result View 44 A heat map is a graphical representation of data that enables you to quickly identify the intensity readings and to find patterns in the data Each column in the heat map corresponds to an analyte each row to a sample and each cell to a data point The cells are color coded according to their median or midpoint compared to other samples for that analyte You can copy heat maps and tables to the clipboard and paste them into the application of your choice See Copying Plots and Graphs on page 35 To switch from table to heat map view e Click Heat Map Result View in the toolbar The Results table data appear as a heat map Manage Data Sets File Edit Data View Chart Options Help fk cl a ba HY E Sample Label Field b Sample Label Group by O Ascending Data Set pes Descending c Edit Samples Add Columns Show in sample label Then by 9 Ascending S le Label v ampe ae Descending Perform Analysis Create Charts L Show in sample label Then by Ascending lt none gt X Descending Show in sample label 5 X MXxwxxxxx Qoo oot b amp b m b wN 0o PUN RMR MK KKK KK KK KK KK KK KK KK KK KR OK JW Wo 0 0 0G SS 8S M N A o o o o
24. Filtering Data in the Sample and Results Tables Contents of each column including custom columns can be filtered using the drop down list in its column heading By default all rows are included Note Filters cannot be applied to the Multi Analyte table in the View Results View Heat Map view Filtering Data To filter data 1 Click the triangle in the column header to open the drop down menu In the Filter section there is a list of the different sample types for that column Sample Type sease plasma 1 Unknown Manage Data Sets Normal sease plasma 1 Unknown Normal l Reset Sample Lists to Default Sort sease plasma 1 Unknown Normal 9 None sease plasma 1 Unknown E Normal Ascending sease plasma 1 Unknown Edit Samples Normal Descending sease plasma 1 Unknown Add Columns Normal bease plasma 1 Unknown Breast Ca pease plasma 1 Unknown Breast Ca Clear All pease plasma 1 Unknown Breast Ca Name E Unknown Breast Ca E Unknown Breast Cad fg c E Unknown Il Breast Ca q ee 5 Unknown Unknown Perform Analysis Create Charts Unknown Unknown Unknown Colon Can sease plasma 1 Unknown Colon Cancer Plasma Female Normal vs Disease plasma 1 Unknown Lung Cancer Plasma Female Normal vs Disease plasma 1 Unknown Lung Cancer Plasma Female Normal vs Disease plasma 1 Unknown lt 2 Select the checkboxes of the samples you want to include and c
25. If the project contains data the project is opened in the Manage Data Sets view It displays all the data sets contained in the project See Importing Data Sets on page 15 for more information about data sets Fite Edit Data Wew Help A cedar Bu ee Name User Hame Acquistion Time Description Imported Date Imported by Comment 1 Marma v Droste plata l vupta Aa 2011 dele PA Ara 2011 amp 3M PM byeck tm Ee R2 Pormal vi Disate perum D vaupta TLL 4 26 PM 217 2011 530 PM tyeck If the project does not contain data the Import Bio Plex Results File dialog box appears See Importing Data Sets on page 15 for information on importing data into your project 12 Editing a Project Editing a Project You can edit project attributes in the Edit project dialog box To edit a project 1 Click Open Project The Open Project dialog box appears displaying a list of projects 2 Right click in the row of the project you want to edit and click Edit Data Set TIP Do not click the project name this will open the project Fle Ex Date Wem Help A cues By ti The Edit project dialog box appears 2 Edit project Co JCE Jm Name Test Owner jtenenbaum Team Supervisor Description Cancel 3 Edit the project settings and click OK 13 Bio Plex Data Pro Software User Guide Organizing Your Projects Organizing Your Projects 14 Over the course of time you will create multiple projects
26. Inthe plot or graph right click to open the context menu and then click Copy Plot to Clipboard 2 Paste the table into the application of your choice 35 Bio Plex Data Pro Software User Guide Exporting Plots and Graphs Exporting Plots and Graphs Plots and graphs can also be exported as Portable Network Graphics png JPEG bitmap or TIFF files These exported files are of higher quality than the ones produced using the copy and paste tools To export a plot or graph 1 In the plot or graph right click to open the context menu and then click Export Plot 2 Inthe Export Plot dialog box enter the name of the file 3 Select the type of file in which you want to save the plot or graph 4 Click Save 36 Bio Plex Data Pro Software User Guide 5 Viewing the Data There are several different ways you can view your results in Bio Plex Data Pro software You can look at the results for one analyte at a time You can view all the analytes at once and compare the results You can view the results graphically in a heat map In addition you can merge multiple rows of data for the same sample to simplify your view of the data Viewing Results In the View Results View Heat Map view the Results table lists data by individual samples from every RBX file imported into the project It presents a view of the aggregates of all the results across plates 37 Bio Plex Data Pro Software User Guide Viewing Results 38
27. OUPING DATA IN THE HEAT MAP You can specify how data in the heat map are grouped You can group your data by any column in the Results table Note The Group by drop down list includes all visible predefined and custom columns in the Results table Hidden columns do not appear in the list Using the controls to the left of the heat map you can apply up to three levels of sorting to the heat map data The primary sort is set using the Group by control The second and third level sorts are applied using the Then by controls Each subsequent sort is done within the constraints of the previous sorts For each sort level you can also select whether the data appear in ascending or descending order To sort the heat map data 1 Click Group by and select a column from the list 2 Select Ascending or Descending 3 If you want the sort option to appear in the sample label click the Show in sample label checkbox 4 f you want to apply another level of sorting click Then by and select another column from the list Follow the procedure in Step 2 and Step 3 Adding a Column You can add columns to the Results and Sample tables See Adding a Column on page 27 47 Bio Plex Data Pro Software User Guide Adding a Column 48 Bio Plex Data Pro Software User Guide 6 Viewing the Results and Basic Statistics In the Perform Analysis Create Charts view you can view the graphs and tables that present
28. Plex Data Pro the data are displayed in a Sample table Pp x u paenail Unbnan DELE purus Do Uningan p The Sample Table The toolbar in the Edit Samples Add Columns view displays additional icons ER Select Sample Type for Analysis opens a dialog box in which you select the sample types to use in your analysis These sample types are the standards controls blanks and unknowns imported from Bio Plex Manager This selection is a global setting Merge Samples run on different panels assays Undo Merge Samples 9 qu Customize Sample Table opens a dialog box in i which you can select columns that will be visible in the Sample table m Add Column opens a dialog box in which you can select a column to add or type a column heading The toolbar also displays a drop down list of all the data sets in the project Data Set 3 Diabetes 12 Plex Plate 1 1 35 E Left and right arrows facilitate scrolling through the list This list enables you to view just the data set you want to edit The choice of data set persists for the duration of the session The Sample table displays the following columns by default Sample ID this field can be used to assign a unique ID to a sample and its replicates Data Set one source data set if the sample belongs to multiple data sets the sample is listed multiple times Sample Type Unknown Control Standard Blank or External Standard Description dis
29. able and the dependent variable established by the model does not actually exist Plate Assay plate Bio Plex assay plates have 96 or 384 wells The plate contains assay data Sample A solution of analyte standards or unknowns that when added to an assay will bind to the microspheres in that assay and be identified and quantitated by the array reader UOLQ Upper limit of quantitation Index Symbols CV 39 A Active project definition of 14 Adding columns 27 28 Analysis Configuration pane 50 51 Analyte detail views 54 Analyte Charts and Tables 51 Analyte table 50 52 toolbar 52 Bar graph 54 56 Bio Plex Manager importing data from 17 18 Box and whisker plot 54 55 55 56 Bio Plex Data Pro Software User Guide C Color Gradient command 45 Column custom adding 27 hiding 28 29 Compare by 51 Conc CV column 39 Concentration of the coefficient of variation 42 Control 51 Copying data 34 39 graphs 35 heat maps 44 plots 35 Creating a project 9 10 Data copying 34 39 filtering 24 26 importing 15 18 65 Bio Plex Data Pro Software User Guide importing from Bio Plex Manager 17 18 importing status of 16 merging 40 43 row copying 34 rows combining 40 sorting 29 34 unmerge 41 Data set deleting 18 editing 18 import status 16 17 name changing 18 Data sets number of imported 15 Data Sets table 10 copying 35 description of 11 Data Transform command 45 Description column
30. ages ree 61 Bio Plex Data Pro Software User Guide 1 Introduction Bio Plex Data Pro software provides tools for annotating and analyzing the results of experiments conducted in Bio Plex Manager software When you import Bio Plex Manager data into Bio Plex Data Pro you can edit samples view results and perform statistical analysis on data These actions take place within a Bio Plex Data Pro project Basic Workflow The basic workflow in Bio Plex Data Pro consists of the following steps 1 2 3 4 o Create a project Import your data sets Bio Plex Results files rbx Edit your samples View your results Analyze your data This guide explains how to use Bio Plex Data Pro to perform the steps in this workflow Bio Plex Data Pro Software User Guide The Workspace The Workspace Fee Ede Data Vee Help Araure The Bio Plex Data Pro software Home view displays buttons to create a new project or to open an existing project You can also open a recent project by clicking the project name in the Recent Projects list The left pane displays the project workflow starting at the top with Manage Data Sets and ending with Perform Analysis Create Charts Once a project is open clicking each button in the left pane takes you to the view where you complete the step Toolbar Commands Toolbar buttons give you quick access to basic commands You can also access these commands from the menu
31. ble similarly the column order you create in the Results table also applies to the Sample table Note however that the Results table with multiple analytes has fixed columns 2f Bio Plex Data Pro Software User Guide Hiding a Column To add a column to the Sample table 1 Click Add Column in the toolbar to open the drop down list Other Time Dose Concentration Compound Drug Cancel 2 Select a column from the list or select Other and type a name for the column in the Header box You can select from the following predefined column headings e Time numeric units only select seconds minutes hours days or months Dose numeric Concentration numeric Compound text Drug text The type of data that is valid for each column appears in parentheses 3 Optional Type a unit in the Unit field 4 Click OK The column is added to both the Sample and Results tables Hiding a Column 28 Any column can be hidden from view Columns you hide in the Sample table are hidden in the Results table and the reverse also is true Hiding columns enables you to exclude data from being exported Only data visible in the table are exported To hide a column in the Sample table 1 In the Edit Samples Add Columns view click the Customize Sample Table in the toolbar 2 In the Customize Sample Table dialog box clear the Visible checkbox for each column you do not want to display 3 Click OK So
32. cludes this column to display the sample annotation for the group In addition this table includes the sample value for the currently selected measure type Hu Leptin 24 a La E Sample Label Data Set X14 X15 X16 X17 X18 X19 X20 Normal vs Disease plasma 1 Normal vs Disease plasma 1 Normal vs Disease plasma 1 Normal vs Disease plasma 1 Normal vs Disease plasma 1 Normal vs Disease plasma 1 Normal vs Disease plasma 1 Normal vs Disease plasma 1 Normal vs Disease plasma 1 Normal vs Disease plasma 1 Normal vs Disease plasma 1 Normal vs Disease plasma 1 Normal vs Disease plasma 1 Normal vs Disease plasma 1 Normal vs Disease plasma 1 Normal vs Disease plasma 1 Description breast cancer breast cancer breast cancer breast cancer breast cancer breast cancer breast cancer breast cancer colon cancer colon cancer colon cancer colon cancer colon cancer colon cancer colon cancer colon cancer Condition Breast Cancer Breast Cancer Breast Cancer Breast Cancer Breast Cancer Breast Cancer Breast Cancer Breast Cancer Colon Cancer Colon Cancer Colon Cancer Colon Cancer Colon Cancer Colon Cancer Colon Cancer Colon Cancer FI 6914 25 8459 00 4843 25 1316 75 9326 75 4106 50 1661 50 3817 50 6539 00 1071 25 696 00 4639 50 2370 00 8102 00 2581 75 2234 50 Bio Plex Data Pro Software User Guide Analyte Charts and Tables 60 Bio Plex Data Pro Software User Guide
33. cluding the mean value for the analyte the P value fold change and the statistical method used On each bar an error bar plots one standard error from the mean Pibe PVs Method Fold Change Ara Site OV DHOMA Kraska pi WHS iiih 6017 4234 DAJ Krakal iaig Bae 3019 2017 TT Dipi Krantas ies SPL 630 IE Seber Krasta Wy Lot sey A O 104 Erais Wal L ena aay F Fb DH recia Wall QAT Krasta Wallis f6h Kratak aig 01101 tad al sde roe 197141 Breast Cancer Colon Cancer Cord of Bio Plex Data Pro Software User Guide Analyte Charts and Tables 58 Summary Table The Summary Table displays descriptive statistics for each sample group Each group is displayed as a row in the table If you select a Control group you will see the following statistics e P Value the P value is the result of comparing the selected group with the control e Fold Change the fold change is the base 2 logarithm of the ratio between the control group and group being compared The minus sign indicates the down regulation of the group compared to the control No sign indicates the up regulation of the group compared to the control For example if the mean of the control group is 1 and the mean of the group being compared is 1 then the fold change is O If the mean of the group being compared is 2 then the fold change is 1 The following table shows the calculated fold change for different control groups and compare groups Mean Value
34. d filtered In this example Y the down pointing arrow indicates descending order 33 Bio Plex Data Pro Software User Guide Copying Data in Tables Basic Sorting in Bio Plex Data Pro Software This section describes how to sort Bio Plex Data Pro data if you are not using the sort tool available in the Sample and Results tables Sorting that is done without the sort tool that is basic sorting does not persist between sessions The data return to their pre sorted order For information on sorting in the Sample or Results table see Defining a Sort in the Sample or Results Tables on page 30 To sort data e Click the column header once to sort the data in ascending order A triangle appears in the column header indicating an ascending sort is applied on the data e Click the column header a second time to sort the data in descending order The triangle in the column heading is now inverted indicating a descending sort is applied on the data e Click the column header a third time to return the data to its unsorted state The triangle disappears indicating the data are unsorted TIP To sort on multiple columns hold the SHIFT key down and click on the column header You can sort on as many columns as you like Copying Data in Tables 34 Copying a Data Row When you copy a data set row to another application only the selected rows are copied The column heading is not copied To copy a row from the data set tabl
35. dth of the individual sample categories and the Margin the width between sample categories can be adjusted manually Hovering over either plot displays detailed information about the object under the cursor Note Plots display only samples with valid values Bar Graph Bar graphs make it possible to view the graphs of different analytes simultaneously You can choose to display selected analytes and filter out others Doing so makes it easier to find analytes that have different expressions across different groups For example you can compare one drug to another to look for a different expression between the drugs You can view the results for more than one analyte The first analyte in the table is selected by default You can select other analytes To select an analyte e Inthe analyte table click anywhere in the row to select a single analyte To select additional analytes press CTRL and select additional rows e Inthe analyte charts and tables pane select one or more analytes in the Analytes drop down list Analyte Charts and Tables When you select the Bar Graph icon a Legend column appears in the analyte table Each selected analyte corresponds to a color These colors match the colors of the bars in the bar graph You can also display a legend in the analyte charts and tables pane by clicking the Legend checkbox in the toolbar When you hover over the bar an annotation appears with information about the analyte in
36. e 1 Select the rows you want to copy 2 Right click and click Copy in the context menu 3 Right click and click Paste in the application of your choice Viewing Data Values with Full Precision Copying the Data Set Table You can copy the data set table to the clipboard and paste it into another application Doing so copies the entire table including the column headings To copy the data set table to the clipboard 1 In the data set table right click to open the context menu and then click Copy Table to Clipboard 2 Paste the table into the application of your choice Viewing Data Values with Full Precision Values in the data tables are rounded off You can see the data values with full precision by copying the data to another application for example Microsoft Excel or Notepad This is useful for verifying the software s calculations To view the data with full precision 1 In the data set table right click to open the context menu and then click Copy Table to Clipboard Full Precision 2 Paste the table into the application of your choice Note Check to see that the application is displaying the values with the level of precision you desire For example the default in Microsoft Excel is to display two decimal places Use Format gt Cells to change the level of precision Copying Plots and Graphs You can copy a plot or graph to the clipboard and paste it into an application To copy a plot or graph 1
37. e being quantitated Attribute Custom field relevant to customer research entered manually or copied in from another list Excel for example Concentration The amount of substance present in a sample usually expressed in weight per volume Data set The sample and analyte information available after a run has been performed and analyzed A data set is roughly equivalent to a plate and is imported via a file import for example from Bio Plex Manager software via an RBX file Fl Fluorescent intensity A relative measurement of fluorescent intensity based on the median a robust statistical measure Heat map Graphical representation of peak expression data Each column of a heat map represents a spectrum each row represents a peak cluster and each cell represents a specific peak that is color coded to reflect its expression level Displays relationships between samples and analytes LLOQ Lower limit of quantitation LOD Limit of detection Outlier Sample concentration derived from Fl below the background value Sample concentration that meets a user defined criteria including CV concentration above or below a certain range 63 Bio Plex Data Pro Software User Guide 64 P value Statistical representation of the chance that the relationship being observed is observed by pure chance For example a P value of 0 01 means that there is a 1 chance that the relationship between the independent vari
38. ected it controls the distance between the bands in the bar and whisker plot and in the bar graph In the scatter plot it controls the distance between the sets of dots e Legend when selected a legend appears identifying the analytes in the bar graph Scatter Plot and Box and Whisker Plot The scatter plot and box and whisker plot display each group of samples as a category band along the horizontal axis The scatter plot displays each sample as a point Points within the same group are artificially spread out along the horizontal axis in order to minimize overlapping samples Hu Cates T 9 a T P m Fem aue Aub Hu Osteopontin A box and whisker plot is a quick way of graphically examining the distribution of points in each group This plot is particularly useful for comparing distributions between several groups or sets of data The box and whisker plot displays a box from the first quartile to the third quartile The bottom and top horizontal bars are the minimum and maximum sample values respectively The horizontal bar within the box represents the median sample value 55 Bio Plex Data Pro Software User Guide Analyte Charts and Tables 56 Hu Osteop at m La m Ei Y Auto Band Width Maron Hu Osteopontin When the Auto checkbox is selected the plot displays all groups inside the view port and maximizes the use of the view port When Auto is not selected the Band Width the wi
39. ee Sed hae o ota e RR C e 34 Copying the Data Set Table oooooooomoonomo 35 Viewing Data Values with Full Precision o o oooooooomommo 35 Copying Plots and Graphs o oocococoocooo ees 35 Exporting Plots and Graphs 2 23 ES a ce Oa Cx CE ee BAS 36 Chapter 5 Viewing the Data 37 ME WINO HesullS 5n x DONI IR hed ie Rs als eye baw Kap eee ae 3 View Results for All Analytes 0 00000 ee eee 39 VICTOING Datos asia a Pe GE Ce oe Eee id Ce 40 Viewing Merged Data in the Results Table 42 Viewing Merged Data in the Sample Table 43 Heat Map Result View 2 0 00 eas 44 Adding a COMME eS PY xs 47 Chapter 6 Viewing the Results and Basic Statistics 49 Analysis VWOPKEIO Wind dite We a etary ic as neh a ge 50 AMAIVSIS VV OIKSD ACS 5 2 uh Seen ede tints dd ace dose ee at uos 50 Analysis Configuration Pane 000 cee eee eee eee 51 Anaie Tables iaa da ed da 52 Interpreting P Value Statistics llle 52 Understanding Fold Change Statistics 0 0 0 0 00 cee eee ees 54 Analyte Charts and Tables 0 0 00 eee eee 54 Scatter Plot and Box and Whisker Plot 99 Ale CAA Miers a edad bs 56 ourmriaary T able a ss ae eh acy a 58 Export to Statistics Software c oooommmmm orrisrsaza 59 Results by Sample iu eoru ces ane d vac dad duco a 59 Appendix A Error Mess
40. es in the Perform Analysis Create Charts view are generally unaffected depending on your experimental setup The Merge samples run on different panels assays command allows you to combine the rows of data so that each sample appears only once in the table Merging data reduces the complexity of the data Data can be merged based on a matching Sample ID Description or Sample Label or a combination of these fields You can also merge the data using custom fields Another use for merged data is to average the data in the grouped samples You can always unmerge merged data Merging data does not change the original data Merging Data To merge results data 1 In the Results table choose Merge samples run on different panels assays in the toolbar 2 In the Merge Samples Dialog dialog box select the columns to match and merge samples 2 Please select the columns used t coenpane and menge matching samples Merged samples are uie hic all rr viec Mergen ampli can be undant Mege Mame Cata type Unit Sample 1D Text n ento Sample Label Tea Records with matching names in the selected column are merged appropriately When data are merged a column is added to the left side of the table Merge column and this column is used to sort the table in ascending order To unmerge results data e Select Undo Merge Samples in the toolbar In the Results table all merged data are separated 41
41. he sale or acquisition of this Bio Plex system containing Luminex technology the System to you the end user By using this System you agree that i the System is sold only for use with fluorescently labeled microsphere beads authorized by Luminex Beads and ii you obtain rights under Luminex s patents to use this System by registering this System with Bio Rad in accordance with the instructions accompanying this System and by purchasing a kit containing Beads Bio Rad Laboratories Inc 2000 Alfred Nobel Drive Hercules CA 94547 1 800 424 6723 Copyright 2001 2011 by Bio Rad Laboratories All rights reserved Bio Plex Data Pro Software User Guide Table of Contents Chapter 1 Introduction 00 cee ee 1 Basic WOFKIIOW 223 53 93 5 A E IAS dea 1 de VVOLKSDaGOG s rta ao ci tas ta a a ah 2 Toolbar Commands adi ee t2 edo a aie Decade SC HS DE a 2 Overflow Toolbar Commands eee eee eee 3 File Menu Commands llle 4 Edit Menu Commands aaa res 4 Data Menu Commands celeres 6 View Menu Commands leen 7 Chapter 2 Working with Projects 9 Creanga ProjeCl ss cdas aca eae E RR AE A ya we eee ee tees 9 Data Sets Tables pita a a a mee aa nda 10 Openinga PIoOIeeE odium a eI EUR Geese Du CR ACRI ED EACH ce 11 lA EC aet qo ke aide tana Sale c dieit tame ia Scr AEN ow sa 13 Organizing Your Projects ni aana a ew RU RO
42. including the path to the file The Status displays as Ready LE Import Bio Plex Res Import Into Sample Project 1 New Project Data Set Files P Status Data File 5 Click Import The system imports all data files in the list Each file displays a status as it is imported Done the data set was successfully imported Exists if you try to import a data set that was previously imported into the project Bio Plex Data Pro does not reimport the data It displays the status Exists to indicate the data has already been imported into the project Importing Data Sets Ext Standards the data were successfully imported The unknown values were calculated using an external standard curve Renamed the data were successfully imported The name of the file being imported is the same as a file previously imported into the project but the data are different To distinguish between the two data sets the file name of the data being imported is appended with a number for example 007 is appended at the end of the file name Bio Plex Phosphoprotein 11 Plex 007 Note If the data being imported are identical to data previously imported but they are exported from a different version of Bio Plex Manager Bio Plex Data Pro imports the data and displays the Renamed status 6 You can click Browse and import additional data sets or click Close if you are done importing data Importing Data Directly from Bio Plex
43. inted down descending sort A black arrow indicates the first sort also Known as the primary sort All subsequent sorts are indicated with a grey arrow TIP You can sort a single column by clicking the column header Clicking multiple times cycles through ascending sort descending sort and no sort When you click on a column header any existing sort settings on the table are cleared You can clear sorting on a single data column or you can clear sorting on all table columns at one time 31 Bio Plex Data Pro Software User Guide Sorting Data Clearing Data Sorting To clear sorting on one column e To clear sorting on the column click the button in the column header to display the drop down list and select None When you clear sorting on one column the sorting on any other column is retained The data will resort based on the remaining sort settings Fee Ede Data Vite Help f ew e ER Y LET De Cura dan 24d data seti Condioa Metro Gender Data Set j Semple Type lA Demnpeon ampie Label Horral tir wee para Unkncen Horral wee puma Urnkncen wee Glee Unkncen wae Glee l Unkncen wee Glee Unkncen uns puma Unkncen inp puma Unkncen uns puma Uningan Mae plea Unknosn Horrral N ace puma Unkncen Norma wae pum Unkncen Hiorral wee puma Unkncen Parral j Mae piuma y Uda ient Lana wee piama Unkncen creat carer Great Dare wee para Unkncen A anaj amp Great Lara 5 M
44. iti Unknown Normal24 C9 C10 33 96 Ovarian Cancer Female CDG kiti Unknown OC1 D9 D10 57 57 EX L4 Manage Data Sets Edit Samples Add Columns 0 Ou un Pp lt Use this table to review the results of the experiment you imported You can specify how you want to view the results by filtering or by adding custom columns Columns and the order you select in the Results table persist until you change them You can also select different analytes and switch from the table to a heat map view Heat maps help you quickly identify the intensity readings for an analyte The following additional buttons appear in the toolbar Each option affects the data shown in the Results table E Results Table Single Analyte EL E Heat Map Result View Eg Multi Analyte Table o e Analyte drop down list with left and right arrows Fluorescent intensity of the selected analyte Observed concentration of the selected analyte ae Observed concentration that falls within the usable range of the standard curve o Viewing Results The normalized ratio fold expression or gene expression value Ratio calculated in Bio Plex Manager software cv Coefficient of variation Std Dev Standard deviation over the mean of a group of replicates zz Customize Result Table add comar Add Column The Results table displays the following columns by default You can add columns by clicking Add Column in the toolbar
45. kflow comprises the following actions e Choose the variable for comparison In order to do this you must first annotate the data e Optional Choose the biological replicate group that represents a control for your experiment if one exists to do pair wise comparisons between the control and all other groups e Select the statistical test to apply to the data e Use the filter to exclude data or refine group selection If the desired samples are not available in any of the Filter options add a new custom column in the Sample table or edit the annotation of an existing column Filter e For each analyte visually inspect the differences between the groups Analysis Workspace Analysis configuration Analyte table Analyte charts and tables There are three panes in the workspace e Analysis configuration the series of controls on the left most side of this view that define the specifics of your desired analysis e Analyte table table at the top of this view It shows all analytes in the selected samples with descriptive statistics for each analyte 50 Analysis Workspace e Analyte charts and tables charts and tables that provide the results to the analysis you have configured using the controls in the analysis configuration pane Analysis Configuration Pane The area on the left of the Perform Analysis Create Tables view is called the analysis configuration pane It has the following con
46. lear the checkboxes of the samples you want to exclude 25 Bio Plex Data Pro Software User Guide Filtering Data Filtering Data in the Perform Analysis Create Charts View The filter tool allows you to filter your data in the same way that you filter your data in the Results tables To filter data in the Perform Analysis Create Charts view 1 Click Filter by and choose the column you want to use to filter your data The list of samples is displayed in the Filter section m l 52 gii 5Io Plex L Pre File Edit View Help E aw i eal RA A E Start Here a Analyte P Value P Value Met MUR EE EEUU Hu FGF basic Manage Data Sets Pe Hu Follistatin Control sone y Hu G CSF Hu HGF Statistical Test Hu Leptin t test one way ANOVA Hu Osteopontin Hu PDGF BB Edit Samples Add Columns Mann Whitney Kruskal Wallis PAM a 119 1 id Filter Results a b xd gt Filter by Condition View Results View Heat Map Clear All Name Breast Cancer Colon Cancer Lung Cancer Obs Conc Normal 2 Select the checkboxes of the samples you want to include and clear the checkboxes of the samples you want to exclude 26 Adding a Column Removing Filters To clear filtering e To clear filtering on a column click the button in the column header to display the drop down list and select Select All e To clear all filters on a
47. lears all sort settings in the project Merge samples run on different panels assays opens a dialog box that allows you to merge samples from different data sets rbx files that share the same identity Choose the column that contains the information you need to match samples TIP Use Merge Samples when you notice samples listed several times that you want analyzed as a single sample Undo Merge Samples restores the sample row settings The Workspace View Menu Commands From the View menu you can access the following e Imported data sets in the Data Set Table e Individual sample data in the Sample Table e Results in the Results Table Single Analyte Multi Analyte Table and Heat Map e Analyze data in Scatter Plot Box and Whisker Plot Bar Graph Summary Table Export to Statistics Software table and Results by Sample table Bio Plex Data Pro Software User Guide The Workspace Bio Plex Data Pro Software User Guide 2 Working with Projects A project is the container for your data After you create your project you import data into the project You can have as many projects as you like Creating a Project Follow the procedure for creating a new project To create a project 1 From the Home view click New Project The New Project dialog box appears Name Owner Team Supervisor Description Note You can also create a new project by selecting New Project from the File menu or clicking New
48. ll columns in the table click Clear All Analysis Filters There are two places where you can do this You will find it in the drop down list for any column or in the toolbar Bio Plex Data Pro Sample Project O x File Edit Data View Help f cad Manage Data Sets Condition v Matrix Gender P Data Set Lung Canc Sorting isease Lung Canc IG E Reset Sample Lists to Default Sort Lung Canc Lung Canc 9 None isease Clear All Analysis Filters Edit Samples Add Columns Lung Canc Ascending isease Lung Canc Descending Lung Canc Filter isease Lung Canc isease Y Select All Clear AIl Y Name 8 Breast Cancer 0 Oi nm d Uu N nm View Results View Heat Map 8 Colon Cancer 8 Lung Cancer 13 Normal Perform Analysis Create Charts Adding a Column In addition to the predefined columns you can add up to ten custom columns to the Sample or Results tables When a custom column is added to a table you must specify its attributes A column that is added to the Sample table is also added to the Results table and vice versa Columns are added to the left of existing columns You can reposition a custom column by clicking the column heading and dragging it to a new position in the table or by using the Customize Sample Table dialog box The column order you create in the Sample table also applies to the Results ta
49. mal Manage Data Sets Sorting Normal li Reset Sample Lists to Default Sort Normal Normal None Normal D Ascending Normal Descending Normal Breast Canc Breast Canc Clear All Breast Canc L3 TUE O O JO ui d WN Hn View Results View Heat Map Breast Canc 16 Breast Cancer Breast Canc Breast Canc yi 16 Lung Cancer Ilis Breast Canc s 26 Normal Perform Analysis Breast Canc Create Charts Colon Canc Colon Canc A m m m m m m m m m im m f m f f m m Colon Canc Colon Cancer Serum Female Normal vs Dise Lung Cancer Serum Female Normal vs Dise Lung Cancer Serum Female Normal vs Dise Lung Cancer Serum Female Normal vs Dise Lung Cancer Serum Female Normal vs Dise Normal Plasma Female Normal vs Dist _ 4 b Note The icon that appears on the button varies depending on whether the data are sorted filtered or neither See Sorting and Filtering Icons on page 33 for a description of the icons Sorting Data 2 Inthe Sorting section select Ascending or Descending to sort the data in the column Sorting Se Je Reset Sample Lists to Default Sort 9 None Ascending Descending Filter H 16 16 16 26 2 a al Clear All t Mame Breast Cancer Colon Cancer Lung Cancer Normal After a sort is applied to a column an arrow appears in the column either pointed up ascending sort or po
50. olumns 28 merged data in 43 synchronized with Results table 24 Sample Type column 39 Samples blank 21 excluding 21 excluding from analysis 24 26 selecting 21 standard 21 Scatter plot 54 55 Sorting basic 34 clearing 32 33 default 32 33 heat map data 47 icons 33 in ascending order 33 in descending order 33 on multiple columns 34 Statistical Test 51 Std Dev 39 67 Bio Plex Data Pro Software User Guide Summary table 54 T Toolbar Analyte table 52 Bio Plex Data Pro 2 Edit Samples Add Columns view 23 Overflow 3 t test 53 V View Results View Heat Map view 37 W Well Locations column 39 Workflow Analysis 50 Bio Plex Data Pro 1 2 Workspace Analysis 50 Bio Plex Data Pro 2 68 Life Science Group 10022871 Rev A US EG Bio Rad Laboratories Inc Web site www bio rad com USA 800 424 6723 Australia 61 2 9914 2800 Austria 01 877 89 01 Belgium 09 385 55 11 Brazil 55 31 3689 6600 Canada 905 364 3435 China 86 21 6169 8500 Czech Republic 420 241 430 532 Denmark 44 52 10 00 Finland 09 804 22 00 France 01 47 95 69 65 Germany 089 31 884 0 Greece 30 210 777 4396 Hong Kong 852 2789 3300 Hungary 36 1 459 6100 India 91 124 4029300 Israel 03 963 6050 Italy 39 02 216091 Japan 03 6361 7000 Korea 82 2 3473 4460 Malaysia 60 3 2117 5260 Mexico 52 555 488 7670 The Netherlands 0318 540666 New Zealand 64 9 415 2280 Norway 23 38 41 30 Poland 48 22 331 99 99 Portugal 351 21 472 7700 Russia 7 495 721 14 04 Singapore 65 6415 3
51. on Cancer Doon Cancer Colon Cancer Colon Lancer Lung Larc r Lung Cancer Female Female Female Female Female Female Matre Plasma Plasma Plasma Plasma Plasma Plasma obs Conc In Range Ratio sv sta De B Add Columa HusEGFA1 Hu HoF bara Hu Fallcibaler F Debs tom Cone WV Obs tere Com WV Obg tern Lora WV ObsLo 473431 18 0844 Loe eqn ae el as 1S 1483 5 0 22A 7B H poe oa 26 riu 1361 60 Qin 45 Sia 574 92 19 nu KEH 45 Y3541 12 1 9503 138 150 76 E25 22 a 212 6198 Be 551 14 413 4 1145 44 VALLA 15 GERTI a 5 34 135 1a T6540 LBE 215 40 4 33 ES 0545 Ada ii x 14731 18S EaD 136 732 a a usur ALS 13 257 30 ory LE 2154821 0 341 ILALL 639 41129 Lal TH ZIAL 0501 137 68 T3 517 25 450 229 lE EE 20809 533 M331 el THN 132 12 esi 125 48 1 35 Teal al 554 104 28 72 161 Lip Tid 514 x0 5 34 T4 12154 53 52 ile dd tHe 23 08 508 472 12211 53 123 238 67 307 201 46 422 758 19904 97 331 24219 130 EE P ir BOS be Ose 14 64 DAI 55i 75 165 2135 46 45 16 35 bourse ala 5 555 Lad A ZB 1143 4 5 447 91 556 481 3345073 152 12188 5 95 Rial 275 12 18133 49 A5 W581 1 ES ign 1e e Bead regions for the assays may overlap e Assays from different kits cannot be mixed together for other reasons The outcome of this is that results for a particular sample appear in several data sets When the data files are imported the sample is listed multiple times in the Sample and Results tables The tabl
52. ot plea Unkncan creat cipe Great Lara wee Glee Unkncen Era carne Breet Lar L wee puma Unkncen Irae ceret Great Lancer we aene parra Q Unica creat cipe Breet Lancer we asse para l Uningan creat cep Great Lancer we Cheese para Unekncen creat corer Lotion Career we Cheese pima l Uningan qolon EA Lotion Career we Dasare parra Uningan molon cancer Lotion Career we Cheese para Unknoen ion cancer Lotion Cange we asata para Uningan qolon Cancer ad Lotion Career we asse para Unknoen colon cA 32 Sorting Data To clear sorting on all columns e To clear sorting on all columns in the table click Reset to Default Sort in the drop down list for any column or click the Reset to Default Sort button in the toolbar Fie Ede Data Wee Help fic AAA O a siiedeciediedigieciedieiidg rent L want au L salon L chon Cu chon Cu chon Cu chon EL gion Cu chon Cu chon EL ung ta ung ta ung ta ung ta LA ung ta nata ung ta TORT Md SATT aal SORTING AND FILTERING ICONS For the Sample and Results tables a button on each column displays a triangle by default The icons on the button change depending on the combination of sorting and filtering applied on the column v Column is not sorted or filtered This is the default e Column is filtered only y Ex Column is sorted only The arrow pointing up indicates ascending order An arrow pointing down indicates descending order Column is both sorted an
53. plays information that was entered in the sample description in the originating Bio Plex Manager Results file You can edit this field Well Locations the well or wells on the plate where the experiment was run for example A1 A2 or C1 D1 Sample Label name of the sample for example S1 Sn X1 Xn C1 Cn eS1 eSn Replicates identified in Bio Plex Manager are merged 23 Bio Plex Data Pro Software User Guide About the Sample and Results Tables About the Sample and Results Tables The Sample and Results tables are synchronized so changes you make to one table are immediately reflected in the other For example e Filtering data from one table filters it from the other e Sorting data in one table sorts it in the same order in the other e Adding a column of data in one table adds it to the other e Changing the order of the columns in one table changes the column order in the other Filtering Data 24 Data can be included or excluded from the project dynamically This is referred to as filtering You can choose the types of samples being used in your analysis at any time by using the filter tools These filters are available in most views throughout the software All filters apply across the entire project For example filter settings you apply to the Sample table are reflected in the Results table and Perform Analysis Create Charts view They can be cleared at any time using the Clear All Analysis Filters command
54. pply e lf there are two groups the Mann Whitney test is applied e If there are three or more groups then the Kruskall Wallis test is applied Bio Plex Data Pro does not assume that data are in a normal distribution and uses the Mann Whitney or Kruskal Wallis test by default 53 Bio Plex Data Pro Software User Guide Understanding Fold Change Statistics Understanding Fold Change Statistics Fold change is calculated for each analyte Samples are grouped by the field specified in the Compare by list The mean is calculated for each of the groups The fold change is calculated by dividing the highest mean value by the lowest mean value and then calculating the base 2 logarithm of the result Note The values used in statistical calculations and plots depend on the measure type FI Obs Conc observed concentration In Range concentration in range or Ratio selected in the main toolbar All statistics including P values fold change values and descriptive statistics incorporate input only from valid sample values If a sample value is out of range or below range it is not included Analyte Charts and Tables The analyte charts and tables pane displays the following detailed information about the analyte selected in the analyte table These views can be accessed from the toolbar in the analyte charts and tables pane 94 F qu F yl xi Scatter Plot displays the data for the selected analyte a
55. rting Data To hide a column in the Results table 1 Inthe View Results View Heat Map view click Customize Result Table in the toolbar 2 Inthe Customize Result Table dialog box clear the Visible checkbox for each column you do not want to display 3 Click OK Sorting Data You can sort any number and combination of columns of data in ascending or descending order You apply a sort to the first column and the rows of data are resorted based on the order you select If you then apply a sort ona second column the first sort persists and the second sort is applied to the sorted data You can always return your data to its original unsorted state All tables in the application can be sorted by clicking on the column header In addition you can sort the Sample and Results tables by clicking on the triangle in the column header For more information on sorting see the following e Defining a Sort in the Sample or Results Tables on page 30 e Basic Sorting in Bio Plex Data Pro Software on page 34 29 Bio Plex Data Pro Software User Guide Sorting Data 30 Defining a Sort in the Sample or Results Tables Sorted data in the Sample or Results tables persist between sessions To define a sort in the Sample or Results tables 1 Click the button in the column header to display a drop down list Bio Plex Data Prot S e m File Edit Data View Help a amp orwatar BYE A Condition Matrix Gender Data Set Nor
56. s A Home at New Project Open Project The Workspace Import Bio Plex Results File Undo Redo Select Sample Type for analysis Clear All Analysis Filters Reset Sample Lists to Default Sort Merge samples run on different panels assays Undo Merge Samples Overflow Toolbar Commands Clicking the triangle displays an overflow toolbar of hidden commands Overflow toolbars are enabled whenever the screen size does not allow all toolbar commands to be displayed Bio Plex Data Pro Software User Guide The Workspace da De ks Vane E bh Ese in ga Rta B TI 3 Overflow toolbar Overflow toolbar File Menu Commands The following commands can be accessed from the File menu New Project opens a dialog box in which you specify project details Open Project opens a dialog box in which you can open an existing project and organize your projects Import Bio Plex Results File opens a dialog box in which you can browse to a data set and import it Exit closes Data Pro software Edit Menu Commands The following commands can be accessed from the Edit menu Undo removes the effect of an action you performed Redo restores the effect of an action you undid Cut copies contents of selected cells to the clipboard and deletes contents of selected cells that are not read only Copy copies the contents of selected cells to the clipboard Paste pastes clipboard contents
57. s a scatter plot The x axis in this plot does not represent a value Dots are distributed in each group to facilitate visualization of each sample Box and Whisker Plot displays the data for the selected analyte as a box and whisker plot Bar Graph displays the data for the selected analytes as a bar graph with standard error bars Summary Table displays descriptive statistics for each sample group in a separate row Export to Statistics Software displays the data grouped as defined in the configuration pane in a format that is convenient for export to statistical software Each column displays the data for one group Results by Sample displays the data used to create the plots and other tables along with descriptive data for the samples of the selected analyte Analyte Charts and Tables The following are additional options available on the toolbar e Analytes drop down list of analytes in the data set e Fill toggle between displaying the dots in the scatter plot as outlines or filled in e Point Size changes the size of the dots in the scatter plot e Auto when selected controls the band width and margin of the scatter plot bar and whisker plot and bar graph e Band Width when Auto is not selected it controls how close the dots are to one another in the scatter plot For the bar and whisker plot and bar graph it controls the width of the bands e Margin when Auto is not sel
58. t m View Resums lo Perform Analysis Creste Charts The following information is displayed for each data set Name displays the name of the data set by default this is the name of the imported Bio Plex Results file rbx file User Name displays the name associated with the data set as specified in the general information page of Bio Plex Manager Acquisition Time displays the date and time the data were collected on the Bio Plex instrument Description displays the data file description entered in the Bio Plex Manager RBX file Imported Date displays the date the file was imported into the system Imported by displays the name of the Windows user logged in when import occurred Opening a Project You can open a project in the Home view by clicking on the project name in the Recent Projects list or by clicking Open Project To open a project 1 Inthe Home view click Open Project 11 Bio Plex Data Pro Software User Guide Opening a Project Acte Projects inactive Projects Project Chener Team Supenvipor Description Modified Arcoerped Sample Prospect Broad Laboratories Not Required Net Required Data created ung Eso Per PrcB 17 2011 523 PM 8 27 2011 431 PA Sample Precec 2 BioRad Laboratories Not Required Mot Required Samples were run by an anony E 17 2011 523 PM B 17 2001 824 PAL Desctivate J 2 Inthe Open Project dialog box click on the project name
59. the top of this view It contains a list of all analytes in the selected samples Each row in the table contains the analyte name and descriptive statistics for the population of samples for this analyte ANALYTE TABLE TOOLBAR COMMANDS Cota In the main toolbar you can select one of the following measurements for the analyte table If all toolbar commands are not visible click the triangle on the right side of the toolbar to view hidden commands Fluorescent intensity Fl a relative measurement of fluorescent intensity based on the median a robust statistical measure Observed concentration observed concentration of the analyte calculated from its fluorescent intensity and the standard curve Observed concentration within range observed concentration that falls within the range of standard concentrations given a specified recovery range Ratio normalized ratio fold expression or gene expression value calculated in Bio Plex Manager software Analyte P Value P Value Method Fold Change Avg Std Dev CV f Valid Samples Total Samples Outliers Groups Nor e 0 A 2 Hu Follistatin 0 048 one way ANOVA 0 592 27019 10475 38 77 37 37 0 4 3 Hu G CSF 0 244 one way ANOVA 166 510 18 700 90 137 38 37 37 0 4 4 Hu HGF 2 25e 05 one way ANOVA 107 36282 144 27 39 76 37 37 0 4 5 Hu Leptin 0 303 one way ANOVA 0 709 4017 72 2789 96 6944 37 37 0 J 6 Hu Osteopontin 0 028 one way ANOVA 0 620 10540 93 3967 89 37 64 37 37 0
60. trols to filter your data indicate how data are compared and specify which statistical test to apply for analysis Compare by specifies the variable used to group the data for presentation in the charts and to apply statistical tests The list displays the columns in the Results table Control specifies the control group for your analysis The list displays the groups for the variable selected in the Compare by drop down list Statistical Test specifies the statistical test applied e lf the data are normally distributed choose t test one way ANOVA e Ifthe data are not normally distributed or you are unsure how the data are distributed choose Mann Whitney Kruskall Wallis This is the default Filter by includes or excludes groups from your analysis By default it displays the same column heading selected in the Compare field You can change this setting Clear All Analysis Filters selects and includes all items that were cleared with any filter setting Note This feature removes any filters that were set in any table in the product Any filters you set in any of the other views will be cleared Select All selects all items in the list therefore including them in the analysis Clear All clears all items in the list therefore excluding them from the analysis o1 Bio Plex Data Pro Software User Guide Interpreting P Value Statistics Analyte Table The analyte table is the table at
61. ts When you import data into a merged project the new data are merged automatically fica te Bly Bee i41 El Condition Matrix v Gender Data Set Manage Data Sets E Edit Samples olumns View Results View Heat Map Perform Analysis Create Charts Breast Cancer Breast Cancer Breast Cancer Breast Cancer Breast Cancer Breast Cancer Breast Cancer Breast Cancer Colon Cancer Colon Cancer Colon Cancer Colon Cancer Colon Cancer Colon Cancer Colon Cancer Colon Cancer Lung Cancer Lung Cancer Lung Cancer Lung Cancer Lung Cancer Lung Cancer Lung Cancer Lung Cancer Plasma Plasma Plasma Plasma Plasma Plasma Plasma Plasma Plasma Plasma Plasma Plasma Plasma Plasma Plasma Plasma Plasma Plasma Plasma Plasma Plasma Plasma Plasma Plasma Female Female Female Female Female Female Female Female Male Male Male Male Female Female Female Female Male Male Male Male Female Female Female Female Normal vs Normal vs Normal vs Normal vs Normal vs Normal vs Normal vs Normal vs Normal vs Normal vs Normal vs Normal vs Normal vs Normal vs Normal vs Normal vs Normal vs Normal vs Normal vs Normal vs Normal vs Normal vs Normal vs Normal vs Disease p Disease E Disease E Disease E Disease E Disease E Disease E Disease E Disease E Disease E Disease E Disease E Disease E Disease E Disease E
62. wn list of the types of data that can appear as the sample label to the immediate left of the heat map Intensity Type opens a drop down list of the types of data that can be displayed Data Transform opens a drop down list of the ways the data can be calculated Color Gradient lists colors and color combinations you can select to change the appearance of the heat map TOOLBAR COMMANDS Intensity Type Color Gradient and Frame Cells controls also appear in the toolbar In addition Width and Height controls make it easy to resize the heat map Intensity Type Obs Conc Color Gradient Greyscale Width Height Frame Cells 45 Bio Plex Data Pro Software User Guide Heat Map Result View HEAT MAP SETTINGS The pane to the left of the heat map controls the labeling and sorting of the samples ie Ede Dita View Chat Opies Help A cede Bf OS BIE ue Use the controls to label the heat map and to sort samples LABELING THE HEAT MAP The Sample Label Field drop down list displays the columns in the Results table You can label the heat map with the values of one of these columns To specify a sample label in the heat map e Click Sample Label Field drop down list and select one of the choices in the list Sample Label Field Condition Gender Matrix Sample Label Description Data 5et If lt none gt is selected no sample label appears next to the heat map 46 Adding a Column GR

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