Vector Xpression Tutorial: Latin Squares/Dye Swap Analysis
Contents
1. Figure 18 Chip Design Window with Lover Panel Populated To see the new header you may need to resize column widths Place the cursor on the column divider and drag to the left or right See also that the lower panel of the Chip Design window is now populated with information 4 Click OK to finish the chip design 16 Tutorial Latin SquaresiDye Swap Analysis Vector Xpression retums you to the main Import dialog box Figure 19 sie remm tiv wena sui EET io ems fem o mes Cem EE m 3 tan DE mma Figure 19 Inport Dialog Box 5 Click the Import button circled in Figure 19 to finish loading the data into the database 6 In the Import message that asks you whether you want to remember this file format not shown click No The Finalize Import dialog box that opens Figure 20 displays a summary of the fie features for the database and allows you to change them if necessary Now you will provide new names for fle features Erami Nane Troe Name Tiasa Nome Chanel Channel 2 Channel 1 Channel 2 Channel Chanel 2 cin et 16186 Vm aono ikon dame Vu ik Spote Genee Chip Cmm Figure 20 Finale Inport Dialog Bas Label the Microarray 7 Tutorial Latin SquaresiDye Swap Analysis 7 Under Fie Namo cick in the clicontalinglatinsquare2. Figure 40 Ran Project Viewer ati Squares Gene and Gene Interaction Effects The new spreadsheet tab columns described as follows Column 1 Gene Names Gene names are represented in this fle as numbers Column 2 Gene Effect individual gene main effects in the Latin Squares Design Columns 3 amp 4 Microarray 1 and 2 Microarray and gene interaction effects The sum of these two values for any gene wil always equal zero because of assumptions of the Latin Square model Columns 5 amp 6 Liver and Muscle Tissue and gene interaction effects The sum of these two values for any gene vil also always equal zero for the same assumption 35 Tutorial Latin SquaresiDye Swap Analysis Inthe Text Pane to the left of the spreadsheet click on the next to the Latin Squares Analysis folder to expand it see Figure 41 mm EE Fin Tae 288 a u aaay Baa pee Teo 27 Moog 2 Mask dre of od idle N ent Figure 41 Run Project Viewer Expanded Latin Squares Analysis Folder Result The folder displays the names of the Analyzed Runs as well as all of the values for additional main effects except Gene from the Latin Squares model in the Summary folder circled in Figure 41 Again the sum of these values will be zero 36 Tutorial Latin Squares Dye
3. cr TEES De B Sex Sul a E E Ei 75 sam T T ns ms E S T5 5s sm ri Sx m sa sa E 78 s cur mss s a r1 E 12 wow r1 Ls as z 2 82 P Figure 29 Vector Xpression Rav Data Viewer The Vector Xpression Viewer consists of a menu bar a tool ar a Text Pane that displays the raw data and a Spot List spreadsheet that contains imported data for all spots on the chip Because this Viewer displays Raw Data this screen willbe called the Raw Data Viewer from here forward 2 As mentioned in the introduction you must take the antlog of the data so that itis in the correct format for the Latin Squares analysis tool To do this select Calculations gt Log transform on the menu bar The Log Anti Log Transform dialog box displays Figure 30 mc cl Process both chemels Cio Amie maua New colunn header Logana Figure 30 Log nti Log Transform Dialog Box 3 In the Log Anti Log Transform dialog box click the Anti Log radio button and select Natural in the Base list Make sure the Process both channels check box is selected Click OK to finish 25 Latin SquaresiDye Swap Analysis The spreadsheet now displays in the Raw Data Viewer containing two additional columns entited Log transform one column for each dye see Figure 31 EENET 1 Figure 31 Raw Da
4. me oar osema Gave xam we emo 2 Figure 15 Dialog Box Click Chip Design to Build a Chip Design 2 Inthe Chip Design dialog box Figure 16 make sure that the Current file radio button is clicked and click Chip D atn rape 1 ped oe C Aretha Figure 16 Chip Design Dialog Bax 14 Tutorial Latin SquaresiDye Swap Analysis The Chip Design window Figure 17 displays showing the source fie The lower panel is initially empty but wil be populated once the chip design is built momo arare Caner Conlon Earl Figure 17 Chip Design Window 18 Tutorial Latin SquaresiDye Swap Analysis 3 Right click on the Gene ID column header circled in Figura 17 and click Gene Names on the shortcut menu The term Gene Names is added ta the column header after the term Gene ID Figure 18 and the entire new header now displays in red inia T ai E E Ex ene _ i ane Gono tas 1 OU m emer covet ames Dome E cma DNI Ixxs comes BEEN im Tee es mw Goa
5. Figure 43 Run Projet Viewer Expanded Microarray amp Gone P Talue Folder Result After the calculation completes an additional subfolder Microarray amp Gene P value is added to the Latin Squares Analysis folder in the Text Pane circled in Figure 43 The first line of text in this subfolder displays the observed F value the second line shows the estimate of the P value or the probability that the interaction term should be removed from the model Clearly in this case the P value is highly significant The Randomized F summary exhibits a 95 confidence interval for the F statistics as well as the smallest and largest calculated F statistics Your values may look slightly different than those displayed in Figure 43 and those reported in the Kerr paper They report the lowest value as 81 and largest as 1 27 The explanation for this is that these values are based on randomization in this example alone there are an approximate 107 that is a 1 with almost 17 000 zeros after unique possible randomizations of Which Vector Xpression calculates only 20 000 So there will be a slight difference between any two randomizations Note that the conclusion should always be the same however Significance may suggest problem with the cross hybridization and indicate that you look at the scanned images 39 Tutorial Latin SquaresiDye Swap Analysis Calculating Differential Tissue Expression Overview In this
6. 12 In the Import message dialog box not shown cick Yes to close the Import dialog box 19 Tutorial Latin SquaresiDye Swap Analysis You are returned to the Vector Xpression Database Explorer Figure 25 With the Raw Data table selected you can see the first microarray w Database Todi arso oe gt EA Ran T che Wane I Dye Sw Ready ET A Figure 25 Vector Xpression Database Explorer Result You have successfully built the chip design and associated it with the raw data In addition you have saved the chip and raw data for the first microarray to the Vector Xpression database Importing Data From the Second Microarray Overview Now you will import data from the second microarray of using the method you already used although this time specifying different values Because the import process for the second microarray is the same as the import process for the first microarray with slight changes the applicable steps for the second microarray are summarized in the following table Refer to the sections beginning with Importing Data From the First Microarray on page 6 through page 20 for the related figures and details Action Step Dialog Box Action Next stop if automated 1 Vector Xpression Database Select Tools gt Import Expression Explorer Data 2 Select Expression Data Navigate to the directory where
7. Calculating the ANOVA Table 37 Estimating the Significance of Microarray and Gene Variable 38 Calculating Differential Tissue Expression 40 Calculating Significance of Differential Tissue Expression 41 Reviewing Graphics From the Latin Squares Dye Swap Analysis 43 Histogram of Gene Effects 44 Histogram of Differential Tissue Expressions 45 Scatter Plot of Predicted Expression versus Model Residuals 47 Scatter Plot of Predicted Expression versus Absolute Model Residuals 48 Normal Quartile Plot of Model Residuals 50 Plot of Bootstrap Intervals for Differential Tissue Expression 51 Important Please Read STOP This tutorial assumes that you are familiar with the standard Windows user interface and basic Windows techniques such as maximizing windows selecting objects Zooming in and out on objects switching between panes in a viewer window etc For more information about basic Windows operations see Chapter 3 of the Vector Xpression User s Manual Itis also assumed that you are somewhat familiar with microarray techniques and expression data files generated from microarray data analysis For more information see the Gene Expression Overview section in Chapter 4 of the Vector Xpression User s Manual Before beginning this tutorial complete the following actions if you have not yet performed them 1 Install Vector Xpression For more information about installing Vector Xpression see the Vector Xpression Installation
8. Expression experiments Import Scheme A template that identifies the position of the various types of dala in the expression data file being imported This template provides a map for the expression data in the file so it is correctly parsed and imported into the appropriate fields in the Vector Xpression Database Import Tool A tool that creates the necessary import scheme for an expression data file and then uses that scheme to import the expression data file into Vector Xpression Raw Data Source Files Data processed from an image of a microarray The processed image file contains information about measurements such as Signal Background Signal to Noise ratio etc for each individual spot One Raw Data object can contain data for one or two channels The two channel data is usually collected with the and Cy5 dyes Tutorial Latin Squares Dye Swap Analysis Opening Vector Xpression Database Explorer Overview The Vector Xpression Database is a collection of expression objects information and data organized for easy retrieval and management in the Vector Xpression Database Explorer Similar in functionality to the Windows 95 98 NT Explorer interface the Vector Xpression Database Explorer supports intuitive browsing of databases drag and drop operations and other functions typical of window based database management You will use the Database Explorer to import the data into a new empty database t
9. Guide that can be accessed from the InforMax Inc Web site htipjwww informaxinc com vntilvntisuite Installaion VXpression 100302 p d Be sure that you are not running a demo version of the software The fully licensed version does not limit the number of genes 2 In Windows click Start gt Settings gt Control Panel Click Display In the Display Properties dialog box make sure that Colors is set to a minimum of High Color 16 bit Lower settings will cause scatter plats shown in this tutorial to display black r r If you have completed Steps 1 and 2 listed above proceed with this tutorial Tutorial Suit Other tutorials are available from InforMax to teach you how to use the Vector Xpression Module Analysis of Diauxic Shift Microarray Data Statistical Significance Testing and Analysis of Differentially Expressed Genes Intensity and Spatially Dependent Normalization Tutorial Latin SquaresiDye Swap Analysis Introduction Purpose The purpose of this tutorial is to teach you how to import expression data process the data and perform a Latin Squares Dye Swap Analysis of the data This tutorial involves four major steps 1 Import raw data derived from two microarrays of dye swapped data that has been numerically transformed You will load the data from the same file twice once for each microarray This will produce two Raw Data objects in the dat
10. open the Run Project Viewer see Figure 38 sisi EC iue n d Figure 38 Run Project Viewer Spreadsheet Like the Raw Data Viewer the Run Project Viewer consists of a menu bar a tool bar a Text Pane that displays information about the Expression Runs opened in the Runs Project and a Spreadsheet that contains expression values for the data Result Now that the data is loaded in Run Project Viewer you can do the analysis 33 Tutorial Latin Squares Dye Swap Analysis Estimating the ANOVA Model Variables Overview The Latin Squares Design Model is applied to the dye swapped data and all of the model components are estimated This first step allows you to configure the Expression Runs and specify how they are positioned for the Latin Squares Analysis Action 1 From the Run Project Viewer select Tools gt Latin Squares gt Step 1 Estimate ANOVA Model Variables The Dye Swap Expression Runs dialog box opens Figure 38 EDETCIDTITCITTEENENNNS S x Fistexpeimert Chan 2 Dane TIOE 3 Meca 1 Dye 2 Nue zi 3 Dve 1 Nus 3 Dye Live Figure 39 Dye Swap Expression Runs Dialog Box n this analysis you fit the following ANOVA model AG D Vk Glg VG kg elika where i 1 2 indexes the microarray
11. s to Import Dialog Box 2 Inthe Select Expression Data File s to Import dialog box perform these steps a In the Look in list navigate to the directory where you saved the source data fle that you downloaded see page 2 for the location Do not cick Open yet Because the fie does not have a ixt extension click All files in the Files of type list In the box below the Look in list cick the latinsquare file This enters the fle name in the File Name textbox d Inthe Delimiter area cick Whitespace because the file is a space delimited file Click Open The software searches for an import scheme compatible with your data Because such an import scheme does nat exist the following message appears Figure 5 2 ener oma Wy banat chan rh Fama sem owe Figure 5 Inport Message 3 Click Yes to confirm building an import scheme for this fie type Tutorial Latin SquaresiDye Swap Analysis 4 in the Import dialog box that opens not shown click the Raw Data radio button and click OK The main Import dialog box displays Figure seme fem o Toner wen same Fans zan isi A Figure 6 Inport Diog Bax Data from Source Fl Result You have successfully loaded the raw data for the frst microarray in the Import
12. they are not entered by defaut cor postion o heat ard da Press Guess button to autodetect ace aowa HT E ET 00 Stop on w 3 3 gr c Figure amp Header and Data Dialog Box 3 Click Next to continue 4 In the Coordinates dialog box Figure 9 you need to identify that the Gene IDs are presentin Column 5 of the source To do this select 1 in the Gene IDs in column text box and change it to 5 Entor Gone ID or Spot Cosrainatos M einen 8 Figure 9 Coordinates Dialog Box 5 Click Next to continue Tutorial Latin SquaresiDye Swap Analysis 6 Inthe Channels dialog box Figure 10 cick Two Channels Two color experiment Chereis Enter number of channels chanel costis eere cenian Oro Chanrel ncs edor am LIRE E sba 7 Bets Heb Figure 10 Channels Dialog Bax 7 Glick Next to continue 8 In the Data Enter Data names dialog box Figure 11 select the Signal check box because only signals are given in this file Data Enter Data names ens Doe Heb Figure 11 Data Enter Data names Dialog Box 9 Glick Next to continue n Tutorial Latin SquaresiDye Swap Analysis 10 The Data Enter column number for Signal of Channel 1 dialog box which correctly guesses the column
13. 59 mforMax Vector Xpression Tutorial Latin Squares Dye Swap Analysis November 21 2002 Copyright Information Copyright Notice 2002 InforMax Inc All rights reserved The unauthorized disclosure copying or altering of this document whether in hardcopy personal computer diskette format through any electronic medium or otherwise is strictly prohibited While every effort has been made to ensure the accuracy of this publication InforMax Inc assumes no liability for error or omission from use of the information contained herein Vector Xpression is a registered trademark of InforMax Inc in the United States and other countries Logos of InforMax Inc are also trademarks registered in the United States and may be registered in other countries Other product and brand names are trademarks of their respective owners Table of Contents Introduction 1 Opening Vector Xpression Database Explorer 4 Importing the Raw Data 6 Importing Data From the First Microarray 6 Viewing and Defining Raw Data From the Source File 8 Building the Chip Design 13 Importing Data From the Second Microarray 20 Preprocessing the Data 24 Calculating the Antilog for Microarray 1 24 Creating Expression Runs from the Raw Data for Microarray 1 26 Calculating the Antilog for Microarray 2 29 Creating Expression Runs from the Raw Data for Microarray 2 30 Performing the Latin Squares Dye Swap Analysis 32 Estimating the ANOVA Model Variables 34
14. Swap Analysis Calculating the ANOVA Table Overview After fitting the Latin Squares Model or any ANOVA type model you can calculate an ANOVA table The table displays information about every term in the model including the sums of squares degrees of freedom in estimation mean sum of squares and F stalistic due to the modeling components Each of these bits of information provides insights into the appropriateness of the terms in the model Note that we do not calculate P values for the F statistics in the ANOVA table because we do not make the parametric assumptions these assumptions are rarely true in practice We do use a randomization technique in the next subsection on page 38 Estimating Significance of Microarray and Gene Variable however to estimate the P value for just the microarray and gene interaction term Action From the Run Project Viewer select Tools gt Latin Squares gt Step 2 Calculate ANOVA Table and ensure that the third right most tab in the Spreadsheet Pane is selected so you can view the ANOVA table Figure 42 a Ciara pereat Gita ere Ano attend Fore Menara Dra Hue 22 Menara Sammy of Mada Variaties i i Hl me Figure 42 Run Project Fewer ANOVA Table Tutorial Latin SquaresiDye Swap Analysis Result The ANOVA table calculated in this step summarizes the contributions of each componen
15. a file and then uses that scheme to import the file into Vector Xpression You will create an import scheme and Use that scheme to import data from the same source text file twice each time specifying values from a different microarray This produces two Raw Data objects in the Vector Xpression database Each object has both dye channels of information Instead of the actual expression values the data is represented as the natural log of the observed expression values Because the data is in this format you must import it as Raw Data You can then calculate the antilog to get back to the untransformed data for Vector Xpression to do the analysis Importing Data From the First Microarray Overview You will now import data from the first microarray Action 1 In the Vector Xpression Database Explorer Figure 3 select Tools gt Import Expression Data Tee Eat bees Took Mm e gt ER Ea a ele Desarston chi Nene I Ready Seexed RawDxa _ RawDxa Figure 1 Vector Xpression Database Explarer Tools Menu Tutorial Latin SquaresiDye Swap Analysis The Select Expression Data File s to Import dialog box displays Figure 4 lect preston Daba tev E six Lomi ie ec Decrees Py Computer fy Nelourk Heres 4 Figure 4 Select Expression Data File
16. abase each with both dye channels of information 2 Preprocess the data by calculating the antilog and creating Expression Runs for each microarray dye combination 3 Perform a complete Latin Squares Dye Swap analysis of the data 4 Interpret the graphics that are associated with the Latin Squares Dye Swap analysis if you follow the directions as outlined your tutorial will produce the same answers except where otherwise noted as those published in the paper by Kerr Martin and Churchill that outlined this analysis Information on this paper is presented in the next section Tutorial Latin SquaresiDye Swap Analysis Background Reference This tutorial is based on the complete Latin Squares Dye Swap model as presented by Kerr M K Martin and Churchill G A 2000 Analysis of variance for gene expression microarray data Journal of Computational Biology 7 819 837 To download a preprint of this paper in pdf format click the following link http www jax orgiresearch churchilliresearch expression kerr synteni pdf Note This paper is viewed in Adobe Acrobat Reader which you can download at no charge from this Adobe link http www_adobe com products acrobatireadstep htm Note Although the Kerr paper presents two different analysis approaches for the expression data this tutorial covers only the Latin Square Analysis Because this tutorial uses the same data analyzed in the publication you can down
17. an average 99 bootstrap width of 1 61 The spreadsheet displays a result of about 1 66 To obtain this result manually calculate the difference between the 95 LCI and UCI values and take the average of those values Your figure might be slightly different Again the difference is due to the randomness of the bootstrap method As suggested by the Kerr paper you can estimate the approximate significant fold change at 99 confidence by calculating c Using this formula the paper reports a result of 2 24 as the significant fold change Using the figures in the spreadsheet shown in Figure 46 the result equals 2 30 To obtain this result perform the same manual calculation for the 99 LCI and UCI values You have now run the entire Latin Squares Dye Swap analysis on the data The next section summarizes the results Eton 1982 The Jacknife the Bootstrap and Other Resamolng Pians NSF Regional Conference Series n Apple Matis 38 Society for amp Mathematics 42 Tutorial Latin Squares Dye Swap Analysis Reviewing Graphics From the Latin Squares Dye Swap Analysis Overview Now that you have completed the Latin Squares Dye Swap analysis you can review some of the results They display in graphics panels when the associated options are selected in the Run Project Viewer The title bar in each graphic describes the graphic Action From the Run Project Viewer F
18. dat Now cick the text ecit icon T io edit the fe rame Enter the name Microarray 1in ho cel and press e ENTER key see e ce in Figure 21 Simia change al sical Po cunkewm onte so hel he omation rea as shown in Fgre 21 nore ba Base Channel cours tot arr File Mame Spes Gener Chip m Gree Figure 21 Finalize Inport Dialog Box Name Chip Design B Now you need to name the chip design and assign it to the Microarray 1 data Click the empty space under Chip and click the button Inthe Create New Chip dialog box Figure 22 enter Dye Swap in the Name text box In the Description text box type Data from Kerr Martin and Churchill paper Click Create to create a new chip type name sente Hera pom Physical Chips Ne subset Nee Deseo Bacon Fen Mann and Chai pape 21 Figure 22 Create New Chip Dialog Bax 18 Tutorial Latin SquaresiDye Swap Analysis The Finalize Import dialog box now displays as shown in Figure 23 ELIIOm EAENAAR ES ox Sa EIN D em c uu Dees 1 1 E Figure 23 Finalize Inport Dialog Box for Microarray 1 10 Click Save to DB 11 In the Select subset dialog box Figure 24 specify the subset where the data is to be stored For Simplicity put itin the root directory which is already selected Click OK x 31 Now Subset Figure 24 Select Subset Dialog Bax
19. dialog box Viewing and Defining Raw Data From the Source File Overview Because Vector Xpression cannot find an import scheme for this data type the main Import dialog box has opened This dialog box gives you a chance to review the raw data The Source File Pane of the Import dialog box displays data from the loaded source file This source file will remain open for your referral as you step through each window using the Import Wizard Tutorial Latin Squares Dye Swap Analysis Ration 1 In the Import dialog box Figure 7 click Wizard to build an import scheme Sram c E 1 nes siso 8 umm cma avem 9 Figure 7 Import Dialog Box Click W rd to an Import Scheme The Import and Chip design buttons in this dialog box are for use later in the import process The Test and Cancel this file buttons are not used in this tutorial Tutorial Latin SquaresiDye Swap Analysis 2 Once the Header and Data dialog box opens Figure 8 examine the source fie back in the Import dialog Figure 7 noting the location of the Data start and end rows Here the header is row 0 meaning that there is no header row Then enter the appropriate rows in the Header and Data tex boxes as shown in Figure 8 if
20. e antilog for the second microarray Because the antilog calculation for microarray 2 is the same as the one for microarray 1 the steps are summarized in the following table Action Note Steps 2 and 3 reiterate Steps 2 and 3 on page 25 with slight changes Step Dialog Box Action Next step if automated 1 Vector Xpression Cick the Raw Data table and Database Explorer double click on Microarray 2 z Raw Data Viewer Cick Calculations gt Log transform E TagiAnliLog Transform Cick the Anti Log radio bution and Cick OK dialog box select Natural in the Base lst Make sure that the Process both channels check box is selected The Raw Data Viewer spreadsheet now displays two additional columns entitled Signal one for each dye Figure 35 a Geseit Det entm Ea rre panes v a Fanm EEE Figure 35 Vector Xpression Raw Data Viewer with Additonal Columns 29 Tutorial Latin SquaresiDye Swap Analysis Result Now that you have calculated the antilog of the data for the second microarray you can create the Expression Runs from the raw data Creating Expression Runs from the Raw Data for Microarray 2 Overview You wil now create two Expression Runs for the second microarray dye combination Because the process for Microarray 2 is he same as the process for Microarray 1 with slig
21. ect Tools gt Latin Squares gt Scatter Plot of Predicted Expression versus Absolute Model Residuals Figure 52 displays a E DG ass eg dan ig Figure 32 Scatter Plo of Predicted Expression versus Absolute Model Residuals The new Scatter Plot suggested for checking the modeling assumptions displays in the lower panel of Figure 52 The two plots are related because the Y axis of the lower isthe absolute value of the top plot Your Scatter Plots should match those in Figure 52 These Scatter Plots check for problems with homoscedasicily defined as having equal statistical variances across model covariate values which is an assumption in most ANOVA models Now fit a Lowess line to the lower Scatter Plot A Lowess line is a smooth non parametric line representation of the data in the Scatter Plot that is robust to outers Ifthe line shows a large variance from being straight then there are problems with homoscedasticity in that the model shows heteroscedasticy 4B Tutorial Latin SquaresiDye Swap Analysis 2 Lowess line to this plot right cick in the plot and select Fit Lowess The Fit Lowess dialog box opens Figure 53 E Smoother sper d 31 Coa C ee Figure 53 Fit Lowess Dialog Bax 3 In the Fit Lowess dialog box enter 35 for the smoothing span and click OK The plot displays a hor
22. en associated options are selected under the Tools menu option If you follow the directions as presented your tutorial will produce the same answers except where noted otherwise as those published in the paper by Kerr Martin and Churchill To run the analysis you will use the Run Project Viewer The Run Project Viewer displays on a spreadsheet the exact numerical data imported in all fields associated with a given file format In this viewer you can do the following Generate histograms and scatter plots for each Expression Run for comparisons across selected genes View and analyze multiple Expression Runs from the same chip Find and merge Expression Runs normalize them and convert absolute data to ratio Perform Latin Squares calculations and t tests on Expression Runs Save Expression Runs as Run Projects lt reports and export user selected numerical values to Microsoft Excel 32 Tutorial Latin SquaresiDye Swap Analysis Action 4 Inthe Vector Xpression Database Explorer select all four Expression Run objects by pressing the CTRL key and clicking on each object Figure 37 Rer Dh dee Dine Th sp ve s de X C a ree n r3 gite fasst us Tue uon Tue E I Figure 37 Vector Ypresson Database Explorer All Expression Runs Selected 2 Open the shortcut menu by right clicking on the associated objects and selecting Open to
23. erential issue expression at that level The resulting plot of Bootstrap intervals displays as shown in Figure 57 The blue ine the middie curve is the observed differential expression ordered The traces on each side of the blue ine are the bootstrapped confidence intervals This is the same plot as Figure 4 in the Kerr paper 51 Tutorial Latin Squares Dye Swap Analysis Figure 37 Plot of Bootstrap Intervals Result You have reviewed the Latin Squares Dye Swap analysis results in the following graphical forms Histogram of gene effects Histogram of differential issue expressions Scatter lot of predicted expression versus model residuals Scatter plot of predicted expression versus absolute model residuals Normal quartile of model residuals Plot of bootstrap intervals for differential tissue expression Additionally you confirmed your results against those reached by Kerr Martin and Churchill in the paper cited in the introduction to this tutorial 52
24. ht variations the steps are summarized n the folowing table Repeat the steps for both dye channels Action Step Dialog Box Action Next stop it automated 7 Raw Data Viewer Selec Tools gt Save Columnas Expression Run 2 Save Golumn as Expression For Column select Dye Cick OK z Run transform H Accept Column data type as E Absolute For Target accept Muscle a janan Change he Name to Microaray2 Cick Save Dye 1 Muscle a Vector Xpression Viewer None Tikk message 6 Raw Data Viewer Selec Tools gt Save Camm as Expression Run 7 Save Column as Expression Column select Dye 2Log Gick OK Run dialog box transform Accept Column data type as E Absolute For Target select Liver B e Sapes Ran As Change Namo to Microaray 2 Gick Save dialog box Dye 2 Liver a Vector Xpression Viewer None Cic message 10 In the Raw Data Viewer cick the Go to Database Database Explorer 30 button to retum to the Vector Xpression Tutorial Latin SquaresiDye Swap Analysis 1 In the Vector Xpression Database Explorer select the Expression Runs table in the sin the upper let comer circled in Figure 36 nt x amaw eene LEE kan esca mes tate pe Figure 36 Vector Xpression Database Explorer Expression Runs Inthe Database Objects Pane you can view the E
25. icroarray 1 Overview You will now calculate the antilog for the first microarray dye combination This is necessary because the imported data is represented as the natural log of the observed expression values for example In 3274 8 09 The antilog of each value is the opposite of the logarithm for example 655 3274 See the signal for Dye2 in Figure 31 for this example To do this you will use the Vector Xpression Raw Data Viewer The Raw Data Viewer is the user interface in Vector Xpression designed lo display raw data It allows you to normalize and consolidate raw data into Expression Runs assess the quality and edit the data when necessary You can review the raw data manually or optimize the results by filtering Finally you can save the conversion of raw data into Expression Runs Action 1 In the Vector Xpression Database Explorer in the Database Objects Pane double click on Microarray 1 Figure 28 Hap ae le err Ran Data ive Desarston hi Nene I Cress Ere Roady eecediRawbaa Bhanba 24 Tutorial Latin SquaresiDye Swap Analysis If Microarray 1 is not visible make sure Raw Data is selected in the list in the upper left corner circled in Figure 28 f his screen sill does not display press the FS key to refresh the view The Vector Xpression Viewer opens displaying the raw data as shown in Figure 29
26. igure 47 you wil select Tools gt Latin Squares gt to display a number of plots for the Latin Squares analysis Bana a FH X Gael ee Figure 47 Run Project Viewer Additonal Spreadsheet Columns 43 Tutorial Latin SquaresiDye Swap Analysis Histogram of Gene Effects From the Run Project Viewer select Tools gt Latin Squares gt Histogram of Gene Effects to view a histogram of gene effects displays Figure 48 Figure 48 Histogram of Gene Effects This plot reveals basic information in the average log expression values of the ger is across all other factors dye tissue and microarrays This is the same plot as Figure 1 in the Kerr paper Tutorial Latin SquaresiDye Swap Analysis Histogram of Differential Tissue Expressions 1 From the Run Project Viewer select Tools gt Latin Squares gt Histogram of Differential Tissue Expressions Figure 49 displays laaa Ss leew Bee PLE eee ris Freien Figure 49 Histogram of Diferential Tissue Expressions The new histogram in the lower panel in Figure 49 displays differential tissue expression values in other words the Differential Tissue column from the spreadsheet of gene effects It is the same plot figure 1 in the paper In the paper opened from the Web site listed on page 2 this plot should be labeled 1c n
27. izontal ited Lowess line with 35 smoothing span Figure 54 Because itis almost straight the same result obtained in the Kerr paper the authors conclude that there is no problem wn assuming homoscedastci rar aer a 88 RR SI 42 d 2 033 a Yr eres x Figure 54 Scatter Pos of Predicted Espresion versus Absolute Model Residuals with Lowess Line 49 Tutorial Latin Squares Dye Swap Analysis Normal Quartile Plot of Model Residuals From the Run Project Viewer select Tools gt Latin Squares gt Normal Quartile Plot of Model Residuals Figure 55 displays eo De eee IE TE Sekang Figure 55 Normal Quartile Pot of Model Residuals This plot checks if the residuals in other words the parts of the data that cannot be explained by the model are normally distributed The straight line depicts what perfect normally distributed data would look like Data that varies from the straight line depicts what the observed data looks like You are looking for large deviations from the straight line Small differences can be attributed to random fluctuations and you can assume that the residuals are normally distributed Figure 55 plots the model residuals against the straight black line A small pull away from the line at the tails suggests that the distribution of residuals has a slightly heavier tai
28. j 1 2 indexes the dyes k 1 2 indexes the tissues and Element Definition Natural log of the observed gene expression value n Overall average expression value Effect of the i microarray Di Effect of the dye Effect of the k tissue Gig Effect of the g gene AG ig Interaction term in other words the additional effect of the i microarray and g gene Tutorial Latin SquaresiDye Swap Analysis Element Definition Vera Interaction term other words the additional effect of the tissue and the g gene Error term of the model In this analysis we will only assume that this random value has mean equal to zero constant variance and is independently and identically distributed 2 Inthe Dye Swap Expression Runs dialog box the current Expression Runs are liste in the Experiment lists Verify that the default selections in the dialog box appear exactly as shown in Figure 39 making selection changes in the lists as necessary Click OK 3 Inthe Run Project Viewer click the second right most tab at the bottom of the Spreadsheet Pane if not selected by default Figure 40 This spreadsheet called Latin Squares Gene and Gene Interaction Effects see ttle bar in Figure 40 displays the values of the gene main effects and the two interactions the microarray and gene interaction as well as the tissue and gene interaction
29. l lock like Figure 14 if you have identifed your information correcty I ox dad e Y IH Row SignaliChannel 1 SignaiChannel 2 Gene iD Sa feee acest Em 3 smew 5 RICO mme atta ems 2 auam seme imam os Spa Figure 14 Import Dialog Bax Assigned Data Names Result You have successfully viewed and defined the raw data source fle Building the Chip Design Overview To continue the import process you must build the chip design and associate it with the raw data Chip designs link the spot locations of Raw Data objects to their gene names There are two ways to configure a chip design through the Chip Design Wizard or in the Chip Design window You have already defined data using the Import Wizard which functions similarly to the Chip Design Wizard To teach you an alternative technique in this section you will configure the chip design directly through the Chip Design window Note that you can also define the data for the import scheme using either method 13 Tutorial Latin SquaresiDye Swap Analysis Action 1 To start building the chip design click the Chip design button circled in the main Import dialog box Figure 15 LI m EI 5 IH Sister sinahanne 2 7
30. l than the normal distribution Note however that normality of the residuals is not a necessary assumption because nonparametric statistics calculated when needed This plot is similar to Figure 2a in the Kerr paper the difference being what the x axis represents 50 Tutorial Latin Squares Dye Swap Analysis Plot of Bootstrap Intervals for Differential Tissue Expression 4 From the Run Project Viewer select Tools gt Latin Squares gt Plot of Bootstrap Intervals for Differential Tissue Expression The Differential Tissue Expression Plot dialog box displays as shown in Figure 5 EZ Select confcence irtervals to chew onthe plot Boosten CI 1295 Bonttap 288 Boottap CI Figure 56 Diferential Tissue Expression Plot Dialog Bax 2 in the Differential Tissue Expression Piot dialog box you can specify the bootstrap confidence intervals you want to view In this case be sure all three of the possible intervals are selected Click OK The diferential tissue expression and the calculated bootstrap confidence intervals are then plotted The colors match for the upper and lower limits of the confidence intervals The further apart the intervals the wider the confidence interval This means that a 99 confidence interval wil be wider than a 95 interval and a 95 interval will be wider than 90 interval Confidence intervals that do not include show genes with significant diff
31. less you need to correct Click Next any data using the Back button 18 Additional Spot Data None Glick Next 34 Fiag Information dialog None Click Next 15 Final Message dialog box None Click Finish 36 Main Import dialog box Check that data is correct Click the Chip design button 17 Chip Design dialog box Make sure that Current file rado Click OK button is clicked 38 Chip Design window Rightclickonihe Gene ID column Click OK and click Gene Names on the shortcut menu 38 Main Import dialog box None Click the Import bution 20 Import message None Click No 21 Tutorial Latin SquaresiDye Swap Analysis Step Dialog Box Action Next stop if automated Zia Fimalzeimpotdilogbox Under File Name cickin thecell containing latinsquares dat Now click the text edit icon 2 to edit the file name Enter Microarray 2in the File Name cell and press the ENTER key Enter Dye 1 for Channel Name Channel 1 Dye 210 Channel Name Channel 2 Musclo for Target Name Channel 1 Liver for Target Name Channel 2 Muscle for Tissue Name Channel 1 Liver for Tissue Name Channel 2 215 Click the down arrow under Chip gt select Dye Swap The Finalize Import dialog box should look like Figure 26 when you ve entered all your data for Microarray 2 LE 4 mnamo Spa Name Tissus Mame Name Spo
32. load the data from the author s Web page Click this link http www jax orgiresearch churchill datasets expression synteni index him Then under the Synteni Arrays section right click on the link titled latinsquare dat In the shortcut menu click the option Save Target As and save this file to a convenient location such as your desktop on your computer In the Download Complete dialog box click Open Open Folder or Close depending on whether you want to access the data now or later IMPORTANT This tutorial covers several major Vector Xpression functions you will perform during this analysis However you should use this tutorial in conjunction with the Vector Xpression User s Manual for clarification of all functionality VECTOR XPRESSION DEFINITIONS Chip Designs When loading raw data required elements that link the spot locations of Raw Data objects to their gene names Expression Database Explorer A component of Vector Xpression used to manage data Expression Run An array of numbers equal in length to the number of Expression Genes that were measured that corresponds to the expression values obtained when a microarray is hybridized with an Expression Sample whose identity abundance is being detected Tutorial Latin SquaresiDye Swap Analysis Expression Viewer A component of Vector Xpression used to analyze and manipulate raw data Expression Runs Run Projects and or
33. o Steps 1 through 4 to reference figures 27 Tutorial Latin SquaresiDye Swap Analysis Stop Dialog Box Action Next step if automated 5 Raw Data Viewer Select Tools gt Save Column as Expression Run Save Column as In the Column ist select Dye 2 Click OK Expression Run dialog box Log transform Ensure the Absolute radio button is clicked Select Muscle in the Target ist 7 Save Expression Run As Change the Name to Microarray Cick Save dialog box 1 Dye 2 Muscle a Vector Xpression Viewer None Click Yes message After completing these steps you are returned to the Raw Data Viewer 9 Switch to the Vector Xpression Database Explorer by clicking the Go to Database TE button in the tool bar 10 In the Database Explorer select the Expression Runs table in the lis in the upper left comer circled in Figure 34 gm EIN T LIEGE ESTE a aen ei 29 EI Figure 34 Vector Xpression Database Explorer Expression Runs In the Database Object Pane you can view the Expression Runs you just created from the raw data Your screen should appear as shown in Figure 34 Result You have created the Expression Runs for Microarray 1 28 Tutorial Latin Squares Dye Swap Analysis Calculating the Antilog for Microarray 2 Overview Now you will calculate th
34. o store the products and objects in this tutorial Action 4 From the Windows Start button select Start gt Programs gt InforMax gt Vector Xpression gt Vector Xpression Database to open Vector Xpression Database Explorer Figure 1 Tle Edt Vow Too feos e SIE ideato an cats m xpi cene Ready E Figure 1 Opening Vector Xpresion Database Explorer Tutorial Latin SquaresiDye Swap Analysis 2 From the Vector Xpression Database Explorer menu bar select Database gt New Empty Database The Select a Location of New Database dialog box displays Figure 2 lect a location of new database alx Savo in Cd oo ee JE presi Fiename Dye Swap Save Save as po Access Fies Fmd Carca A Figure 2 Select a Location of a New Database Dialog Box 3 In the Selecta Location of New Database dialog box type Dye Swap Analysis in the File name text box to name the database and cick Save 4 In the Vector Xpression Database confirmation message click OK Result You have successfully opened Vector Xpression Database Explorer and a new database Tutorial Latin Squares Dye Swap Analysis Importing the Raw Data Overview Vector Xpression provides a tool called Import that creates the necessary import scheme for an expression dat
35. or the Signal value of Channel 1 to be Column 1 displays as shown in Figure 12 Click Next to continue Data Enter column number for Signal of Channel ow Figure 12 Data Enter column number for Sigal of Channel 1 Dialog Box Referring back to Figure 7 on page 9 column 1 is signal data for the first channel Column 2 is signal data for the second channel which you wil identify in the next step 11 Another Data Enter column number for Signal of Channel 2 dialog box which correctly guesses the column for the Signal value of Channel 2 to be Column 2 displays not shown Click Next to continue 12 The Data Check dialog box displays Figure 13 showing a summary of your selections so you can verify that the information is correct If necessary click Back to adjust any incorrect entries Make sure your dialog box looks ike Figure 13 p Tene 2 we Ties Heb Figure 13 Data Check Dialog Box 12 Tutorial Latin SquaresiDye Swap Analysis Click Next to continue and continue clicking Next in the Additional Spot Data and Flag Information dialog boxes These dialog boxes are bypassed because the fie does not contain this information After clicking the last Next button you wil reach the Final Message dialog box Click Finish The Import dialog box now displays the column and row titles you specified in the Import Wizard It wil
36. ot TTo in ie pubiched paper er tne Joumal of Computatona te rap are labled 45 Tutorial Latin SquaresiDye Swap Analysis 2 In Figure 47 on page 43 the bootstrap width is estimated to be 1 66 see the first bullet on page 42 Now divide this figure by 2 Any differential tissue expression larger than 8 1 66 2 or smaller than 83 1 662 represents at 99 confidence significantly up or down regulated genes respectively You can tag these genes on the histogram by selecting and dragging the region on the histogram that is displayed in Figure 50 IX E ECT Thid UT E Prien 8 o pue Figure 50 Histogram of Differential Tissue Expressions Bootstrap Width Divided by 2 From the shortcut menu you opened by right clicking on the selected region select Tag Selected and the folder color of your choice The tagged group subfolder now appears under the Tagged Genes folder in the Text Pane To rename the Tagged Genes folder select Properties from the shortcut menu opened with a right click on the folder Enter Upregulated Genes in the Name text box NOTE You could have also accomplished this by sorting the Latin Square Gene 42 gt and Gene Interaction Effect spreadsheet sorting the Differential Tissue Eroresstn cou and ooking at values above and below of rest 46 Tutorial Latin Squares Dye Swap Analysis Sca
37. ron and Tibshirani the fathers of the bootstrapping method See the end of this section for the reference Action 1 Select Tools gt Latin Squares gt Step 5 Calculate Significance of Differential Tissue Expression Figure 45 step 5 Sionificance of Diferential xl Calculation of cenicence intervals fer diferential expression might take seniican tme dependert your Hardware and nuber ef genes Cauta for AI T286 cence TT Figure 45 Step 5 Significance of Diferential Ex reson Dialog 2 Inthe Step 5 Significance of Differential Expression dialog box select All 1286 genes and click OK This calculation can also be somewhat time consuming Result Afer the Significance of Differential Tissue calculation has finished you are returned to the Latin Square Gene and Gene Interaction Effects spreadsheet 2 tab see Figure 46 Tutorial Latin Squares Dye Swap Analysis re EXERCI E t 5 75 o o Figure 46 Run Project Viewer Additonal Spreadsheet Columns This table now contains six new columns These columns are the lower and upper bounds to the 90 95 and 99 bootstrap confidence intervals LCI is the lower confidence interval bound and UCI is the upper confidence interval bound Inthe paper Kerr Martin and Churchill reported
38. step the differential tissue expression between the two treatments or two tissues is calculated It controls the other covariates in the model such as chip differences dye differences as can be seen when some dyes show different saturation points tissue or treatment effects and baseline gene level Although you might be tempted to sort this list and find the largest and smallest differential expression values you should continue to the next subsection Calculating the Significance of Differential Tissue Expression to actually estimate the P value of significance Action Select Tools gt Latin Squares gt Step 4 Calculate Differential Tissue Expression and click the second tab again to review the Latin Square Gene and Gene Interaction Effects spreadsheet Figure 44 I MXM MED Qo Figure 4 Run Project Viewer Additonal Spreadsheet Column Tutorial Latin SquaresiDye Swap Analysis Result An additional column displays in the right most position showing the differential tissue expression values This value is merely the difference between the Liver and Muscle columns Calculating Significance of Differential Tissue Expression Overview an attempt to avoid making unnecessary or untrue modeling assumptions significance is calculated using a bootstrapping method as suggested by Ef
39. t in the Latin Squares Model If you compare this table with Table 3 in the Kerr paper you will note that they similar but additionally Vector Xpression has calculated observed F vaiues If you calculate the R value using the formula 1 sum of squares error sum of squares total you wil find the same answer that the paper reports 1 82 75 3851 99 977 Estimating the Significance of Microarray and Gene Variable Overview Considered optional but recommended the randomization technique in this step estimates the P value for the microarray gene interaction variable in the Latin Squares Model As a randomization technique it does not require the typical distribution assumptions associated with estimating the P value In theory this variable is not needed in the model because there should be no interaction between individual genes and the microarray Action 1 Select Tools gt Latin Squares gt Step 3 Estimate Significance of Microarray and Gene Interaction Tem 38 Tutorial Latin SquaresiDye Swap Analysis 2 Click Yes to proceed Because of the large number of randomizations needed to estimate the P value with some confidence this calculation can be time consuming monitor displays the tools progress After the calculation completes Figure 43 displays ET Hite Ba wa EE ba jaaa PEFEA e TENET al E 0
40. ta Viewer with Additional Columns Result Now that you have calculated the antilog of the data you can create an Expression Run from the transformed data of the first microarray Creating Expression Runs from the Raw Data for Microarray 1 Overview You will now create two Expression Runs for the first microarray dye combination 26 Tutorial Latin SquaresiDye Swap Analysis ation 1 Inthe Raw Data Viewer Figure 32 select Tools gt Save Column as Expression Run Figure 32 Raw Data Viewer Select Tools gt Save Column as Expression Run The Save Column as Expression Run dialog box displays Figure 33 cac BENEERESES E mle Ratio cet Loar z Figure 33 Save Column as Expression Run Dialog Box 2 Inthe Column iist select Dye 1 Log transform Ensure the Absolute radio button is clicked for Column data typo Accept Liver in the Target list Click OK 3 Inthe Save Expression Run As dialog box not shown change the Name to Microarray 1 Dye 1 Liver and click Save 4 In the Vector Xpression Viewer message that says the Expression Run is saved click No do not open Expression Run so that you can repeat the steps to convert the other dye channel raw data object to Expression Run Now you wil create an Expression Run with the other dye channel for Microarray 1 Steps 5 through 8 reiterate Steps 1 through 4 with slight changes Refer back t
41. ts Genes ChP Chanol 1 Channel Cham Channol2 Channo 1 Channo 2 Mere TUE NE Deer Dye De2 Musse Mace ber Cem Is Figure 26 Finalize Inport Dialog Bax for Microarray 2 22 nthe Finalize Import dialog box click Save to DB 223 In the Select subset dialog box click OK 24 Inthe import message click Yes 22 Tutorial Latin SquaresiDye Swap Analysis 25 If you have completed all the steps in the table and everything goes well the Vector Xpression Database Explorer now displays Microarray 2 as shown in Figure 27 ex mlar Date Edt Datsbaso Tods gt E ES Soa I y cote Dyes Dre Swan Ready ___ E Figure 27 Vector Xpressian Database Explorer import Complete for Both Microarays If your screen does not look like this make sure you select Raw Data in the table list circled and then press the F5 key to refresh the view Result This completes the import process for your dye swap microarray data You have successfully imported the raw data for the two microarrays into Vector Xpression 23 Tutorial Latin Squares Dye Swap Analysis Preprocessing the Data Overview To preprocess the raw data you have imported you will now calculate the antilog and create two Expression Runs for the each microarray dye combination Calculating the Antilog for M
42. tter Plot of Predicted Expression versus Model Residuals From the Run Project Viewer select Tools gt Latin Squares gt Scatter Plot of Predicted Expression versus Model Residuals Figure 51 displays E TE TE a Figure 51 Scatter Plot of Predicted Expression versus Model Residuals The Scatter Plot of Predicted Expression versus Model Residuals plots the model predicted values versus the model residuals In an observed expression value made up of two parts the first part is what the proposed model can explain the predicted expression and the second part is what the model cannot explain the mode residuals In the Scatter Plot note that the vertical dispersion of points is roughly the same across the horizontal span of the plot The lack of a trend in the plot supports the appropriateness of the Latin Squares Model An apparent trend would possibly suggest a different data model or missing covariates in the experiment such as spatial effects contamination bad hybridization bad pin etc The scale of the y axis of the pilot in Figure 51 varies from the scale in Figure 2c in the Kerr paper because the authors re scaled the image to compare the two plots ar Tutorial Latin Squares Dye Swap Analysis Scatter Plot of Predicted Expression versus Absolute Model Residuals 1 From the Run Project Viewer sel
43. xpression Runs you just created from the raw data Your screen should appear as shown in Figure 36 The following table summarizes the analysis combinations for both Expression Runs as illustrated by the Expression Run objects in the Database Explorer Dye 1 Dye2 Microarray Liver Muscle Microarray 2 Muscle Liver Result You have successfully calculated the antilog and created Expression Runs for both microarray combinations E Tutorial Latin SquaresiDye Swap Analysis Performing the Latin Squares Dye Swap Analysis Overview The Latin Squares analysis was first applied to Dye Swap experimental design by Kerr Martin and Churchill The analysis accounts for the observed covariates in the experiment microarray dye tissue and genes at the same time allowing for differential expression significance testing Note that this model can only be recommended for data that is dye swapped as other types of data may not meet the assumptions inherent in this approach You will perform a Latin Squares Dye Swap analysis on the expression data you have imported and preprocessed There are five steps in a complete analysis Estimate ANOVA model variables Calculate ANOVA table Estimate significance of microarray and gene interaction term Calculate differential tissue expression Calculate significance of differential tissue expression Results of Latin Squares analysis display in graphics panels wh
44. you Fie s toImportdialogbox saved the source data fie Z5 CickAlflesinteFlesoftype iet Ze Click the latinsquare fie in the area under the Look in list This action populates the File name box 20 Tutorial Latin SquaresiDye Swap Analysis Step Dialog Box Action Next stop if automated 24 Gick Whitespace in the Delimiter Click Open 3 message None Tick Yos Import dialog box Click Raw Data Glick OK 5 Main Import dialog box None Cick Wizard Header and Data dialog If not defined by default cick Guess Click Next to instruct the Wizard to detect the Header row and Data start and end rows automatically Be sure it selects 0 for At row 1 for Start at t and 1286 for Stop at row Coordinates dialog box Select Tin the Gene IDs in column Click Next text box and change it to 5 Channels dialog box CickTwo Channels Two color Click Next experiment E Data Enter Datanames Select the Signal check box Tick Next dialog box 10 Data Enter column number None Enter Column 3forthe Click Next for Signal of Channel 1 Signal value for Channel 1 dialog box Ti Data Enter column number None Dialog boxcomeclyguesses Click Next for Signal of Channel 2 the Column 4 for the Signal value dialog box for Channel 2 12 Data Check dalogbox None un
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