Down syndrome
Contents
1. Add Statistics on Chip Statistics Calculated per cel File Raw data M Median M Skewness Logged raw data M Mean Kurtosis Resulting data Standard Deviation Norm l Trimmed Mean l0 1 0 9 Coefficient of Variation Select All Clear All Quality Assess of Gene Expression This option is used to generate the median of NUSE to QC metrics spreadsheet and a boxplot on NUSE to QC Graphs NUSE igure 6 Specifying Advanced Import Options to create chip images of and extract the scan date from the CEL files e Select Import to exit the Import Affymetrix CEL Files dialog Figure 3 Gene Expression Down s Syndrome Using Partek Genomics Suite After importing the CEL files has finished the postImportQC spreadsheet which summarizes intensity values of the Affymetrix control probe sets is shown Also a window with graphical representations of the control probe sets values appears Figure 7 E Partek Genomics Suite Version 6 11 0512 1 postimportQc posti e Edit Trar nn ua n T y Custom sr postimportQC Dc rile td anst View Stat Filter ols Window S DEUX bee AD Mie HQ Workflows Gene Expression X 1 Down_Syndrome GE _ Current Selection Down Syndrome Astrocyte 748 1 U133A CEL a Gene Expression jostimporto postimporto i 4 6 JS QC graphs of spreadsheet I postimporQc T H File Window i Line Graph Box Plot J Hybridization Labeling 3 5 Other 4 AFFX r2 P1 cre avg 7 AFF2. and configure the plot as shown in Figure 14 e Color the points by column 7 Tissue and Size the points by column 6 Type e Select Apply amp Plot Properties sp1 v1 X Slyi Ellipsoids Labels Box amp Whiskers Titles Axes Color Legend Text M Color mom Size mas C Fixed 4 0 Shape ame p Connect res ail auto Manual None Outline v A Style direct Line Width 1 pixels M Lighting Effects mmc Drawing Mode W Fogging i Normal O Mixed Transparency 0 00 Figure 14 Configuring the PCA scatter plot Color by T issue Size by Type Notice now that the data are clustered by different tissues Figure 15 File Edit View Window Help keg BOE Cobr 7 7 Tissue 7 20130 q PCA Mapping 56 9 3 H e Astrocyte e Cerebellum Cerebrum Soh EA i i aon a a pap ye ESS ASEEN SSS DDN PSS pes Ss SEES a ize pape er A HAP Fe H HEH HRH HHHH 3 ie lt lt SS HH ame ne SS SSS D TA e w e e Aig T O 1 S Figure 15 Down syndrome data colored by Tissue and sized by Type Gene Expression Down s Syndrome Using Partek Genomics Suite 12 e Another way to see the cluster pattern is to put an ellipse around the Tissue groups Select the Ellipsoids tab on the Plot Properties dialog Select Add Ellipse Ellipsoid Select the
3. 24 8 Figure 13 Viewing the PCA scatter plot of the Down syndrome data Each dot represents a chip the color of the dot represents the Type Down s or normal of the sample In the scatter plot each point represents a chip sample and corresponds to a row on the top level spreadsheet The color of the dot represents the type of the sample red represents a Down syndrome sample and blue represents a normal sample Points that are close together in the plot have similar intensity values across the probesets on the whole chip genome and points that are far apart in the plot are dissimilar e Left click on any point in the scatter plot and the corresponding row will be highlighted in the spreadsheet e While pressing the mouse wheel down drag the mouse to rotate the plot or choose the Rotate Mode option on the left side of the Scatter Plot window Press and drag the left mouse button to rotate the plot to examine the grouping pattern or outliers of the data on the first 3 principal components PCs e Scrolling the mouse wheel up or down will zoom in and out As you can see from rotating the plot there is no clear separation between Down syndrome and normal samples in this data since the red and blue samples are not separated in space However there are other factors that may separate the data Gene Expression Down s Syndrome Using Partek Genomics Suite 11 e Inthe Scatter Plot viewer select the Plot Properties icon a
4. Ellipse radio button Double click on Tissue to move it to the Grouping Variable s panel Select OK Figure 16 to exit the Add Ellipse Ellipsoid dialog and OK again to add the ellipse Add Ellipse Ellipsoid sp1 v Standard Deviation 2 0 Elipse Line Segments 200 2 Label Elipse s S Ellipsoid Density max Subdivision B 4 Base Shape Auto Categorical Variable s Grouping Variables s 7 Tissue Ellipses Ellipsoids to draw BB Astrocyte Hi Cerebellum Cerebrum Heart Figure 16 Adding an Ellipse on Tissue By rotating this plot you can see that the data is separated by tissues and within some of the tissues the Down s samples and normal samples are separated For instance in the Astrocyte and Heart tissues the Down syndrome samples small dots are on the left and the normal samples large dots are on the right Figure 17 Gene Expression Down s Syndrome Using Partek Genomics Suite 13 Scatter Plot gt wk N File Edit View Window Help BOmColr 7 Tissue v 20 30 PCA Mapping 56 9 e Astrocyte Down Syndrome e pecan Normal m SEES ebrum by a ag N N i N O a PC 1 24 8 Figure 17 Viewing a scatter plot of data colored by Tissue sized by Type and grouped by Tissue PCA is an example of exploratory data analysis and is useful for identifying outliers and major effects in the data From the scatter plot you can see that the
5. Figure 8 Sample information must be added in order to define the grouping and the goals of the experiment e Select Add sample attributes in the mport section of the workflow e Choose the option Add attributes from an existing column as shown in Figure 10 and select OK Specify type Select a type of sample attribute to add to the spreadsheet Add attributes from an existing column Add a categorical attribute H Add a numeric attribute ox Ecaneat igure 10 Configuring the Add Sample Attributes dialog In this tutorial the file name e g Down Syndrome Astrocyte 748 1 U133A CEL contains the information about a particular sample and is separated by hyphens Choosing to split the file name by delimiters will separate the categories into different columns as shown in Figure 11 Gene Expression Down s Syndrome Using Partek Genomics Suite 8 e Inthe Sample Information panel specify the column labels Labels 1 4 as Type Tissue and Subject respectively as categorical and skip the other columns as shown in Figure 11 Select OK tf Sample Information Creation Please specify how you want to split the column Choose to split your sample by delimiters or fixed widths Press the Update button to preview results Choose By delimiters if your fields are separated by certain characters such as commas or underscores Choose By fixed widths if your fields are aligned with fixed widths By delimite
6. outline Selection color Orientation Normal Transpose rows and colun Legend O Vertical legend Horizontal legend Numeric legend width Legend text size 10 v Show category legend kL OT 8 70 6 96 5 22 3 48 1 74 0 00 LLLA O m lo D fad N a wW O a a a N ap oO te O D Type Down Syndrome Normal Figure 26 Hierarchical Clustering results Adding Gene Annotation In the previous steps after the data was imported the GeneChip annotation file was linked to the data so now the results spreadsheets will automatically be linked to the annotation file More information can be added to the genes in new columns if the genes are laid out on rows like in the ANOVA or gene list spreadsheets For example if you want to add additional annotation to the gene list A then e Right click on the second column header 2 ProbesetID in the Down_Syndrome_vs_Normal A spreadsheet and select Insert Annotation from the pop up menu Figure 27 e Select the Chromosomal Location under the Column Configuration panel Leave everything else as default and click OK Figure 28 Gene Expression Down s Syndrome Using Partek Genomics Suite Hierarchical Clustering ATP50 C210rf33 EFEMP1 IFNGR2 SOD USP16 CSTB PIGP LOC10012936 PTTGIIP SUMO3 WRB Pemo D File Edit Transform View Stat Filter Tools Window Custom Help 1 Down_Sy
7. that gene Figure 29 Probe set name 203635_at Chromosome 21 Base Pairs 38595725 38639833 Name Down syndrome critical region gene 3 Description igb NM_006052 1 DEF Homo sapiens Down syndrome critical region gene 3 DSCR3 mRNA FEA mRNA GEN DSCR3 PROD Down syndrome critical region protein 3 DB_XREF gi 5174424 UG Hs 26146 Down syndrome critical region gene 3 FL gb D87343 1 gb NM_006052 1 HTML Report Figure 29 Getting gene information for Probeset_ID 203635 at e To get a dot plot of a specific gene right click on the row header and select Dot Plot Orig Data from the pop up menu Figure 30 File Edit Transform View Stat Filter Tools Window Custom Help Osx tkhhetS SW Mie AQ Bar Chart Orig Data Sources of Variation 1 Down_Syndrome GE _jCurrent Selection 3171 x ANOVA 3way ANOVAResults f 2 Down_Syndrome_vs_Normal A 1 Gene Symbol postImportQC postImportQc Do s eE r21q22 2 DSCR3 Down NM_OOM 4 Co Groupby 6 T X HV momCobkr 6 T py r21q22 3 PTTG1IP pituitary NM_0O sad s ype ype E 21q21 1 ATP5J ATP NM_00 r21q21 ATP ATP M_00 sate i N own syndrome critical region gen Filter Include r21q22 3 SUMO3 SMT3 NM_OO D synaro critical egion ge e3 Filter Exclude r21q22 3 CSTB cystatinB NM_OOM 3 Filter Include Orig Data r21q22 11 USP16 ubiquitin NM_ooi A Select Orig Da
8. 952 bytes 712x712 Figure 9 Viewing a chip image HG U133A 12 17 02 8 59014 5 40818 6 32312 Gene Expression Down s Syndrome Using Partek Genomics Suite aa 4 39536 e Click on the button to zoom in on the image click on the button to zoom out The scroll bars on the right and across the bottom of the window may be needed to navigate around the image at higher zoom levels These pseudo chip images may be used to identify any anomalies with the chip such as scratches hybridization errors and scanning errors if you suspect there might be a problem with the chip This check is usually performed on outliers in the QA QC step of the gene expression workflow For additional information on importing data into Partek Genomics Suite see Chapter 4 Importing and Exporting Data in the Partek User s Manual The User s Manual is available from the Partek Genomic Suite menu from Help gt User s Manual The FAQ Help gt On line Tutorials gt FAQ may also be helpful As this tutorial only addresses some topics you may need to consult the User s Manual for additional information about other useful features It is recommended that you are familiar with Chapter 6 The Pattern Visualization System of the user manual before going through the next section of the tutorial Adding Sample Information Twenty five CEL files samples have been imported into Partek Genomics Suite as shown in
9. A will be created as a child spreadsheet under the Down_Syndrome GE spreadsheet This gene list spreadsheet can now be used for further analysis such as hierarchical clustering gene ontology integration of copy number data or exportation into other data analysis tools such as pathway analysis You should take some time creating new gene list criteria of your own to become familiar with the List Manager tool in Partek Genomics Suite For more information you can always click on the 8 button Hierarchical Clustering The gene list in spreadsheet Down Syndrome vs Normal A can now be used for hierarchical clustering to visualize patterns in the data e Under the Visualization section in the Gene Expression workflow select Cluster based on significant genes e The next dialog asks you to specify the type of clustering you want to perform Select Hierarchical Clustering and select OK e Choose the Down Syndrome vs Normal A spreadsheet under the Spreadsheet with differentially expressed genes Figure 25 Gene Expression Down s Syndrome Using Partek Genomics Suite 22 e Choose the Standardize shift genes to mean of zero and scale to standard deviation of one under the Expression normalization panel This option will adjust all the gene intensities such that the mean is zero and the standard deviation is 1 e Select OK Cluster genes using hierarchical clustering on the expression values using only the genes present in the
10. Gene Expression Analysis of a Down s Syndrome Study Using Partek Genomics Suite 6 6 This tutorial will illustrate how to e Import Affymetrix CEL files and check quality Add attributes describing the sample groups Perform exploratory analysis using the PCA scatter plot Find differentially expressed genes using ANOVA Generate a list of genes of interest Add annotations to the gene list Note the workflow described below is enabled in Partek Genomics Suite version 6 6 Please contact the Partek Licensing Team at licensing partek com to request this version or update the software release via Help gt Check for Updates from the main command line The screenshots shown below may vary across platforms and across different versions of Partek Genomics Suite Description of the Data Set Down syndrome is caused by an extra copy of all or part of chromosome 21 it is the most common non lethal trisomy in humans The study used in this tutorial revealed a significant up regulation of chromosome 21 genes at the gene expression level in individuals with Down syndrome this dysregulation was largely specific to chromosome 21 only and not to any other chromosomes This experiment was performed using the Affymetrix GeneChip Human U133A arrays It includes 25 samples taken from 10 human subjects and 4 different tissues The raw data for this study is available as experiment number GSE1397 in the Gene Expression Omnibus http www nc
11. X r2 Ec bioD avg Select All Clear All AFFX r2 Ec bioC avg i i AFFX r2 Ec bioB avg Metrics Metrics listed below are in expected order from high to low J AFFX r2 P1 cre avg Y AFFX r2 Ec bioD avg Y AFFX r2 Ec bioC avg AFFX r2 Ec bio8 avg Log 2 expression NININININININININININISINININININIP ININISIN NININIFG et hl ie m i b i din z p oo io l oo op Ww gt Ou MOLD Tree ee NAN __ Sample Do not automatically show this dialog after import QC Limits B e e Rows 25 Cols 24 7 a Figure 7 QC metrics are shown in the QC graph as well as in the postImportQC spreadsheet OC metrics of the QA QC section of the workflow provides quality control information from control and experimental probes on the Affymetrix chips to provide confidence in the quality of the microarray data or to identify samples that do not meet QC criteria A more detailed user guide for the Affymetrix QC module is available here or from Help gt On line Tutorials gt User Guides When you close the QC graphs window the result file will be automatically opened in Partek Genomics Suite as spreadsheet I named Down _Syndrome GE You will see 25 rows representing 25 chips and more than 22 000 columns representing genes in this spreadsheet Figure 8 Gene Expression Down s Syndrome Using Partek Genomics Suite 6 me metho h_synerome GE Tools Window Custom Help tie AQ
12. _Syndrome GE rr Information File Optional Using RMA jmpoct settings Figure 3 Configuring import files window Gene Expression Down s Syndrome Using Partek Genomics Suite 3 e Select Customize to configure the import options Figure 4 Algorithm Outputs Probes to use in the import Probes to Import V Interrogating Probes l Control Probes Probe Filtering Skip C Include From File Exclude From File Filter File Normalization configuration Pre background Adjustment Adjust for GC Content Adjust for probe sequence Background Correction Skip RMA Background Correction C GCRMA Background Correction Quantile Normalization Quantile Normalization Distribution File Browse Log Probes using Base 2 Probeset Summarization Median Polish Use Partek Defaults Use RMA Defaults Use GCRMA Defaults Figure 4 Configuring the Advanced Import Options e Select Library Files to specify the location of the library folder to be used and to specify the annotation files to use Figure 5 Specify File Locations ES Please Specify Files For Hi6 W1334 Files Affymetrix Library File C Microarray Libraries HG U1334 cdf File Up to date Probeset Annotation Optional Microarray Libraries HG U133A na32 annot csy File Up to date Download Default Library File Folder C Microarray Libraries Change Search and Copy Ge
13. bi nlm nih gov geo Data and associated files for this tutorial can be downloaded by going to Help gt On line Tutorials from the Partek Genomics Suite main menu The data can also be downloaded directly from http www partek com Tutorials microarray Gene_Expression Down Syndrome Down_ _S yndrome GE zip Gene Expression Down s Syndrome Using Partek Genomics Suite l Importing Affymetrix CEL Files Download the data from the Partek site to your local disk e For this tutorial unzip the files to C Partek Training Data Down_ Syndrome GE or to a directory of your choosing Be sure to create a directory or folder to hold the contents of the zip file e Start Partek Genomics Suite and select Gene Expression from the Workflows panel on the right side of the tool bar in the Partek Genomics Suite main window Figure 1 Partek Genomics Suite Version 6 12 0202 1 empty File Edit Transform View Stat Filter Tools Window Custom Help Osx ith P Mie AA a Current Selection 1 empty Import samples Add sample attributes Edit sample information Choose sample ID column P aa p Analysis gt Visualization p Biological Interpretation gt Genomic Integration mi Figure 1 Selecting the gene expression workflow e Select Import samples under the mport section of the workflow Select Import from Affymetrix CEL files and th
14. e set up e Select the Contrast button Figure 19 to invoke the Configure dialog e Choose 6 Type from the Select Factor Interaction drop down list All of the levels in this factor are listed on the Candidate Level s panel on the left of the dialog Figure 20 e Select Down Syndrome from the Candidate Level s panel and move it to the Down Syndrome Group panel by selecting Add Contrast Level gt in the top half of the dialog Label 1 will be changed to the subgroup name automatically but you can also manually specify the label name e Select Normal from the Candidate Level s panel and move it to the Normal Group 2 panel in a similar manner Since the data is logy transformed Partek Genomics Suite will automatically detect this and will automatically select the radio button Yes in the Data is already log transformed at the top right hand corner Partek Genomics Suite will use the geometric mean of the samples in each group to calculate the fold change and mean ratio for the contrast between the Down syndrome and Normal samples Gene Expression Down s Syndrome Using Partek Genomics Suite 17 Data is already log tre Select Factor Interaction 6 Type f Yes Base 2 0 Candidate Level s Down Syndrome Label Down Syndrome Normal Add Contrast Level gt Down Syndrome Remove Contrast Level Label Normal lt Remove Contrast Level H er e e a C Estimate E F ratio Ci T sta
15. en click OK Click the Browse button to select the C Partek Training Data Down_Syndrome GE folder By default all the files with a CEL extension are selected Figure 2 Gene Expression Down s Syndrome Using Partek Genomics Suite 2 M CEL File Selection Specify Folder s that contain CEL files C Partek Training Data Down_Syndrome GE CEL Files to Process 0 Down Syndrome Astrocyte 748 1 U133A CEL Down Syndrome Astrocyte 1478 1 U133A CEL Down Syndrome Cerebrum 847 1 U133A CEL Down Syndrome Cerebrum 1218 1 U1334 Down Syndrome Cerebrum 1478 1 U133A CEL Down Syndrome Heart 1218 1 U133A CEL Down Syndrome Heart 1478 1 U133A CEL Normal Astrocyte 1479 1 U133A CEL Normal Astrocyte 1521 1 U133A CEL Normal Cerebellum 1390 1 U133A CEL NormalCerebellum 1411 1 U133A CEL Normal Cerebellum 1521 1 U133A CEL Normal Cerebrum 1390 1 U133A CEL Normal Cerebrum 1390 2 U133A CEL Yormal Cerebrum 1411 1 U133A CEL Normal Cerebrum 1411 2 U133A CEL Normal Cerebrum 1521 1 U133A CEL Normal Cerebrum 1521 2 U133A CEL Normal Cerebrum 1565 1 U133A CEL Normal Heart 1390 1 U133A CEL Normal Heart 1411 1 U133A CEL Figure 2 Selecting the folder and CEL files for the experiment e Select the Add File gt button to move all the CEL files to the right panel Twenty five CEL files will be processed e Select Next the Import Affymetrix CEL Files dialog is shown in Figure 3 Output File C Partek Training Data Down_Syndrome GE Down
16. h 0 10 That is 10 of the genes 1n that list are expected to be false positives that is do not demonstrate significant fold change of less than 1 3 or greater than 1 3 Save the list as A select the Create button and Close Gene Expression Down s Syndrome Using Partek Genomics Suite 21 Spreadsheet Name 1 Down_Syndrome GE Single factor find genes that vary across all samples upon single factor 1 ANOVA 3way ANOVAResults 1 postImportQC postImportQC Dow Name Setting Type Tissue Interaction find genes that vary across all samples upon interaction 2 Name Setting Type Tissue Contrast find genes that change between two categories Name Setting Down Syndrome vs Normal Have Any Change v Configuration for Down Syndrome vs Normal Pass 16 Z Include size of the change Fold change gt 1 3 OR Fold change lt 1 3 Z Include significance of the change p value with FDR y lt o1 16 genes passed the specified criteria You are about to create a list of genes that Have Any Change in Down Syndrome relative to Normal with fdr step up lt 0 1 Fold ch ange gt 1 3 or Fold change lt 1 3 Z Save list A Browse Create Configure Figure 24 The List Manager dialog for creating a gene list with fold changes above 1 3 or below 1 3 with a False Discovery Rate FDR lt 0 10 The spreadsheet Down Syndrome vs Normal
17. hip Type foe ae RA emoe Down Syne Astrocyte l1 07 02 02 a J j a 4 c Am Ei Ji j 3 a J L mm zi T 7 7 g J i Down Syndrome Astrocyte 748 1 U133A CEL HG U133A Down Syndrome Astrocyte 748 OF 035 02 Down HG Ui334 DownSyndrome Cerebellum 1218 12 17 02 Sa a a oe bad GET II 55a cl Syndrome Cerebelum 1218 1 U133A CEL Dow HG Ui334 DownSyndrome Cerebellum 1389 Maaza Si Syl nd ome Cerebellum 1339 1 U1534 CEL M Tal ny Figure 12 Viewing the spreadsheet with added sample T Exploratory Data Analysis At this point in analysis you would explore the data preliminarily Do the genes you expected to be differentially regulated appear to have larger or smaller intensity values Do similar samples resemble each other The latter question can be explored using Principal Components Analysis PCA an excellent method for reducing and visualizing high dimensional data e Select Principal Components Analysis PCA in Q4 QC section of the Workflows dialog Select 6 Type from the panel on the right side The Scatter Plot dialog box with your PCA plot will appear as shown in Figure 13 Gene Expression Down s Syndrome Using Partek Genomics Suite 10 r Scatter Plot sp1 v1 File Edit View Window Help BOM Color 6 Type PCA Mapping 56 9 Down Syndrome e Normal PC 2 22 5 PC 1
18. ich factors to include in the ANOVA may be an iterative process while you decide which factors and interactions are relevant as not all factors have to be included in the model For instance in this example Gender and Scan date were not included The Sources of Variation plot is a way to quantify the relative contribution of each factor in the model in explaining the variability of the data e View the sources of variation for each of the factors across the whole genome by clicking Plot sources of variation from the Analysis section of the workflow with the ANOVA result spreadsheet active e lt A Sources of Variation Plot dialog box will appear You have a choice of viewing the sources of variation as a bar chart Signal to Noise Ratio or as a pie chart Sum of Squares e Select the Bar Chart tab and Apply The Sources of Variation plot is shown in Figure 23 Gene Expression Down s Syndrome Using Partek Genomics Suite 19 Sources of Variation 1 ANOVA 3way File Window Bar Chart Signal to Noise Pie Chart Sum of Squares Data Labels Average mean i Sources of Variation Mean F Ratio ok ice oO on Subject Type Type Tissue Factors Apply Reset Figure 23 Viewing the Sources of Variation plot which shows Tissue is the biggest source of variation overall This plot presents the mean signal to noise ratio of all the genes on the microarray All the factors i
19. ing Y axis The genes that have larger and more significant changes between the Down syndrome and normal groups are on the upper right and upper left corner Figure 32 Gene Expression Down s Syndrome Using Partek Genomics Suite 2i Volcano Plot sp1 v File Edit View Window Help K BOE Colbr 10 p value Down Syndrome vs N 2D 3D Volcano Plot of 1 ANOVA 3way p value Down Syndrome vs Normal Fold Change Down Syndrome vs Normal Figure 32 Viewing the volcano plot of Down syndrome vs Normal contrast Each dot represents one gene The X axis represents fold change and the Y axis represents the p value In order to select the genes by fold change and p value we will draw a horizontal line to represent the p value 0 05 and two vertical lines indicating the 1 3 and 1 3 fold changes cutoff lines e Select Edit gt Plot Properties or the icon o within the Volcano Plot viewer e Choose the Axes tab e Select the Set Cutoff Lines button and configure the dialog as in Figure 33Figure 33 Vertical Line s X Axis Value s Horizontal Line s Y Axis Value s 0 05 Line Color Line wiath 3 Figure 33 Setting the Cutoff Lines for the volcano plot Gene Expression Down s Syndrome Using Partek Genomics Suite 28 e Check Select all points in a section to allow Partek Genomics Suite to automatically select all the points in any given section e Select OK The plot will be divided into six sectio
20. iscussed The decision to discard any samples would be based on information from the PCA plot pseudo chip images sample histogram plot and QC metrics To discard a sample and renormalize the data without the effects of the outlier start over with importing samples and omit the outlier sample s during the CEL file import Identifying Differentially Expressed Genes using the ANOVA Analysis of variance ANOVA is a very powerful technique for identifying differentially expressed genes 1n a multi factor experiment such as this one In this data set the ANOVA will be used to generate a list of genes that are significantly different between Down syndrome and normal with an absolute difference bigger than 1 3 fold The ANOVA model should include Type since it is the primary factor of interest From the exploratory analysis tissue was found to be a large source of variation therefore tissue Gene Expression Down s Syndrome Using Partek Genomics Suite 15 should be included in the model In the experiment multiple samples were taken from the same subject so Subject must be included in the model otherwise the ANOVA assumption that samples within groups are independent will be violated In addition the PCA scatter plot showed that the Down s and normal separated within tissue type so the Type Tissue interaction should be included in the model e To invoke the ANOVA dialog click Detect differentially expressed genes in the Analysi
21. n the ANOVA model are listed on the X axis including random error The Y axis represents the mean of the ratios of mean square of all the genes to the mean square error of all the genes Mean square is ANOVA s measure of variance Compare each signal bar to the error bar if a bar is higher than the error bar it means that factor contributed significant variation to the data across all the variables Notice that this plot is very consistent with the results in the PCA scatter plot In this data on average Tissue 1s the biggest source of variation To view the source of variation of each individual gene right click on a row header in the ANOVA spreadsheet and select the Sources of Variation item from the pop up menu View a few plots from rows at the top of the ANOVA table and some from the bottom of the table Another useful graph is the ANOVA Interaction Plot which 1s also accessed by right clicking on a row header in the ANOVA spreadsheet Select ANOVA Interaction Plot from the options Generate these plots for rows 5 CSTB and row 7 CACNAI1G If the lines in this plot are not parallel then there is a chance there is an interaction between Tissue and Type Look at the p values in column 9 p value Type Tissue Create Gene List Now that you have obtained statistical results from the microarray experiment you can now take the result of 22 283 genes and create a new spreadsheet of just those genes that pass a certain criteria This
22. name for the gene list The list can be saved as a text file File gt Save As Text File for use in reports or by downstream analysis software End of Tutorial This is the end of tutorial If you need additional assistance with this data set contact the Partek Technical Support staff at 1 314 878 2329 or email us at support partek com Date last updated November 5 2013 Copyright 2013 by Partek Incorporated All Rights Reserved Reproduction of this material without express written consent from Partek Incorporated is strictly prohibited Gene Expression Down s Syndrome Using Partek Genomics Suite 30
23. ndrome GE ja ANOVA 3way ANOVAResults Down_Syndrome_vs_Normal A postImportQC postImportQC pm Sort Ascending D0 Sort Descending 30 Fill Column 0 Split Column M Find Replace Select 20 Filter Include 22 Filter Exclude Insert 20 0 20 7 Insert Average Delete 22 20 Select Orig Data 20 Gene View Orig Data 20 Fit Columns Create List from Column Labels Create List With Occurence Counts Create List Properties Add Rows Add Columns Add Annotation Add Average Add to the Right of Column 2 Probeset ID v Maximum String Length 80 Add as string _ Add as categorical Add selected to defaults Column Configuration C AGI C Alignments E gt C Annotation Date _ Annotation Description _ Annotation Notes L Annotation Transcript Cluster E Archival UniGene Cluster J EC C Ensembl C Entrez Gene _ FlyBase _ Gene Ontology Biological Process L Gene Ontology Cellular Component _ Gene Ontology Molecular Function _ Gene Symbol Figure 28 Adding Chromosomal Location to each gene in the gene list spreadsheet Of the 16 genes of the Down_Syndrome_vs_Normal A spreadsheet 14 genes are on chromosome 21 Gene Expression Down s Syndrome Using Partek Genomics Suite e Right click on the first row header and select Probe Set Details to get more information about
24. ndrome and normal 1s at the top of the spreadsheet a Partek Genomics Suite Version 6 11 0512 1 ANOVA 3way ANOVAResults File Edit Transform View Stat Filter Tools Window Custom Help 1 Down_Syndrome GE ANOVA 3way ANOVAResult postImportQC postImportQ Dae Xx tki tS PM Mie _jCurrent Selection 3171 HQ i Column 1 006052 5 83452e 007 104339 8 67164e 007 0036 2 0628e 006 6936 8 60609e 006 9 4108e 006 25015 9 44989e 006 96 1 41822e 005 1010019 1 43177e 005 10393 1 61768e 005 011458 1 67069e 005 1013453 2 64972e 005 1_0017219 3 01629e 005 _017446 3 69853e 005 5 6 fs efSeq p value Type p value Tissue 0 283029 1 0992e 007 0 00930333 1 50898e 005 0 000837349 0 0250471 0 00534947 0 00193717 4 8335e 008 0 0152245 0 000632057 0 000374718 0 0113259 8 p valu Workflows Gene Expression a Gene Expression w Import Import samples Add sample attributes Edit sample information Choose sample ID column Ww Qa ac Principal components analysis PCA Plot sample histogram QC metrics w Analysis Detect differentially expressed genes Plot sources of variation igure 22 Viewing the ANOVA results child spreadsheet For additional information about ANOVA in Partek Genomics Suite see Chapter 11 Inferential Statistics in the User s Manual Help gt User s Manual Viewing the Sources of Variation Deciding wh
25. ne Expression Down s Syndrome Using Partek Genomics Suite Partek Genomics Suite will automatically assign the annotation files according to the chip type stored in the CEL files If the annotation files are not available in the library directory Partek Genomics Suite will automatically download them and store them in the Default Library File Folder e The default library location can be modified at by selecting the Change button in the Default Library File Folder panel By default the library directory is at C Microarray Libraries This directory is used to store all the external libraries and annotation files needed for analysis and visualization The library directory can also be modified from Tools gt File Manager e Select OK Figure 5 to close the Specify File Locations dialog e Select the Outputs tab from the Advanced Import Options dialog Figure 6 e Make sure the output chip images based on Original Summarized and Difference values are selected e Inthe Extract Time Stamp and Date from CEL File panel make sure the Date button is selected to extract the chip scan date This information can help you to detect if there is batch effects caused by the process time e Select OK to exit the Advanced Import Options dialog Advanced Import Option Chip Images During the Import Cycle V Original M Summarized lv Difference Background Adjusted Extract Time Stamp and Date from CEL File V Date Time Hours from Midnight
26. ns By clicking on the upper right section all 271 genes in that section will be selected Figure 34 Volcano Plot sp1 v1 File Edit View Window Help Ke BOE Color 7 10 p value Down Syndrome vs N v 20 3 Volcano Plot of 1 ANOVA 3way X 1 3 2 2929 Y 5 83452e 007 0 05 271 points p value Down Syndrome vs Normal Fold Change Down Syndrome vs Normal Figure 34 Selecting genes that significantly up regulated in Down syndrome compared to Normal e Left click in any region in the plot to select the region of interest Right click on the selected region in the plot and choose Create List to create a list including the genes from the section selected Note that these p values are uncorrected Note If no column is selected in the parent ANOVA spreadsheet all of the columns will be included in the gene list if some columns are selected only the selected columns will be included in the list e Specify a name for the gene list and write a brief description about the list Figure 35 The description is shown when you right click on the spreadsheet gt Info gt Comments Gene Expression Down s Syndrome Using Partek Genomics Suite 29 Create List fro m 5 ajg tic im Save as C Partek Training Data Down_Syndror Browse Description Selected rows and columns of datafile C Partek Training Data Down_Syndrome GE ANOVAResults pvalue lt 0 05 Fold Change 1 3 igure 35 Specifying a
27. on about which inferences are being made Random effects appear in red Gene Expression Down s Syndrome Using Partek Genomics Suite 16 on the spreadsheet and in the ANOVA dialog When the ANOVA model includes both random and fixed factors it is a mixed model ANOVA Here is another way to tell if a factor is random or fixed imagine repeating the experiment Would the same levels of each factor be used again e Type Yes the same types would be used again a fixed effect e Tissue Yes the same tissues would be used again a fixed effect e Subject No the samples would be taken from other subjects a random effect You can specify which factors are random and which are fixed when you import your data or after importing by right clicking on the column corresponding to a categorical variable selecting Properties and checking Random effect By doing that the ANOVA will automatically know which factors to treat as random and which factors to treat as fixed Nested Nesting Relationships The subject factor in the ANOVA model is listed as Subject Type this means that Subject is nested in Type Partek Genomics Suite can automatically detect this sort of hierarchical design and will make adjustments to the ANOVA calculation accordingly Linear Contrasts By default an ANOVA only outputs a p value for each factor interaction therefore to get the fold change and ratio between Down syndrome and normal a contrast must b
28. postimportQC postImportQc Figure 8 Viewing the main or top level spreadsheet with CEL file images e Double click on any of the chip image thumbnails to enlarge the image and to Down Syndrome Astrocyte 748 1 U133A CEL HG U133A 07 03 02 10 2934 5 21408 6 29851 8 83625 4 3246 Down Syndrome Astrocyte 1478 1 U133A CEL HG U133A 07 02 02 10 5145 5 81907 6 27588 i 4 23023 Down Syndrome Cerebellum 12 18 1 U133A CEL HG U133A 12 17 02 9 93234 5 254 6 36288 e 4 56094 Down Syndrome Cerebellum 13 89 1 U133A CEL HG U133A 12 17 02 9 91115 5 29298 6 30654 Down Syndrome Cerebellum 14 78 1 U133A CEL Down Syndrome Cerebrum 847 1 U133A CEL HG U133A HG U133A 12 17 02 05 23 02 10 023 9 93037 5 32463 5 30983 6 35685 6 29661 14 75359 4 61398 4 93154 Down Syndrome Cerebrum 121 8 1 U133A CEL HG U133A 05 23 02 9 61995 5 34307 16 32989 Down Syndrome Cerebrum 138 9 1 U133A CEL HG U133A 05 30 02 9 578 5 4112 6 26629 8 61139 8 63673 m 14 45268 14 4548 Down Syndrome Cerebrum 147 8 1 U133A CEL HG U133A 05 23 02 9 5232 5 37381 6 19998 8 79329 4 40119 Down Syndrome Heart 1218 1 U 133A CEL examine the chip Figure 9 File Brows P 1 0 lt gt Down Syndrome Astrocyte 748 1 U133A CEL pimg 506
29. pproximately 6 0 The line inside the Box amp Whiskers represents the median of the samples in a group Mousing over the Box amp Whiskers plot will show this information Generating Gene Lists from a Volcano Plot Next we will generate a list of genes that passed a p value threshold of 0 05 and fold changes greater than 1 3 using a volcano plot e Ensure that the ANOVA 3way ANOVAResults spreadsheet is selected as this is the spreadsheet we will be using to create the gene list Select View gt Volcano Plot from the Partek Genomics Suite main menu Set X Axis Fold Change to 13 Fold Change Down Syndrome vs Normal and the Y axis p value to be 11 p value Down Syndrome vs Normal e Color by the gene with the 11 p value Down Syndrome vs Normal Figure 31 e Select OK X Axis Fold Change 12 Fold Change Down Syndrome vs Normal Y Axis p value 10 p value Down Syndrome vs Normal Color by 10 p value Down Syndrome vs Normal igure 31 Configuring the volcano plot dialog X axis representing the fold change of Down syndrome vs Normal Y axis representing the p value of this contrast In the plot each dot represents a gene The X axis represents the fold change of the contrast and the Y axis represents the range of p values The genes up regulated in Down syndrome on the right side genes down regulated in Down syndrome are on the left of the N C line The genes become more statistically significant with increas
30. rs By fixed width Hyphen Width specification example 3 5 2 Underscore _ Specify width s 1 Period E Other delimiters Update Sample Information You can specify the column label of each attribute by editing the label above its respective column Change the column s type using the drop down menu above each column Type Tissue Subject Label 4 Label 5 Label 6 skip skip U1334 U1334 U1353A U133A U1534 U1334 U1334 U153A mm categorical categorical categorical Down Syndrome Astrocyte 1478 Down Syndrome Astrocyte 7438 Down Syndrome Cerebellum Down Syndrome Cerebellum Down Syndrome Cerebellum Down Syndrome Cerebrum Down Syndrome Cerebrum Down Syndrome Cerebrum 4 ae ere aa ania 4 Figure 11 Configuring the Sample Information Creation dialog e A dialog window asking if you would like to save the spreadsheet will appear Select Yes to save the spreadsheet with new sample attributes e Make column 8 Subject random by right clicking on the column header and selecting Properties Select the Random Effect check box The spreadsheet containing the added sample information is as shown Figure 12 Note More details on Random vs Fixed Effects can be found later in this tutorial under the section Identifying Differentially Expressed Genes using the ANOVA Gene Expression Down s Syndrome Using Partek Genomics Suite 9 Current Selection HG U133A 5 j C
31. s section of the workflow e Inthe Experimental Factor s panel select Type Tissue and Subject by pressing lt Ctrl gt and left clicking e Use the Add Factor gt button to move the selections to the ANOVA Factor s panel e To specify the interaction select Type and Tissue by pressing lt Ctrl gt and left clicking Select the Add Interaction gt button to add the Type Tissue interaction in the ANOVA Factor s panel Figure 19 Do NOT select OK i ANOVA of Spreadsheet 1 Experimental Factor s ANOVA Factor s 6 Type SS 6 Type 7 Tissue Add Factor gt B 7 Tissue B Subject B Subject 6 Type 9 Scan Date Add Interaction gt B 6 Type 7 Tissue lt Remove Factor z save Model Load Model Contrast Cross Tabs 2 Advanced Response Variable s All Response Variables Specify Output File C Partek Training Data Down_Syndrome GE ANOVAResults Browse Senet Gri o F igure 19 Factors and interactions to be included in the ANOVA model Random vs Fixed Effects Mixed Model ANOVA Most factors in analysis of variance ANOVA are fixed effects whose levels represent all the levels of interest In this study Type and Tissue are fixed effects If the levels of a factor only represent a random sample of all the levels of interest for instance Subject the factor is a random effect The ten subjects in this study represent only a random sample of the global populati
32. selected spreadsheet Spreadsheet with list of significant differentially expressed genes 1 Down_Syndrome_vs_Normal A Expression normalization Standardize shift genes to mean of zero and scale to standard deviation of one C Shift shift genes to mean of zero C None do not adjust the values igure 25 Cluster the significant genes dialog The resulting graph Figure 26 illustrates the standardized gene expression level of each gene in each sample Genes which are unchanged are displayed as a value of zero and colored gray Up regulated genes have positive values and displayed in red Down regulated genes have negative values and displayed as blue Each gene is represented in one column and each sample is represented in one row We can see how the Down syndrome samples clustered together as shown in Figure 27 For more information on the methods used for clustering you can refer to Chapter 8 Hierarchical amp Partitioning Clustering in Help gt User s Manual For a tutorial on configuring the clustering plot please refer to the user guide that can be downloaded from here or from Help gt On line Tutorials gt User Guides Gene Expression Down s Syndrome Using Partek Genomics Suite 23 Mouse Mode GD C5 C amp C Heat Dendrog T R Co Data Range Min 3 2877376079 M Mid E E Max 3 28773760795 v High Contrast Display Options m Show heat map _ Cell
33. ta r2p16 EFEMP1 EGF containir NM_00 Down Syndrome T SS ey a aA Normal r12p13 2 LOC1001293 similar to XM_00 H Insert r21q21 3 MRPL39 mitochondrial NM_01 H Delete a Se r21q22 1 2 SOD1 superoxide NM_OO HTML Report r21q22 3 C21orf33 chromosome NM_09 Dot Plot Orig Data r21q22 2 PIGP phosphatidyli NM_15 J XY Plot Orig Data r21q11 2 USP25 ubiquitin NMO l j r21q22 3 WRB tryptophan NM_0OO r21q22 1 q ATP50 ATP r21q22 11 IFNGR2 interferon NM_OO 634 ANOVA Interaction Plot Profile Orig Data _ Probe Set Details 6 256 H Probe Set HTML Report 6 172 I Browse to Location Create List 6 088 6 004 Dow Syndrome Normal Figure 30 Dot plot of the most differently expressed gene between Down syndrome and normal Each dot is a sample The Y axis represents the expression value of the gene the X axis represents different Types The dots and the box amp whiskers are colored by Type In the plot each dot is a sample of the original data The Y axis represents the log normalized intensity of the gene and the X axis represents the different types of samples Gene Expression Down s Syndrome Using Partek Genomics Suite 26 The median expression of each group is different from each other In this example the median of the Down syndrome samples 6 3 but the median of the normal samples is a
34. tissue is the biggest source of variation There are many genes that express differently between the 4 tissues but not as many genes that express differently between type Down syndrome and normal across the whole chip genome The next step in the workflow is to draw a histogram to examine the samples Select Plot sample histogram in the QA OC section of the Workflow to get the Histogram dialog box as shown in Figure 18 Gene Expression Down s Syndrome Using Partek Genomics Suite 14 File Edit View Window Help te Bom Color 6 Type All Rows of 1 Frequency 6 4 7 6 8 7 9 8 10 9 VE Type m Down Syndrome Normal igure 18 Viewing the histogram of all 25 samples The histogram plots one line for each of the samples with the intensity of the probes graphed on the X axis and the frequency of the probe intensity on the Y axis This allows you to view the distribution of the intensities to identify any outliers In this dataset all of the samples follow the same distribution pattern indicating that there are no obvious outliers in the data As demonstrated with the PCA if you click on any of the lines in the histogram the corresponding row will be highlighted in the spreadsheet Down Syndrome GE You can also change the way the histogram displays the data by clicking on the Plot Properties button Explore these options on your own The last option in the QA QC section is QC metrics which has already been d
35. tistic 2 Add Contrast 2 Add Combinations Contrast Name Factor Interaction Status al igure 20 Adding Down Syndrome vs Normal contrast to the computation e Select Add Contrast to add the Down Syndrome vs Normal contrast Figure 21 e Select OK to apply the configuration Data is already log trz Select Factor Interaction 6 i f Yes Base 2 0 Candidate Level s Label Add Contrast Level gt lt Remove Contrast Level Label Add Contrast Level gt lt Remove Contrast Level Other Statistics I Estimate F ratio I T statistic 3 Add Contrast i Down Syndrome vs Normal igure 21 Configuring the Adding Contrasts dialog Gene Expression Down s Syndrome Using Partek Genomics Suite 18 e By default the Specify Output File is checked in Figure 19 and gives a name to the output file If you are trying to determine which factors should be included in the model and you do not wish to save the output file simply uncheck this box e Select OK or Apply in the ANOVA dialog to compute the 3 way mixed model ANOVA The result will be displayed in a child spreadsheet ANOVA 3way ANOVAResults In the child result spreadsheet each row represents a gene and the columns represent the computation results for that gene Figure 22 By default the genes are sorted in ascending order by the p value of the first categorical factor Type which means the most significant differently expressed gene between Down sy
36. will make managing the data more streamlined by focusing on Gene Expression Down s Syndrome Using Partek Genomics Suite 20 just those genes with the most significant differential expression or substantial fold change In Partek Genomics Suite the List Manager can be used to specify numerous conditions to use in the generation of our list of genes of interest In this tutorial we are going to create a gene list with a fold change between 1 3 to 1 3 with the significance FDR of 10 The following section will illustrate how to use the List Manager to create this gene list To invoke the List Creator select Create gene list in the Analysis section of the workflow Ensure that the ANOVA 3way ANOVAResults spreadsheet is selected as this 1s the spreadsheet we will be using to create our new gene list as shown in Figure 24 Select the ANOVA Streamlined tab In the Contrast panel choose Down Syndrome vs Normal in the name and Have Any Change from the Setting dropdown menu list This will find genes that have a fold change different between the different types of samples In the Configuration for Down Syndrome vs Normal panel check the Include size of the change and enter Fold change gt 1 3 OR Fold change lt 1 3 Select Include significance of the change p value with FDR lt 0 1 The number of genes that pass your cut off criteria will be shown next to the Pass field In this example 16 genes pass the criteria of FDR wit
Download Pdf Manuals
Related Search