Alternative Splice Analysis of Exon Data in Partek Genomics Suite
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1. x on Expression TissueType LUZ X Figure 8 Viewing a gene view of the VEGF gene showing it is up regulated in tumor samples Alternative Splice Analysis of Exon Data in Partek Genomics Suite eoo ChrView cv1 v1 Fac a Mean cyosand Hv i lt D wK ammm vascutar endothetiat growth FactorniA VEGFA a aa i K mind 3 D g E A a MMII yas NHB 131s WEE nt Ni aTa BSR EE AE E e is SEREN NM ARTIA RAT 9 5 3 9 FAQ Why is there so much variability in the intensities of individual probe sets within a transcript cluster gene Answer An exon intensity value is a product of typically only 4 probes and has a constrained probe selection region There is less genomic area to average the intensities so this variability is due to differences in both probe quality performance and physical sequence properties But remember within a given exon across two samples those properties are the same To look for differential expression across a gene monitor the parallel nature of the lines between two sample groups Two of the exon probe sets are drawn translucent they were filtered out of the analysis by opting to remove low intensity exons within the Alt splice ANOVA dialog These exons are drawn as transparent in the gene view to aid in isoform mapping The low expression can also be visualized by adding clustering and a heatmap to th2. 5 Tissuetye i lt D m P w 2456718 a im 6 8 a O 3 33 TissueType e 5 06 e Tumor 4 59 mine 2 O O S 3 65 B 3 18 O O 2 71 2 24 Lif COO i BO o Normal Tumor Figure 20 Differentially expressed exon between tissue types Alternative Splice Analysis of Exon Data in Partek Genomics Suite 18 Case 4 Finding genes with the highest probability of alternative splicing between tumor and normal You can also find genes that have the highest probability of alternative splicing between tumor and normal irrespective of differential expression e Rather than create a list sort the alt splice result sheet by the alt splicing TissueType column by right clicking on the column header and selecting Sort Ascending e The 13 ranked gene is shown here as it is easy to see the individual exons in genes with fewer probes Figure 21 e In contrast invoke Gene View on the 3 ranked COLI12A1 eoo ChrView cv1 v1 K A values Mean Cytoband HV I lt l w collagen triple helix repeat containing 1 CTHRC1 g a ee NM_138455 fa Figure 21 Example of a gene with a high likelihood of alternative splicing End of Tutorial This is the end of the Exon data analysis tutorial If you need additional assistance with this data set you can call our technical support staff at 1 314 878 2329 or email support partek com Alternative Splice Analysis of Exon Data in Partek Genomic
3. are red tumor samples are blue Notice that there is an outlier in the upper left corner of the plot you can examine the original chip image of the outlier e Hold on the Selection Mode button in the viewer s vertical mode bar Figure 2 and choose the User defined Selection Mode Alternative Splice Analysis of Exon Data in Partek Genomics Suite 2 z 260 J ia a Ei MERRENEN T Press amp hold then choose User defined Selection Mode EE ee ee ee ee ee ee ee 160 Figure 2 User defined Selection Mode e Use the default settings to show the Original Image in the viewer e Click on the outlier to select it the corresponding row in the spreadsheet will be highlighted sample is 8_4N e The chip image will open in the image browser Figure 3 800 Partek Image Browser original Browse 0 5 lt gt 8 4N CEL pimg 6553608 bytes 2560x2560 Figure 3 Viewing an individual chip image e Close the chip viewer before proceeding Returning to the PCA plot on the top of the scatter plot viewer click on the Plot Properties icon and configure the plot as follows Figure 4a Alternative Splice Analysis of Exon Data in Partek Genomics Suite 3 e Size the points by Age e Shape by Gender e Whilst still in the Shape section click on the Manual button and select the gender symbols from the drop down list Figure 2b e Connect by PatientNo e Click Apply AOG Plot Propert
4. splice core txt List Info Venn Diagram ANOVA Streamlined Advanced Specify criteria using the buttons in the left panel Use shift or control to selg ultiple criteria Click 1 A 2way alt splice core y p Save to generate a spreadsheet from the selected criteria Specify criteria Criteria Specify New Criteria 2 hama apes Combine cr Sel he alt splice sh Specify New Criteria Close Figure 13a The List Manager e Now select Specify New Criteria we will create a list of differentially expressed genes Alternative Splice Analysis of Exon Data in Partek Genomics Suite 11 e oo Configure Criteria Data source Name Spreadsheet 1alt splice alt splice core txt r Column 6 p value Tissue Type T Configure criteria Include p values unadjusted p value less than or 0 05 pass 5263 Figure 13 Create List dialog Name this Criteria A Ensure the alt splice sheet is selected and choose column 6 p value TissueT ype Set the default criteria to look for differentially expressed genes Do this by choosing unadjusted p value less than or equal to 0 05 from the Include p values drop down this value can be changed but we will use the default settings for the tutorial These criteria will generate a list of 5263 genes showing differential expression 5 of which are likely false positives Now create a second list to eliminate alternatively spliced genes Selec
5. zip e Extract the files to C Partek Example Data Colon Cancer Exon e Select File gt Open to invoke the File Browser and open the file Colon Cancer fmt Alternative Splice Analysis of Exon Data in Partek Genomics Suite 1 Exploratory Data Analysis Start by using Principal Components Analysis PCA to explore the probe set summarized exon level data PCA is a very effective method for exploring very high dimensional data e Under the QA QC section of the workflow select Principal Components Analysis PCA eoo kele Ww 1o pa T pa w 5 5 5 E gt n PLESU Fre ce Re oC Ww 1 i nN nN w co w oOo re an 3 or w wo w e R Oe ee ey o 6 n SSS ES SSS SS SSS SS SSS SS SSS SSS SSS SSS SSS SS SS SS gc re rs a ee ss ee a a ee Se et GEG Ee Ee een Gas See eee Se eee SS ee SS Ss ee ee SS LS SS SS SS n Nm Ar s H De WL PC 1 19 1 Figure 1 Viewing a PCA scatter plot of the data In the PCA plot each sphere represents a single sample chip which corresponds to a row in the spreadsheet Clicking on any sample in the plot will highlight both the sphere on the graph and the corresponding row in the spreadsheet Samples that are close together in the PCA plot are similar and samples that are far apart in the plot are dissimilar In the PCA plot shown in Figure 1 the color of the samples represents the tissue type normal samples
6. Alternative Splicing Analysis of Exon Array Data using Partek Genomics Suite 6 6 Overview This tutorial will demonstrate how to e Perform exploratory analysis using a PCA scatter plot e Identify genes that undergo differential expression and alternative splicing e Visualize exon expression patterns in a gene viewer Note the workflow described below is enabled in Partek version 6 6 Please contact the Partek Licensing Team at licensing partek com to request this version The screenshots shown below may vary across platforms and across different versions of Partek Description of the Data Set This experiment was performed using the Affymetrix GeneChip Human Exon 1 0 ST Array It includes 20 paired normal and colon cancer samples taken from 10 subjects Data and associated files for this tutorial can be downloaded by going to Help gt On line Tutorials from the Partek main menu Note it 1s recommended that you read Chapter 6 Pattern Visualization System chapter in the Partek User s Manual before going though this tutorial Open the Data File For instructions on how to import CEL files follow the Importing Exon Array Data into Partek Genomics Suite Import Tutorial tutorial from the Partek Tutorial and Data Repository Help gt On Line Tutorials To proceed with tutorial data open the Partek pre imported tutorial data that already exists in a Partek format FMT file e Download Colon_Cancer_DataAndImages Exon
7. atively Spliced Genes to invoke the Alternative Splice ANOVA dialog e Choose TissueType and PatientNo from the Experimental Factor s panel and move them to the ANOVA Factor s panel by clicking the Add Factor gt button e Select TissueType from the ANOVA Factor s panel and click the Add Factor gt button to move it to the Alternative Splice Factor s panel Figure 5 Alternative Splice Analysis of Exon Data in Partek Genomics Suite 6 e090 Alternative Splice ANOVA of Spreadsheet 1 Experimental Factor s ANOVA Factor s Alternative Splice Factor s perre Add Factors U 5 PatientNo 6 PatientNo 7 Gender 8 Age Add Interaction gt 2 11 Scan Date lt Remove Factor lt Remove Factor 2 Save Mode Load Model Contrast Advanced Exclude Skipped Probe Sets V Exclude probe sets with Max v signal lt 3 V only exclude probe sets with differential expression p value s gt 0 05 2 Restrict analysis to transcripts with fewer than markers 2 v Specify Output File Users Scott Partek Tutorial Data Exon Colon_Cancer_DataAndimages Exon alt splice core txt Browse OK Cancel Apply Figure 5 Configuring the alternative splicing analysis dialog Specify ANOVA model the maximum number of probe sets to be detected and the result file that will be generated e Check Exclude probe sets and differential expression p value s gt to filter out probe sets which do not express in any
8. ding to the specified category In the alt splice table each row represents a transcript cluster gene The first column is the number of exon probesets in that transcript cluster the second column is derived from the Alternative Splice Analysis of Exon Data in Partek Genomics Suite 7 meta probeset file and is equal to the transcript_cluster_id The values in this column act as keys for the transcript annotation file The remaining columns are the ANOVA results such as p values of the factors and interactions in the subsequent columns PartekGS File Edit Transform View Stat Filter Tools Wind e00 D a ll a l HQ 1 Colon_Cancer Current Selection 20 alt splice alt splice core txt 4 3 ANOVA 2way alt splice core exon level txt of markers Tran Clus 1 20 395 3807 2908 Figure 6 Reviewing the Alt splice result spreadsheet genes e are sorted by the p value of TissueType By default the results are sorted by the first p value which in this case is TissueType The p value TissueType shows genes that are differentially expressed between normal and tumor tissues are at the top with small p values Despite the label of alt splice for this table gene level results are displayed and the p values in this column are consistent with the p values of tissue type at gene level analysis not shown in this tutorial e Right click on the row header that corresponds to the VEGF gene 4 row of the alternativ
9. e plot Figure 12 e Seelct the Plot Properties icon a in the view tool bar e Select the Clustering tab and check Show clustering e Color dendrogram by TissueType e090 ChrView cv1 v1 E g Values Mean Cytoband Hv I Pl w yascitar endethetiat rowth facta A Nik AWEGFEAGJ aaa a nI Bal eI a s EEO S G G Amt 2 ooo e LEERI II l I Iul EE He iii ee af A Pee a x TissueType 9 Exon Expression Sf UZ Z a 7 5 3 Exon Expression l C E Figure 9 Viewing a gene view with clustering by tissue type Alternative Splice Analysis of Exon Data in Partek Genomics Suite 10 Common Analysis Scenarios In Partek the list manager can be used to specify numerous conditions to use in the generation of a list of genes or exons of interest The following scenarios will illustrate how to use the list manager to create commonly sought lists Case 1 Finding genes that are differentially expressed but NOT alternatively spliced e Open the alt splice sheet and invoke the List Manager Figure 13a dialogue by pressing Create Gene List under the Alternative Splicing Analysis section of the Exon workflow e The active sheet will be used as the default source you can manually choose which sheet to create the list from in the list manager e Click on the Advanced tab List Manager Spreadsheet Name 1 Colon_Cancer 1 alt splice alt
10. e splicing result spreadsheet e Select Gene View Orig Data from the pop up menu Figure 7 Alternative Splice Analysis of Exon Data in Partek Genomics Suite 8 1 2 3 4 5 of markers Transcript gene_assignm Gene Symbol RefSeq Cluster ID ent 1 lad 4958658 NM_004737 LARGE NM_ 0047 2 15 9163982 NM_00104027 ADAMTSL1 NM_ 0010 3 19 3807809 NM_00110165CXXC1 NM_0011C A 4010253 VEGFA NM_0010 Copy Pacte 103382 VIPR2 NM 003 102126 MHLE o NM_0021 ate o 0107991 LARIQ1 NM_ 0010 ilter Exclude 53274 BEST4 NM_ 15327 Filter Include Orig Data Filter Exclude Orig Data 114182 ORMDL2 _ NM_o141 101169 AQP NM_o011 Insert 02747 MAPK4 NM_ 0027 Delete 10 Right click select Gene View HTML Report joz522 WINFTAT NM_UU25 Sources of Variation 01704 NBAP NM o017 Dot Plot Orig Data ossaa EGM NM 0O56 Profile Orig Data pren k i 14294 MTRF1 NM_o042 Gene View Orig Data 15236 LPHN3 NM_0152 Transcript HTML Report j17639 DCHS2 NM_0176 vintwlolelolalololelelelelololelwlale a PE 0104816 STIL NM_ 0010 robe Set Keport 121101 CLDN1 NM 0211 Send to IPA ahd 73674 DCBLD1 NM_ 1736 Browse to Location 00194 HPRT1 NM_o0015 Create List 3599 ANOS NM_ 2135 Figure 7 Selecting a Gene View to visualize VEGF gene that is differentially expressed between two tissue types This gene is up regulated in the tumor samples Figure 8
11. ia 2 Name _ pass Criteria A 3437 p value TissueType unadjusted p value greater than 0 5 Combine criteria B 3393 alt splicing TissueType unadjusted p value less than or equal to 0 05 Union Or C 546 AandB Q Intersection And 2 Manage criteria Edit 2 Remove 2 Save List 2 t AhsS Close Figure 17 List creation for Case 2 genes with alternative splicing but without differential expression And example of a gene in this list can be seen in Figure 18 Alternative Splice Analysis of Exon Data in Partek Genomics Suite 15 eoo ChrView cv1 v1 D chromosome 9 open reading frame 140 C9orf140 zj 3 C9orf140 NM_178448 5 TissueType NN aN 3 5 OQ o B aa Figure 18 An example of a gene which is not differentially expressed but is alternatively spliced Here the gene follows a very different pattern between the two tissue types the most obvious being that the three exons at the 5 end of the gene are expressed in greater amounts in Normal tissue while the exon at the 3 end is expressed at a much greater level in the Tumor tissue It should also be noted that this is consistent across both probes in the exon Advantage of exon analysis e A 3 based expression strategy would detect this gene as increased in tumor relative to normal due to missing the vital differences at the 5 end e A whole transcript approach might miss this due to the compensating increase i
12. ies sp1 v1 Style Ellipsoids Labels Box amp Whiskers Titles Axes Color Legend Text Color s0 a _ _ IIID_ _ Size O Fixed 4 0 CT S Tissuetype Manual app h Shape Connect a a E mana sss l 6 PatientNo Outline s 5 a Style direct Line Width 1 pixels Mj Fogging Transparency 9 00 e Normal _ Mixed 2 ES ss Ce cot ey fe Figure 4a Configuring the scatter plot s Plot Properties dialog eoo Manual Shape spl Ral Use Manual Shapes F keme oo M Figure 2b Manually shaping the samples using gender symbols e The PCA plot is now configured to visually display the sample attributes Figure 3 Alternative Splice Analysis of Exon Data in Partek Genomics Suite 4 e090 Scatter Plot sp1 v1 Fair vices BOB Color 5 TissueType v 2030 a PCA Mapping 35 3 TissueType iai m Tumor S a 210 110 Qs PNI HHH x tae Ja 7 PC 2 9 33 A axe po 240 A r e E N e A a 1 440 363 287 211 135 60 16 92 168 244 320 PC 3 6 8 7 PC 1 19 1 Figure 3 Viewing a scatter plot of the 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 Notice that in this graph most of the lines that connected the two tissues from each subject are almost parallel to PC1 w
13. ith the exception of the subject that has an outlier normal tissue This means a lot of exons are differently expressed between the two tissue types To more clearly see the separation between normal and tumor tissue press the mouse wheel and drag it to rotate the plot or choose the Rotate Mode option s and press and drag the left mouse button Rotate the plot so to bring the Z axis PC 3 labels to the front Figure 4 Alternative Splice Analysis of Exon Data in Partek Genomics Suite 5 Scatter Plot sp1 v1 O i i cT A SSSSS Sas Pee eT eer ty LL cE EET N Y NJ Se er ee SS SIS ace 390 332 2 218 161 105 47 9 66 N Fas q ed E D db a gt N gt HA 123 Ke KA BOG Color 7 5 TissueType 2030 ils Q PCA Mapping 35 3 TissueType W Tumor SBS ooo f ee i pal LA HHH AHH OOS TIT TTT PANETE ee 22 il 3253535 S 5 a s HHH 39 A 253633 25555 S52 SSS SSS SHS 2s SSS SS ae ae nS SS SS SS ee ee ee Se E an p Ama 6 35 Figure 4 Viewing the rotated scatter plot to see tissue separation Notice that with the exception of the selected outlier the tumor tissue is on the left and the normal tissue is on the right e Close the scatter plot before continuing Alternative Splicing Analysis e Select Exon from the Workflows drop down e Select the Alternative Splicing Analysis button e Select Detect Altern
14. n normal relative to tumor in the 5 end e Only a detailed exon analysis properly enlightens the changes in exon specific gene expression within this transcript cluster Alternative Splice Analysis of Exon Data in Partek Genomics Suite 16 Case 3 Finding differentially expressed exons between tumor and normal The alternative splicing ANOVA outputs exon level information in addition to gene level information For this reason Partek is not limited to detecting differentially expressed genes and can also detect differentially expressed exons which can potentially be used as biomarkers e Open the ANOVA 2 way sheet and open the list creation dialogue by pressing Create Gene List under the Alternative Splicing Analysis section of the Exon workflow Figure 19 e Change the Include p values to Significant with FDR of and leave the value at 0 05 and select Create Figure 19 800 Configure Criteria Data source Spreadsheet TIANOVA 2way alt splice core exon level txt Column 3 p value TissueType ii Configure criteria Include p values significant with FDR of 0 05 g pass 9 Figure 19 List creation for exons to be used as bio markers e Visualize the output by right pressing on a row header and choosing dot plot Orig Data from the contextual menu Figure 20 Alternative Splice Analysis of Exon Data in Partek Genomics Suite 17 e080 Dot Plot sp1 v1 ik cw 5 TissueType av mom coor
15. ntensities to the gene lists Figure 15 Create List saving window e Select OK And example of a gene in this list can be seen in Figure 16 as before refer to Figure 10 find a gene of interest in the spreadsheet right click and select Gene View GALR1 in this example Alternative Splice Analysis of Exon Data in Partek Genomics Suite 13 e090 ChrView cv1 v1 le Roan cytoband wv ike PI D galanin receptor 1 GALR1 a SI g GALR1 NM_001480 B fay 18 5 xon Expression TissueType Figure 16 Example of differentially expressed but not alternatively spliced genes The parallel nature of the expression shows the GALR1 gene being expressed higher in Normal tissue than Tumor tissue but having the same expression profile across all exons Alternative Splice Analysis of Exon Data in Partek Genomics Suite 14 Case 2 Finding genes that are alternatively spliced but NOT differentially expressed This is often described as the cassette exon use case e Follow the same steps as described in Case 1 but choose to use a p value Tissue Type gt 0 5 and an alt splicing TissueType lt 0 05 Figure 17 List Info Venn Diagram ANOVA Streamlined Specify criteria using the buttons in the left panel Use shift or control to select multiple criteria Click Save to generate a spreadsheet from the selected criteria Specify criteria Criteria Specify New Criter
16. of the transcripts of these samples these should be checked by default e The Exclude Probe Sets option will remove any probe set exon that meets the specified limit Using the default options this will remove low expression non responsive exons In subsequent visualizations these exons will be displayed as translucent rather than black e The sub checkbox differential expression p values provides an override to the low expression limit Here an exon will be included in the analysis despite a low expression value if the exon displays a p value below the specified limit suggesting that the exon is differentially expressed e Leave the Restrict analysis box unchecked This option would limit the results to only transcript clusters genes that have fewer than the specified number of exons Sometimes this helps to ease interpretation as transcript clusters with many exons can be difficult to interpret The resulting ANOVA table has a column with number of probe sets thus enabling this same level of filtering to occur at a later point using the Partek Interactive Filter e Use the default file name for the Result file which will be generated in the same folder and the data file Alternatively the output file can be renamed as desired The ANOVA results will be displayed in two spreadsheets alt splice and ANOVA Figure 6 The ANOVA table displays each exon as a separate row and displays p values showing differential exon expression accor
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18. t Specify New Criteria again and use the same sheet Name this Criteria B Choose Column 8 alt splicing TissueType representing our alternative splicing p values To eliminate alternatively spliced genes change Include p values to unadjusted p value greater than and Value to 0 95 This second list now includes 501 genes showing NO alternative splicing 5 of which are likely false negatives To find the genes that meet both sets of criteria we now combine the lists Select both lists A and B and use the Intersection And option under the Combine Criteria header call this list C Alternative Splice Analysis of Exon Data in Partek Genomics Suite 12 e Beside each criterion the number of genes that pass is displayed along with the actual criteria used Figure 14 Specify criteria Criteria Specify New Criteria 2 Name pass Criteria A 5263 p value TissueType unadjusted p value less than or equal to 0 05 ombine criteria 501 alt splicing TissueType unadjusted p value greater than 0 Combi iteri B 501 licing Ti Type dj d han 0 95 Union Or 7 a 0 Intersection And E Manage criteria ct JG Remove 2 Save List 2 Figure 14 Viewing the criteria and results for each list e Select Save List e Check the lists you wish to save in this case select C Figure 15 8 OGO List Creator Check the criteria that you would like to save as lists A Output Options _ Add signal i
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