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1. 90 Doubplesclick Method ccc aan aU E BGRGoREP POSESSSS ORE POOLE ESE R AS RHEE 90 Contents 6 Filtering Column Data 0 0 eee 90 Editing Filtering Properties iussa sek ks RR RRGG 84 uk m Rex ow ene 3 Rx ER RA ESS 91 4 MC MERRRERERTRTTERRITIQOOSOTOTITI TTD DOTT ae eeeaedeeaue as on 91 hende R I 91 PONS ann eit oo dp Aan oa wee oa oe Re eds Oh oe See Ree as an aa a 92 Table OPUONS 2 2 55 sen 4 ark cate a bone eho aoa ee ee eee ee ae eentacnbetacehaees 92 Rearranging Column Orders 0 0 eee 92 Resetting Table Defaults 2 0 eee eee 92 Searching Keywords 0 0 0 0 e 92 Changing Condition vs Condition Palfitig5 sssaaa acce tees buen eI Ren 92 EXDORIFIOODEIORIS amp 2372 9 17 5 271 9939 eos 99293 59 2209 5 199 035 2 ete eta us oes 93 Exporting the Current Table with 1st Gene Symbols 0 0 020 002 0005 93 Exporting the Current Table 0 s 93 POOMMG AD M P C C 94 Saving Table Information wd ou lt x ipn ds beeen CR x Re e seule ay 94 Copy Selected ROW S 0 eee eee 94 Copy Selected ID s ee eee 94 Copy Selected Row s Gene Symbols 0 0 00 000 eee 95 Copy Exon INIOMMaUON La nc e acm neh bones aa deb RE ti ach e6 5 5 08emtenanGeeees 95 The selected exons are now copied to the Windows Clipboard for pasting 96 Accessing External Databases Internet Connection Required
2. E E Bl MAQCA Gene Avg Signal log2 8 73 gt gt 7 3 H H Bl Liver vs MAQCA Gene Fold Change linear 1 21 Genomic View pu Ej Show Junction El Truncate Intronic Region mm Using the Zoom Feature 1 Zoom Method 1 To zoom in on a region of interest left click hold then move the mouse to frame your selection Release the mouse button Figure 2 39 2 Zoom Method 2 Left click and hold onto the slider bar m then move it right to magnify your view Use the horizontal scrolling bar to center your view as this zoom method is not based on your selection Alternative Splicing Analysis 40 3 Undo Click to return to the default view Figure 2 39 Zoom View 7 1 TC10000779 hg 1 Find in Table Gene Symbol SLK Location chr10 105 726 959 105 788 991 Strand Positive Splicing Index MY eal Save PNG a Print Intensity Structure View A _ Show Junction Combine Neighboring Psrs Enable Tooltip lll Liver Gene Avg Signal log2 9 01 SEER EEE EEE EEE EEE EET eT E TET EE EEEE 99999990000000000009090009099000009090000009900000900000 E B Muscle Gene Avg Signal log2 9 15 lee Lidididdiddiidddddihidddddiiilli lilii LLL LLL LLLI Bl Liver vs Muscle Gene Fold Change linear 1 10 PSR10009289 hg 1 Using the Show Junction Option The option is checked by default and uses dotted lines to show
3. 96 Searching the Affymetrix NetAffx Website 00 eee 96 Appendix A AIONA r ar cary de qiie Qd bea qae par wy eee doente won dre m ee we 97 1 Tukey s Bi weight Average Algorithm 0 0 0 0c eee 97 2 ANOVA and Standard Deviation are Calculated using NMATH Package 97 3 Chromosome Naming Scheme 0 00 0 eens 97 4 Splicing Index SI Algorithm lllieel RR 98 Performing an Alternate Splicing Analysis lille 98 5 Benjamini Hochberg Step Up FDR controlling Procedure 00004 99 FOUC 42 a occo d 1 303 ua eerste RIS Geass Ead P RE SU Rd a Eq E RUE epa ih dt 100 H Hierarchical CUSTE uo ac a eae ae aan eh GE ee we eee od ob ee weed Bo 100 Introduction Transcriptome Analysis Console TAC TAC performs statistical analysis to obtain a list of differentially expressed genes and alternative splicing events IT also provides the visualization of genes exons junctions and transcript isoforms TAC runs analysis based on Expression Console EC generated CHP files El Note Affymetrix recommends that if you are using GCOS CEL files you should use the Data Transfer Tool DTT provided by Affymetrix to move the CEL files out of the GCOS directory Software and Hardware Requirements The table below shows the operating systems the recommended minimum requirements Table 1 1 P IMPORTANT Larger data file sizes associated with Whole Transcriptome arrays should be ta
4. a E UJ s 60 1E 06 es e E m 2 S a RS us At BOX y 5 go ae S T 4 x CIUS ONDE Toe uaa i X yX x XXX CX x NL f 0 1E 05 x x 40 0 0001 30 0 001 x 20 0 01 10 0 1 Lassoing Genes of Interest The Lasso tool allows you to select genes of interest inside the volcano plot ER If you have already lassoed while in the scatter plot the volcano plot displays the same probe sets in blue as graphs and tables are always in sync 1 Tolasso a gene or a group of genes hold down the left mouse button and use the cross hair cursor to encircle the gene s of interest Figure 3 35 Once a complete circle is made the genes of interest are highlighted in blue Gene Level Differential Expression Analysis 69 The data for these genes are also highlighted blue inside the table view as shown below Figure 3 35 Figure 3 35 Volcano Plot Graph and Table c2 Analysis 29 tac Analysis Result GESA Ed Split View Summary able Scatter Plot ano P Chromosome Summary Hierarchical Qlustering Comparison Liver vs Muscle Search Show Hide Columns v Export gt Clear Current Filter s Reset to Default 1C01001354 hg 1 T 4 56E 08 APCS 1C10000181 hg 1 7 34E 10 4 54 08 APBBIIP 1C11001966 hg 1 7 32E 10 4 53E 08 SLC29A2 1C02000260 hg 1 6 35 A 7 25E 10 4 49E 08 EML4 1C01001486 hg 1 E 648 7 24E 10 449E 08 FMO3 TC02004094 hg
5. smEg amEm sui sam amem sabm mug sabm DeEn impm Pepe pene ne All Gene Symbols First Gene Symbol Only View in UCSC Genome Browser Search Affymetrix NetAtix Copy Selected Row s ii Ctrl c TCiz0022g2 Copy Selected ID s Tcoinnn708 Copy Selected Row s Gene Symbols TCO4002793 hg 1 The selected gene level information shown on the left side of the splicing table are now copied to the Windows Clipboard for pasting Copy Selected ID s 1 Click to highlight light blue a ID or Ctrl left click to highlight multiple rows 2 Right click then click Copy Selected ID s to copy Transcript Cluster IDs Figure 2 29 Figure 2 29 Copy Selected ID s option TCO1003388 7 TCO4001274 TC12002283 TCO2004095 TC10002643 TCOADO1662 TCO1004981 Search NCBI Entrez Databases Search NCBI Gene Database Search Ensembl Databases CE sme sam ELEC INN CEE e aceea veneno All Gene Symbols rc View in UCSC Genome Browser Search Affymetrix NetAtix Copy Selected Row s Copy Selected ID s Copy Selected Row s Gene Symbols TC12002282 TCO1000 08 m 24 gt JN The selected TC IDs are now copied to the Windows Clipboard for pasting Copy Selected Row s Gene Symbols 1 Click to highlight light blue a row or Ctrl left click to highlight multip
6. Available Columns Description Eligible True or False statement regarding whether this PSR presents in all the transcript isoforms based on criteria specified in the Algorithm Options Use an eligible PSR to detennine gene expression if it is present in greater than or equal to 50 oof all the transcript imaforms As an example True 65 0096 means this PSR is eligible for determining gene intensity because it presents in 65 of all the transcript isoforms when the cutoff is 50 False 21 4296 means this PSR is not eligible for determining gene intensity because it only presents in 21 4296 of all the transcript isoforms when the cutoff is 5096 Note If no PSR is eligible based on the specified criteria all PSRs are considered as eligible Condition1 PSR JUC Expressed Condition2 PSR JUC Expressed True or False statement regarding whether this PSR or Junction is expressed in a condition based on percentage of samples that meets the DABG cutoff specified in the Algorithm Options A FRI ee m exphidsed m a condimen y geeaber than or equal ta 50 55 of Ehe hnapin haee DADO p value lepp tha 005 As an example True means this PSR or Junction met the criteria gt 50 samples has DABG 0 05 False means this PSR or Junction didn t meet the criteria gt 50 samples has DABG 0 05 Example only 2596 samples has DABG 0 05 Note Only PSR Junctions that are expressed in at least one condition can be in the table default
7. Check this box to show genes that have at least 1 PSR that passed your current filter criteria a Uncheck this box to show genes that have at least one PSR JUC that passed the current filter criteria Showing All Data Click the Show AllData button top right to show all data including the data that did not pass the default Splicing Index algorithm criteria and default table filter criteria A warning appears advising you that all current filters will be cleared Figure 2 23 Click Yes to proceed Figure 2 23 Show All Data Warning Show All Data All data including not expressed TCs PSRs JUCs will be shown and all filters will be cleared Are you sure you want to proceed Hu NOTE At any time click Reset to Default to auto check all 3 data options Rearranging Column Orders 1 Click on a column you want to move 2 Drag it left or right to its new location 3 Release the mouse button The column is now in its new position Resetting Table Defaults 1 Click Reset to Default to return the table to its factory setting Searching Keywords m NOTE The Search Tool is limited to finding matching strings It is not a full search engine 1 To search for a keyword within your table click inside the Search field then type your keyword 2 Click the Prev or Next buttons to search Figure 2 24 Figure 2 24 Search Tool Search SLK Prev Next Lr Alternativ
8. cele les 42 Using the Up and Down Regulated on Top Buttons 0 00 00 220 43 Using the Show Junction ORTON ua dtr rmm dodo gate debe n o dudebbe wee ooo we 43 Using the Truncate Intronic Region Option 0 0 0 ee 44 Using tne Enable TOOIUD OOUON xa s dote dia ge R440 ER oe Res BAGS REGE e He 44 Using the Zoom Feature 22 eee 44 Selecting Different Transcript Isoforms IDs Internet Connection Required 44 Linking Isoforms to an External Source Internet Access Required 44 How Transcript Isoforms are Sorted usus x edi Reed e Ree RU X RR hd 45 Linking Out to the UCSC Genome Browser Internet Access Required 45 Using the Get Score Button 0 eee 46 Chapter 3 Gene Level Differential Expression Analysis 20000 eee 47 Setting Up an Analysis Using Gene CHP Files 0 0 0 ee 47 Parsing Imported Data File Names Optional 0 20 00 0000 0 eee 49 Importing CHP Files into Different Condition Groups 0 0 0 0 00000 50 Importing Files using Drag and Drop 1 eee 51 Column Headers 0 0 s 56 Showing or Hiding Table Columns 0 0 00 000 56 xenueldeu dn 4 4 57 Double Click Method s saxorum qw eae dio ugar dev dor rod ave PERRA a pwned gs 57 P ANO COUNT OA MER 57 Editing Filtering Properties aucucx and acs ud QR aaaea 58 A P errre ranae E e E E ENSA 58 heres Aa PP 58 AIFI nuo neeqmdcdderdvmgex Rp 2 S
9. Transcriptome Analysis Console New Analysis Open Existing Result Library path C Users ppavic Desktop TAC_Library Installed Array Types Array Type Description Download Array Type Files v Internet Settings v Algorithm Options Parts of the Preferences Window Setting Up a Library Path IMPORTANT The first time you install TAC you must assign a path to your library folder The library folder contains the library and annotation files required to run the TAC software Figure 1 2 Library Path New Analysis Open Existing Result Library path Users ppavic Desktop TAC Library Do the following to create a new Library Path Figure 1 2 1 Click Browse right side of library path to create a new library folder path Chapter 1 Introduction 9 The following Browse For Folder window appears Figure 1 3 Figure 1 3 Browse For Folder Browse For Folder Please define a library path EE Desktop 153 Libraries B Pavich Peter j Computer hi Network A Control Panel Recycle Bin TACScreens 2 Click New Folder to choose a new Library folder Name the new folder then click OK Installed Array Types Displays the currently installed array types inside your library folder Figure 1 4 Figure 1 4 Installed Array Types Installed Array Types Array Type Description HTA 2 0 Alternative splicing and gene level
10. m E m E 06 E 05 001 0 01 X0 1 512 256 128 B4 32 18 B8 4 2 1 2 4 8 18 32 64 128 256 C02004093 hg 1 Signal 12 43 4 22 FC 294 96 pVal 3 39E 10 chr2 21225293 21225747 genesymbol TIP The best way to view individual probe sets is to make the graph full screen by un checking oan Chromosome Summary Graph Overview a The Chromosome Summary graph is a visual summary of your results on chromosomes Probe Sets are plots based on their chromosomal positions however Probe Sets without chromosomal positions are not plotted on the graph Probe Sets with non standard chromosome assignments are also not plotted Keep this in mind as you may see less probe sets in the chromosome summary graph than the scatter and volcano plots In the example below Figure 3 39 up regulated probe sets in Liver Conditionl vs Muscle Condition2 are plotted on the top of a chromosome in red The down regulated probe sets in Liver Condition vs Muscle Condition2 are plotted at the bottom of a chromosome in green Gene Level Differential Expression Analysis 72 The size of each chromosome summary square represents its gene size therefore the larger the square the larger the gene In some instances a square appears larger because it may contain more than one probe set due to the minimal pixel limitation Also a square might contain multiple probe sets due to minimal pixel limitation Figure
11. Gene Level Differential Expression Analysis 78 To Export Probe Sets See Exporting Probe Sets of Interest on page 78 Figure 3 49 Mouse Zoom Tool Result TCOY000342 hg 1 TC08001053 hg 1 FAM166B TC17001321 hg 1 COROS TC410001428 hg 1 DUSP13 TC11000051 hg 1 TNNI2 TC17001448 hg 1 CACNB1 TC01003817 hg 1 PTPN14 TC17000087 hg 1 SLC2A4 TC03000074 hg 1 CAND TC12001583 hg 1 ITGAT TC10000883 hg 1 TACC2 Exporting Probe Sets of Interest 1 Right click on the probe sets that are shown in the current window The following menu appears Figure 3 50 Figure 3 50 Right Click Mouse Menu Green Red Text file Blue Red Gray Print Save as PNG o Export names to Clipboard Copies your export data to the Windows Clipboard for pasting Text file Saves your export data as a txt file Changing Graph Colors 1 Right click on the graph The following menu appears Figure 3 51 Figure 3 51 Right Click Mouse Menu Clipboard Green Red Text file Blue Red Gray Print Save as PNG Gene Level Differential Expression Analysis 79 2 Click to select a desired color combination Green Red Colors range from green to red Blue Red Colors range from blue to red Gray Tones range from black to light gray Obtaining Information Related to Individual Probe Sets 1 Mouse over position the cursor over a probe set to show its details In the
12. 12 R8235090 PN A701107 6 10 5N rma exon all dabg 7 4TTma exon al dabg HuE l O stv2 rma exon all dabg CAUsersip4T Colon Total RNA S 25 2005 7 R8235090 PT A701098 4 9_ST rma exon all dabg_ HuEx 1_0 st v2 rma exon all dabg C Users p 5T Colon Total RNA S 25 2005 o R8235090 PT A701105 5 1 1T Tma exon all dabg 15 ST rma exon all dabg HuEl O stv2 rma exon alldabg CAUserstp ST Colon Total RNA5 25 2005 15 R8235090 PTA701090 e 13 7T rmz exon all dabg 1l GL rma exon all dabg HuEl O stv2 rma exon alldabg CAUserstp 6T Colon Total RNA S 25 2005 111 R8235090 PT A701107 6 Condition1 Condition2 Each condition must have at least one file Analysis File C Users ppavic Documents TAC AnalysisResults Analysis_33 tac Browse s Click Show Grouped Files checkbox Figure 4 4 to display sample file names and attributes even they have been added to various conditions Samples that have been added to different conditions get grayed out a Click Figure 4 4 to remove a file s from the Sample File window Parsing Imported Data File Names Optional This option gives you the ability to parse attributes from the sample file names and helps you set up conditions See Parsing Imported Data File Names Optional on page 49 Importing CHP Files into Different Condition Groups m IMPORTANT Customize your condition names first then add the CHP files into each condition 1
13. The L Maus dus option is checked by default It truncates the intronic regions to allow the exons to be more visible Uncheck this box to show the true genomic locations of PSRs exons within a gene including relative size and genomic coordinates Figure 2 46 m NOTE The Genomic ruler bottom Figure 2 46 only appears when the Truncate box is unchecked Figure 2 46 Un truncated Intronic View Genomic View Up Regulated on Top Down Regulated on Top 3 EUPTTBT E B uei ura ser aire Fere UCSC TR10005657 hg IH E H HHHH TR10001881 hg E HEI HH TR10004184 hg fo EY TR10000675 hg He HHHH TR10008732 hg H E H HH TR10001295 hg E H H H EX100129 l l l l l LL J 105 726 959 105 735 821 105 744 683 105 753 545 105 762 406 105 771 268 105 780 130 105 788 992 Using the Enable Tooltip Option The BEBE option is checked by default Mouse over position the cursor over a PSR or Junction to see the details related to that PSR or junction Using the Zoom Feature 1 Zoom Method 1 To zoom in on a region of interest left click hold then move the mouse to frame your selection Release the mouse button 2 Zoom Method 2 Left click and hold onto the slider bar m EM then move it right to magnify your view Use the horizontal scrolling bar to center your view 3 Undo Click to return to the default view Selecting Different Transcript Isoforms
14. 13 7T uma exon core dabg chp 14 7M ma exon all dabg chp 14 7M ma exon core dabg chp 15 8T ma exon all dabg chp 15 ST ma exon core dabg chp 15 8N ma exon all dabg chp 16 8N ma exon core dabg chp Files of type Exon aray CHP Files Cancel Total RNA Total RNA Total RNA Total RNA Total RNA Total RNA Total RNA Total RNA Total RNA Total RNA Total RNA Total RNA Total RNA Total RNA A a a a a a a 2233272343428233 4 7 E amp Ed Co EB P9 Eo amp bo o be be E 3 Single click Ctrl click or Shift click to select multiple files as shown above IMPORTANT You MUST only import 1 type of file into your conditions In the example below ma exon all dabg files were selected m NOTE To optimize the analysis Affymetrix recommends importing more than 1 sample per condition 4 Click 2 The selected files are now populated in the Sample File Window Figure 4 4 Chapter 4 Exon Level Differential Expression Analysis 83 Figure 4 4 Import Data into Sample File Window Transcriptome Analysis Console New Analysis Open Existing Result Preferences 4m Exon Level Differential Expression Analysis Remove Selected Parse File Names _ Show Grouped Files Name Array Type File Type File Path Sample Name Sample Type Sample Date sample number part number lot number pa 12 6N rma exon all dabg HuEx 1 0 st v2 rma exon all dabg CAUsersNpioN Colon Total RNA 5 25 2005
15. 55 mS aa sa 2m an 38 3m TC15002793 hg 1 CAPN3 Homo sapiens calpain 3 p94 Coding JUC15013945 hg 1 7442 5 63 345 0 65 2 28 7 60 RP11 164 J CAPN3 transcript variant 1 PSR15002552 hg 1 6 53 5 60 1 90 0 23 232 4 26 mRNA Homo sapiens calpain 4 3 p94 CAPN3 transcript JUC15013928 hg 1 7 96 7 07 1 85 112 0 89 4 03 variant 1 mRNA Homo JUC15013938 hg 1 6 51 ses 181 0 31 227 3 90 sapiens caipain 3 ps4 PSR15002554 hg 1 537 457 174 140 3 36 3 89 CAPN3 transcript variant 2 i mRNA Homo sapiens calpain JUC15013966 hg 1 6 14 5 36 1 72 0 60 2 56 3 88 7 1D TC10000779 hg 1 a in Table Gene Symbol SLK Location chr10 105 726 959 105 788 991 Strand Positive Splicing Index MY Ga Save PNG s Print 9 Intensity Structure View Show Junction Combine Neighboring Psrs Enable Tooltip Ea B Liver Gene Avg Signal log2 9 01 lll MAQCA Gene Avg Signal log2 8 73 Bl Liver vs MAQCA Gene Fold Change linear 1 21 i E E D BH Genomic View 4 __ Show Junction J Truncate Intronic Region J Enable Tooltip esaioo02 e r P Pfps efe 5m1000925359 9 7 e e b TEE TL Isoform Ensembl Transcripts Alternative Splicing Analysis 37 Visualization Tab Parts of the Visualization Tab Figure 2 34 Information header JALG i AW Find in Table Gene Symbol SLK Location chr10
16. 665 236 2249 0 000337 0 609967 015 0 5573 3 4 Gene Rows 10963 Exon Rows 21795 Selected Rows 0 Chapter 4 Exon Level Differential Expression Analysis 88 Parts of the Table Column Headers Table Options Column Headers The factory default columns and 2 preset filters bd are as shown Figure 4 9 See Table 4 1 for definitions of these columns Figure 4 9 Default Table Column Headers Chapter 4 Exon Level Differential Expression Analysis 89 Showing or Hiding Table Columns 1 Click the Show Hide Columns drop down menu to show or hide columns in the Exon table 2 Click outside the Show Hide Drop down menu to close it Table 4 1 Exon Table Columns and their Definitions Column entries in BOLD are factory defaults Available Columns Description Transcript Cluster ID ID of Transcript Cluster TC Gene Symbol Gene symbol for this transcript cluster Note RefSeq gene symbol is listed as the first gene symbol if there are more than 1 gene symbol Note A TC with no gene symbol may be auto assigned a public gene ID Description Gene Description for this TC Chromosome Chromosome for this transcript cluster See Chromosome Naming Scheme for a detailed description Genomic Position Genomic Start Stop position for this TC Public Gene IDs Public Gene IDs for this TC PSR Junction ID ID of Probe Selection Region PSR and Junction Probe Sets Condition1
17. Clear Current Filter s Reset to Default raeno aseo Jars TC10000181 hg 1 6 18 7 34E 10 4 54E 08 APBB1IP TC11001966 hg 1 7 02 907 7 32E 10 4 53E 08 SLC29A2 TC02000260 hg 1 923 635 725E 10 449E 08 EML4 TC01001486 hg 1 1034 648 7 24E 10 449E 08 FMO3 1C02004094 hg 1 12 60 4 94 724E10 449E 08 TC03000191 hg 1 9 05 642 7 23E 10 4 49E 08 XYLB 1C02004572 hg 1 3 93 7 20 716E 10 447E 08 TC17000165 hg 1 9 51 6 61 716E 10 447E 08 HS3ST381 TC04001090 hg 1 833 627 710E 10 4 44E 08 SEPSECS TC03002093 hg 1 8 99 610 707E 10 443E 08 MASP1 TCOYOO0342 hg 1 653 9 83 698E 10 4 40E 08 TCO8001599 hg 1 644 8 65 698 10 4 40E 08 TMEM6S TC15000442 hg 1 922 481 694E 10 4 38E 08 UPC TC12001429 hg 1 1136 7 56 6 90E 10 4 36E 08 SLC38A4 TC01001820 hg 1 8 56 6 30 6 89E 10 4 36E 08 MARCI M TC01001731 hg 1 648 8 65 6 88E 10 4 36E 08 PFKFB2 TC10002092 hg 1 8 00 1219 18 2 6 88E 10 4 36E 08 TC04001132 hg 1 10 37 7 67 6 84E 10 4 35E 08 C4orf34 TC11001465 hg 1 9 52 6 67 6 83E 10 4 35E 08 HPSS TC03001414 hg 1 9 07 7 03 6 82E 10 4 35E 08 MST1 AC TC11000238 hg 1 1191 8 23 6 76E 10 4 32E 08 SAA1 55 r 1C11000718 hg 1 10 61 7 04 6 76E 10 4 32E 08 CCND1 1C10000347 hg 1 546 8 01 674E 10 4 32E 08 PRKG1 TC22001430 hg 1 1028 657 671E 10 4 31 08 CYP2D6 TC04000828 hg 1 10 00 6 58 6 70E 10 4 31 08 MSMOL S 1C08001282 hg 1 5 66 8 18 6 70E 10 4 31 08 PDE7A TC10002971 hg 1 8
18. Click Open The selected files are now populated in the Sample File Window Figure 3 4 Figure 3 4 Import Data into Sample File Window Transcriptome Analysis Console Open Existing Result Preferences 4m Gene Level Differential Expression Analysis Remove Selected Parse File Names Show Grouped Files Name Array Type File Type File Path HTA2 Liver BetaSamplePool 1 v03 rma gene full HTA 2 O rma gene full C Users p HTA2 Liver BetaSamplePool 2 v03 rma gene full HTA 2 0 rma gene full C Users p C HTA2 Liver BetaSamplePool 3 v03 rma gene full HTA 2 0 C Users p HTA2 Liver BetaSamplePool 4 v03 rma gene full HTA 2 0 rma gene full C Users p I HTA2 Muscle BetaSamplePool 1 v03 rma gene full HTA2 Muscle BetaSamplePool 2 v03 rma gene full C Users p HTA2 Muscle BetaSamplePool 3 v03 rma gene full rma gene full HTA2 Muscle BetaSamplePool 4 v03 rma gene full rma gene full Condition1 Condition2 Add File s Here Add File s Here Click to Create New Condition Each condition must have at least one file Analysis File C Users ppavic Documents TAC AnalysisResults Analysis_13 tac a Click _ Show Grouped Files checkbox Figure 3 4 to display sample file names and attributes even they have been added to various conditions Samples that have been added to different conditions get grayed out m Click Remove Selected Figure 3 4 to remove a file s from the Sample File window Gene Level Di
19. Click on the Condition1 window header field to rename it to an appropriate Condition name Figure 4 5 2 Click to select and highlight the data you want to use for Conditionl 3 Click in the Condition window to add your selected files to the Condition window 4 If needed click to move selected files back to the Sample File window 5 If needed click to delete your current Condition and move all its files back to the Sample File window 6 Repeat the steps 1 3 above for Condition2 To create more than 2 conditions click Click to Create New Condition Figure 4 5 then repeat steps 1 3 above for your 3rd Condition Chapter 4 Exon Level Differential Expression Analysis 84 8 If needed edit your Analysis result file path and or name by clicking inside the Analysis File text field Figure 4 6 or click Browse to select a new file destination Importing Files using Drag and Drop Click Shift or click Ctrl to select a group of files 2 Click and hold onto the last file in the group then drag them into the appropriately labeled Condition window 3 Release the mouse button The Condition Normal window now contains your files 4 If needed click to move a selected file back to the Sample File window 5 If needed click to delete your current Condition name and move all its files back to the Sample File window Figure 4 5 Edited Condition name with samples Normal E eS 09 Colon Cancer rma exon
20. TCO1003388 7 amp A9E 13 TCO4201274 Search NCBI Entrez Databases 259E 11 548E 09 TCi2002283 Search NCBI Gene Database 830E 10 4 92E 08 EE TC10002643 TCOADO1662 View in UCSC Genome Browser Search Affymetrix NetAtix TCO2004085 Search Ensembl Databases 94E 10 5 45E 08 4 74E 10 3 43E 08 Copy Selected R TCO4002784 COPY Selected Row s 7 636 11 104E08 To12002282 COPY Selected ID s cea Seal Copy Selected Row s Gene Symbols All Gene Symbols TCO1000 08 TCOADO2793 hg 1 T Your selected rows with gene symbols are now copied to the Windows Clipboard Accessing External Databases Internet Connection Required 1 To link out to various external databases right click on a TC of interest The following menu appears Figure 3 28 Figure 3 28 Search Database menu 6 49E 13 875E 10 CRP smEm amEm smi sam mem saem Search NCBI Entrez Databases Search NCBI Gene Database Search Ensembl Databases Re C02004035 TC10002643 TCOADO1662 View in UCSC Genome Browser Search Affymetrix NetAtix TCO1004981 474E 10 3 43E 08 EL Copy Selected Row s TC04002794 7 63E 11 1 04E 08 E 4 TCiz00z2g2 Copy Selected ID s laser 31 z amp nena Copy Selected Row s Gene Symbols All Gene Symbo
21. felefex ele ex1ooozo3sng feje e E E E de 1 1 10 0 0 5umouzv05 8 8 8 j TRIOQQ4184hQ TR10000675 hg E e je ejex e e Exto003038hg JEJE PEF ESE E Jee E TR10008792 hg PEEP HEfEpexfefe amp a000 038n9 Efe feE F jeje je jeje 1111 15a000 9559 TR10001295 hg Ex10012906 hg Je felefexfejef 00 89 ee fe jeje jefe fet 1 1 1 mooz 0 0 0 0 0 0 0 0 0 j Up Regulated on Top button a Down Regulated on Top button a Show Junction checkbox Truncate Intronic Region checkbox Enable Tooltip checkbox UCSC button Alternative Splicing Analysis 43 Get Score button Isoform drop down menu Using the Up and Down Regulated on Top Buttons 1 Click to highlight a PSR then click button to bring the isoforms with high SI value PSR red to the top based on the Splicing Index and p value values of your highlighted PSR Note the sorting order of the PSR and Isoforms Figure 2 44 In the example below Figure 2 44 the top 2 isoforms plus the TR10004184 hg some or all of them are the likely isoforms in Liver Condition1 because these isoforms contain 2 PSRs with a high Splicing Index It also shows significant p values in the Liver Condition compare to Muscle Condition2 The bottom 2 isoforms plus TR10008792 hg some or all of them are the likely isoforms in Muscle Condition2 because these isoforms do not contain these 2 PSRs Fi
22. the contig name 1s appended to the regular chromosome name as in chrl g1000191 random If the chromosome is unknown the contig is represented with the name chrUn followed by the contig identifier as in chrUn_gl000211 Note that the chrUn contigs are no longer placed in a single artificial chromosome as they have been in previous UCSC assemblies See the sequences page for a complete list of hg19 chromosome names a The 9 haplotype chromosomes are as follows Name Accession UCSC chr Name HSCHR6 MHC APD CTG1 GL000250 1 chr6 apd hap1 HSCHR6 MHC COX CTG1 GL000251 1 chr6 cox hap2 HSCHR6 MHC DBB CTG1 GL000252 1 chr6 dbb hap3 HSCHR6 MHC MANN CTG1 GL000253 1 chr6 mann hap4 HSCHR6 MHC MCF CTG1 GL000254 1 chr6 mcf hap5 HSCHR6 MHC QBL CTG1 GL000255 1 chr6 qbl hap6 HSCHR6 MHC SSTO CTG1 GL000256 1 chr6 ssto hap7 HSCHR4 1 CTG9 GL000257 1 chr4 ctg9 hap1 HSCHR17 1 CTG5 GL000258 1 chr17 ctg5 hap1 Appendix A Algorithms 98 4 Splicing Index SI Algorithm Splicing Index algorithm is a way to measure of how much exon specific expression differs between two conditions after excluding gene level influences The algorithm first normalizes the exon and junction expression values by the level of gene expression and creates a ratio of normalized signal estimates from one condition relative to another Performing an Alternate Splicing Analysis In the Preference window you can customize algorithm parameters by typing values i
23. 105 726 959 105 788 991 Stranc Positive SplicingIndec E fealSave PNG 4 Print Intensity Information about the currently selected active TC Find in Table button Splicing Index scale Intensity scale Save as PNG ua Print Vertical and Horizontal Split View buttons Changing the Factory Set Scale Limits T Click to expand the information header and show the Splicing Index and Intensity scales Figure 2 35 Expanded Information Header E ID TC10000779 hg 1 Find in Table Gene Symbol SLK Location chr10 105 726 959 105 788 991 Strand Positive laa Save PNG s Print Splicing Index Color Scale Intensity Color Scale F Auto Scale Setting New Scale Ranges 1 Click to check the Auto Scale checkbox to use the minimum lower bound and maximum upper bound splicing index values intensities as the min max scale for display Figure 2 35 2 Click to uncheck the Auto Scale checkbox to set a fixed scale then enter your min and max number scales This newly fixed scale is now saved for use with other TCs and genes m NOTE For the Splicing Index scale the lower bound cannot be higher than 1 The upper bound cannot be lower than 1 Changing Scale Colors 1 Use the drop down color menus to change scale color properties Figure 2 35 Find in Table 1 Click Find in Table to restore the current displayed TC in the splicing viewer back to the current table view a Click to retur
24. 1st gene symbol only D All gene symbols Column to Export 1 Click either Gene level information only or All information Gene Symbols to Export 1 Click either 1st gene symbol or All gene symbols 2 Click OK The Save As window appears 3 Click on an existing folder or click New Folder to choose a new save location 4 Type a filename for the table then click Save The table is now saved as a txt file Exporting All Data 1 Click Export AII Data El Note Only currently paired data is exported including data in the hidden columns and the paired data s gene level information The Save As window appears 2 Click on an existing folder or click New Folder to choose a new save location 3 Type a filename for the table then click Save The table is now saved as a txt file Saving Table Information Use this copy feature to save table information to the Windows Clipboard then use this buffered information for pasting into other applications or websites Copy Selected Row s 1 Click to highlight light blue a row or Ctrl left click to highlight multiple rows Alternative Splicing Analysis 33 2 Right click then click to select Copy Selected Row s Figure 2 28 Figure 2 28 Copy Selected Row s option TOOL003389 h TCO4001274 TC12002283 TCO2004095 TC10002643 TCOADO1662 TCO1004981 Search NCBI Entrez Databases Search NCBI Gene Database Search Ensembl Databases
25. 2 48 Your internet browser opens to the appropriate ensembl org or ncbi gov website with detailed information about your selection How Transcript Isoforms are Sorted Isoforms with IDs appear on top Isoforms that fit a Conditionl criteria are sorted on top Isoforms that fit a Condition2 criteria are sorted at the bottom Choosing a different type of transcript isoform ID may leave some isoforms with blank gray boxes because not every transcript isoform has a Ensembl ID or RefSeq ID Figure 2 49 Mouse over any Transcript ID to see all the associated IDs Figure 2 49 Gray Isoform ENSTOO000540617 1 ENSTOO000468526 1 ENSTOO000374829 1 TRO1021818 hg i ucOOLlbeu 4 Linking Out to the UCSC Genome Browser Internet Access Required Gene 1 Click to highlight a gene of interest then click the button Your internet browser opens to the genomic ucsc edu website and displays detailed information about your selection Exons 1 Right click on an Exon A menu appears Alternative Splicing Analysis 46 2 Click View Exon in UCSC EE NOTE You must click the Exon not the PSR in order to link out Your internet browser opens to the genomic ucsc edu website and displays detailed information about your selected Exon Using the Get Score Button 1 Click the button to obtain the sorting scores The scores appear in a Windows Notepad file Figure 2 50 See How Transcript Isofo
26. 3 39 Chromosome Summary window Analysis_35 tac Analysis Result Summary Scatter Plot Volcano Plot mai I I CE t M 651 i tai T L Tee eT ri aT I E E d if L vil I ia mi I i I i I t La I TI T hi ul ui I Td F nl WI Ew I rut Ti T M m d 1 i 1 rie prt z EH NEDLENE S a unt lru t F aud 1 Nr E vF St HN gda PETS a ng 8 aa 1 el 11 A THELL a z E WT Ty wom a 1 ut rf eo 8 m E Split View i Clear Selection ag Save PNG s Print horn 1t aJ ia or g a a mg AP Gene Level Differential Expression Analysis 73 Previously lassoed genes in blue are also reflected in the Chromosome Summary graph Figure 3 40 Figure 3 40 Table and Chromosome Summary Analysis 35 tac Analysis Result SE Split View DEM Table S b Scatter Plot Volcano Plot hromosome Summar Hierarchical Clustering Comparison Liver vs Muscle Search B Up in Liver vs Muscle E Down in Liver vs Musch 73 Clear Selection aa Save PNG s Print Show Hide Columns Export 7 Clear Current Filter s Reset to Default A Wu dP E TUE ERI e di Ttg uy r m qu E A TEE DECOR Tl he e Liver Gene Symbo C iB E INS LEY quu ee NH y 1C010
27. 7 Parse File Name table with 2 attributes Separating Characters Parse Add Attributes Tissue o Attribute 6 o Liver rma alt splice dabg Liver rma alt splice dabg Liver rma alt splice dabg Liver rma alt splice dabg MAQCA v03 MAQTA v03 MAQCE v03 MAQCE v03 Muscle rma alt splice daba 6 To save your parsed attributes to the Same File window click Add Attributes The parsed file name attributes Figure 2 7 are added to the Sample File window as additional attributes Figure 2 8 Figure 2 8 Parsed results reflected in the Sample File window 4m Alternative Splicing Analysis Import Data Remove Selected Parse File Names _ Show Grouped Files Name Array Type File Type File Path Tissue Attribute 6 HTA2 Liver BetaSamplePool 1 v 03 rma alt splice dabg HTA 2_ 0 rma alt splice dabg LAHT Ada rma alt splice dabg HTA2_Liver_BetaSamplePool_2 v03 rma alt splice dabg LA liver rma alt splice daba R HTA2 Liver BetaSamplePool 3 v03 rma alt splice dabg L liver rma alt splice daba BY HTA2_Liver_BetaSamplePool_4 v03 rma alt splice dabg IHTA 2 0 rma alt splice dabg l i liver rma alt splice daba PO HTA2 MAQCA PolyA EC1 Lv irma alt splice dabg HTA 2 0 rma alt splice dabg LA socawo3 OM HTA2 MAQCA PolyA_EC1_2 v03 rma alt splice dabg LA l HTA2 MAQCB PolyA EC1 1 v03 rma alt splice dabg LA HTA2 MAQCB PalyA EC 2 v03 rma alt splic
28. Benjamini Yoav Hochberg Yosef 1995 Controlling the false discovery rate a practical and powerful approach to multiple testing Journal of the Royal Statistical Society Series B Methodological 57 1 289 300 MR 1325392 Appendix A Algorithms 100 6 Fold Change Fold change is a number describing how much the signal changes from an initial condition group to a final condition group Fold changes are represented in linear space 7 Hierarchical Clustering Clustering is performed on both probe sets and CHP files Distance metric used between objects is the Euclidean distance Distances between clusters of objects are computed using the complete linkage method maximum distance between a pair of objects in the two clusters Results are displayed in a heat map and dendrogram
29. Figure 2 17 Right click Column Menu Liver Bi Muscle Bi Fold sprit Bun niht Arr l a mua weight Avg weight Avg lines Transcript Cluster ar Sort By Ascending iL Sort By Descending y He f 72020044 YE Clear Column Filters TC100024 J Hide Column 2 Click Filter The following window appears Fold Change column example shown Figure 2 18 Figure 2 18 Filter Properties Fold Change linear Liver vs Muscle Editing Filtering Properties Alternative Splicing Analysis 1 Click the Or or And button to choose Or or AND logic 90 Oana 2 Click the symbol drop down menu s to select new symbol s Figure 2 19 Figure 2 19 Drop down Menu 3 Click inside the numbering field s 2 4 Click to add filter s 5 Click to remove filter s to enter new value s 28 Alternative Splicing Analysis 29 Clearing Filters Individual Filter 1 Right click on the filtered column you want to clear The following window appears Figure 2 20 Figure 2 20 Right click Column Menu rt By Ascending i Sort By Descending Y Filter Clear Column Filters xw Hide Column 2 Click Clear Column Filters The filter is removed All Filters 1 Click Clear Current Filters remove ALL filters from the table headers Table Options Use the Table Options Menu Figure 2 21 to customize your table view Figure 2 21 Tab
30. Next normalize each PSR or junction intensity using the gene intensity of that sample Figure 1 13 Normalized intensities from Condition is compared to normalized intensities from condition 2 using One way Between Subject ANOVA for the PSRs and junctions within a gene Chapter 1 Introduction 13 Configurable Parameter 5 After running ANOVA multi testing correction is performed using Benjamini Hochberg Step Up FDR controlling procedure for all the expressed genes and expressed PSRs Junctions expressed in at least one condition lA NOTE By default the Alpha level is set as 0 05 in Parameter 5 False Discovery Rate field Warnings If algorithm parameters have been changed to other values than default red warning text appears at the bottom of the Preferences window Below are two warning examples Figure 1 15 Figure 1 15 Warnings A PSR lunction is expressed in a condition if greater than or equal to is not a number of the samples have DABG p value less than 0 02 False Discovery Rate lt 0 01 Y Parameter s are different than the defaults Default 1 A blank non number entry generates a is not a number warning 2 Parameter s entered that are different from the default generate a Parameter s are different than the defaults warning Pm NOTE Warnings are not analysis specific Example If you change parameters for a splicing index then run a gene level analysis any change warnings remain o
31. Region PSR with Splicing Index linear 2 or Splicing Index linear gt 2 The group of genes listed below 2059 2565 and 6360 represent the genes that do not meet the Splicing Index criteria of Genes need to be expressed in both conditions Alternative Splicing Analysis 24 4 Displays the algorithm parameters used to perform the splicing analysis 5 This section displays the factory default filtering criteria results NOTE Only transcript clusters that pass in the Splicing table criteria are summarized 6 Displays each Condition name and the total number of CHP files in it Scroll down to reveal the other Conditions in your analysis Alternative Splicing Analysis Table Window Overview The table results are based on the algorithm parameters applied during the analysis The information shown is divided into 2 parts Figure 2 13 The left side of the table provides gene level information The right side of the table provides PSR Junction information and is organized by each transcript cluster Figure 2 13 Table window Analysis 24 tac Analysis Result Summary Table S Visualization EE M pw Expressed Genes Only Show Expressed PSR JUC Only V Have at least one PSR JUC10002228 hg 1 1155 5 31 5 96 11371 0 000005 PSR10003813 hg 1 1 27 1 81 14 48 0 000027 Cz TC10000327 hg 1 5 83 17 92 C100rf71 TC09000935 ng 1 1145 1193 140 PLIN2 Homo sapiens cDNA F
32. T pue ema pes sum pen ew 3 All Gene Symbols i View in UCSC Genome Browser Search Affymetrix NetAtix Copy Selected Row s ium Copy Selected ID s Copy Selected Row s Gene Symbols First Gene Symbol Only The selected gene level information shown on the left side of the exon table are now copied to the Windows Clipboard for pasting Copy Selected ID s 1 Click to highlight light blue a ID or Ctrl left click to highlight multiple rows Chapter 4 Exon Level Differential Expression Analysis 95 2 Right click then click Copy Selected ID s to copy Transcript Cluster IDs Figure 4 22 Figure 4 22 Copy Selected ID s option Search NCBI Entrez Databases Search NCBI Gene Database Search Ensembl Databases TOOL003389 h TCO4001274 TC12002283 TCO2004095 TC10002643 TCOADO1662 TCO1004981 mem smi sam mem saem z 5 View in UCSC Genome Browser Search Affymetrix NetAtix mug 343606 7 53bE 11 LO4E 08 All Gene Symbols UMEN EVI s aea rand never o OE Nn NES Copy Selected Row s Copy Selected ID s Copy Selected Row s Gene Symbols TC12002282 TCO1000708 The selected TC IDs are now copied to the Windows Clipboard for pasting Copy Selected Row s Gene Symbols Click to highlight light blue a
33. The following window appears Figure 4 11 Figure 4 11 Right click Column Menu Muscle Bi Fold pesi i ga c bres asse m nmm y WEIQTT Awg Limes 2 Click to select either Sort By Ascending or Sort By Descending Double Click Method 1 Double click on a column header to sort its data in an ascending order Double click on the same column header to sort its data in a descending order Filtering Column Data All table columns are filterable 1 Select a column then right click on it The following window appears Figure 4 11 Figure 4 12 Right click Column Menu Liver Bi Muscle Bi weight Avg weight Avg linea ar Sort By Ascending TCO1003 iL Sort By Descending j Y Filter E 72020044 WS Clear Column Filters TC10002t i 2 Click Filter Chapter 4 Exon Level Differential Expression Analysis 91 The following window appears Fold Change column example shown Figure 4 13 Figure 4 13 Filter Properties Fold Change linear Liver vs Muscle x Editing Filtering Properties 1 Click the Or or And button to choose Or or AND logic 90r And 2 Click the symbol drop down menu s to select new symbol s Figure 4 14 Figure 4 14 Drop down Menu 3 Click inside the numbering field s 2 to enter new value s 4 Click to add filter s 5 Click to remove filter s Clearing Filters Individual Filter 1 Right click on the fi
34. The green TCs are the ones down regulated in Liver Conditionl vs Muscle Condition2 The red TCs are the ones up regulated in Liver Conditionl vs Muscle Condition2 If you switch the condition pair or the filter criteria in the table the data in the graph will change accordingly Figure 3 29 Scatter Plot Graph x Analysis 29 tac Analysis Result ll Split View Volcano Plot Chromosome Summary Hierarchical Clustering Summary SC atter Plo E Up in Liver vs Muscle B Down in Liver vs Muscle i Clear Selection ka Save PNG amp Print Gene Level Differential Expression Analysis 65 Lassoing Genes of Interest The Lasso tool allows you to select genes of interest inside the scatter plot 1 To lasso a gene or a group of genes hold down the left mouse button and use the cross hair cursor to encircle the gene s of interest Figure 3 30 Once a complete circle is made the genes of interest are highlighted in blue The data for these genes are also highlighted blue inside the table view as shown below Figure 3 30 Scatter Plot Graph and Table en Analysis 29 tac Analysis Result arc x Split View Summary le Scatter Plot Volcano Plot Chromosome Summary Hierarchical Clustering Comparison Liver v vs Muscle Search E Up in Liver vs Muscle B Down in Liver vs Musch 73 Clear Selection aa Save PNG s Print Show Hide Columns M Export gt
35. chp MAQCA 4 1 HTA2 MAQCA PolyA EC1 1 v03 rma gene full chp 2 HTA2 MAOQCA PolvA EC1 2 vO03 rma aene full chp Gene Level Differential Expression Analysis 54 Summary information per this analysis NOTE Summaries vary between Gene Exon and Splicing analysis Array summary Total number of transcript clusters and numbers of coding and noncoding transcript clusters on this array Summary of total differentially expressed transcript clusters among all conditions union of differentially expressed transcript clusters from all unique condition pairs Also displays the summary of total differentially expressed Coding and Non Coding Transcript Clusters Summary of total differentially expressed transcript clusters per condition pair These are the Coding and NonCoding transcript clusters that pass the default filtering criteria listed in the Default Filter Criteria section below Shows total number of up regulated Coding and Non Coding transcript clusters in this condition pair Shows total number of down regulated Coding and Non Coding transcript clusters in this condition pair Shows the algorithm used to perform the Gene Level Differential Expression Analysis This section displays the factory default filtering criteria results NOTE Only transcript clusters that pass through these criteria are summarized in the Summary section above and listed in the Gene table as default Displays each Condition name and the total number of CHP
36. column header to sort its data in a descending order Filtering Column Data All table columns are filterable 1 Select a column then right click on it The following window appears Figure 3 15 Figure 3 16 Right click Column Menu Muscle Bi Fold weight Avg linea ar Sort By Ascending i Sort By Descending olumn Filters 7 Filter i O20 Clear Ce ers 2 Click Filter Gene Level Differential Expression Analysis 58 The following window appears Fold Change column example shown Figure 3 17 Figure 3 17 Filter Properties Fold Change linear Liver vs Muscle x Editing Filtering Properties 1 Click the Or or And button to choose Or or AND logic 90r And 2 Click the symbol drop down menu s to select new symbol s Figure 3 18 Figure 3 18 Drop down Menu 3 Click inside the numbering field s 2 to enter new value s 4 Click to add filter s 5 Click to remove filter s Clearing Filters Individual Filter 1 Right click on the filtered column you want to clear The following window appears Figure 3 19 Figure 3 19 Right click Column Menu NE mE Ar Sort By Ascending ij Sort By Descending Y Filter Clear Column Filters x Hide Column Gene Level Differential Expression Analysis 59 2 Click Clear Column Filters The filter is removed All Filters 1 Click Clear Current Filters remove ALL f
37. interest m NOTE The NetAffx Query Center is compatible with Windows Internet Explorer and Firefox Chrome is not supported at this time nm NOTE If a Probe Set or Transcript Cluster is not available an appropriate message appears Algorithms 1 Tukey s Bi weight Average Algorithm Tukey s Bi weight average is a method to determine a robust average unaffected by outliers Step 1 The median is determined to define the center of the data Step 2 The distance of each data point from the median is determined This distance is then used to determine how much each value should contribute to the average For example outliers that are far away from the median should contribute less to the average All end result values represented as a Bi weight average are shown in a log2 scale if the CHP files are summarized using RMA For more information go to http media affymetrix com support technical whitepapers sadd_whitepaper pdf 2 ANOVA and Standard Deviation are Calculated using NMATH Package Analysis of Variance ANOVA TAC computes and summarizes a traditional unpaired One Way single factor Analysis of Variance ANOVA for each pair of condition groups and for all condition groups if gt 2 conditions 3 Chromosome Naming Scheme In addition to the regular chromosomes the hg19 browser contains nine haplotype chromosomes and 59 unplaced contigs If an unplaced contig is localized to a chromosome
38. last file in the group then drag them into the appropriately labeled Condition window 3 Release the mouse button The Condition Liver window now contains your files Figure 2 9 Edited Condition name with samples Liver lt Condition HTA2 Liver BetaSamplePool 1 v03 rm HTA2 Liver BetaSamplePool 2 v03 rm HTA2 Liver BetaSamplePool 3 v03 rm HTA2 Liver BetaSamplePool 4 v03 rm 4 Repeat the steps 1 3 above for Condition2 5 To create more than 2 conditions click Click to Create New Condition Figure 2 9 then repeat steps 1 3 above for your 3rd Condition 6 If needed edit your Analysis result file path and or name by clicking inside the Analysis File text field Figure 2 10 or click Browse to select a new file destination Figure 2 10 Editable Analysis Result File Paths Analysis File Users ppavic Documents TAC AnalysisResults Analysis_2 tac Browse 7 After the Conditions have been labeled and populated click Run Analysis ER TAC auto saves your studies At any time click on the Open Existing Result tab to view recent analysis results Alternative Splicing Analysis 22 Please Wait appears then by default the Summary and the Splicing Viewer appear together using a split window view Figure 2 11 Figure 2 11 Summary and Splicing Viewer n x Analysis 28 tac Analysis Result soie Split View Liver vs Muscle vs MAQCA Analysis Typ
39. row or Ctrl left click to highlight multiple rows Right click on the selection then click to select Copy Selected Row s Gene Symbols 3 Click to select either All Gene Symbols all possible gene symbols for a Transcript Cluster or First Gene Symbol Only the first gene symbol that belongs to the Transcript Cluster Figure 4 23 Figure 4 23 Copy Selected Row s gene Symbols options 6 49E 13 8 75E 10 CRP TCO1003388 n7 TCO4001274 TC12002283 TCO2004095 TC10002643 TCOADO1662 TCO1004981 TC04002794 TC12002282 TCO10007 08 TCOADO2793 hg 1 Search NCBI Entrez Databases Search NCBI Gene Database Search Ensembl Databases smEm amEm suem sam pmem sabm View in UCSC Genome Browser Search Affymetrix NetAtix 474E 10 3 43E 08 7 63E 11 1 04E 08 EE l Copy Selected Row s Copy Selected ID s Casee 11 Tene no Copy Selected Row s Gene Symbols All Gene Symbols T Your selected rows with gene symbols are now copied to the Windows Clipboard Copy Exon Information 1 Click to highlight light blue a exon or Ctrl left click to highlight multiple rows Chapter 4 Exon Level Differential Expression Analysis 96 2 Right click then click either Copy Selected ID s or Copy Selected Row s to copy exons Figure 4 24 Figure 4 24 Copy Selected PSR JUC option x EZ A i t g A Bi
40. the relationship of the detected Junctions and PSRs Uncheck this box to hide these junction lines Figure 2 40 Figure 2 40 Show Junction Option ON and OFF Using the Combine Neighboring Psrs Option The Ez fex mE unEESS option is checked by default and link 2 PSRs together if they are physically right next to each other Uncheck this box to turn this option off Using the Enable Tooltip Option The BEBE eJ option is checked by default Mouse over position the cursor over a PSR or Junction to see the details related to that PSR or junction Figure 2 41 Figure 2 41 Tooltip Example PSR10009288 hg 1 Position chr10 105 770 574 105 770 666 Liver Bi weight Avg Signal 1592 9 19 Alternative Splicing Analysis 41 Identifying an Alternative Splicing Event using Structure View In the example below Figure 2 42 the Muscle Bi weight Avg Signal log2 of this PSR PSR10009288 is low 5 47 compared to the Liver Bi weight Avg Signal The left JUC10005163 and right JUC10005171 inclusion junctions signals are low in Muscle while the exclusion junction JUC10005158 signal is high in Muscle The Splicing Index results show a high SI value for this PSR high SI values for inclusion junctions and a low SI value for the exclusion junction This data indicates this PSR is preferentially included in the Liver but not in the Muscle Therefore this PSR is likely to be an alternative Cassette Exon Th
41. vs Condition2 Condition2 FDR p value Condition1 vs FDR adjusted p value based on Benjamini Hochberg Step Up FDR controlling Procedure Condition2 IMPORTANT All ANOVA p values from all genes are sent to FDR for correction ANOVA p value All One Way Between Subject ANOVA p value for all conditions Conditions FDR p value All Conditions FDR adjusted p value based on Benjamini Hochberg Step Up FDR controlling Procedure for all conditions IMPORTANT All ANOVA p values from all genes are sent to FDR for correction Gene Symbol Gene symbol for this transcript cluster Note RefSeq gene symbol is listed as the first gene symbol if there are more than 1 gene symbol Description Gene Description for this TC Chromosome Chromosome for this transcript cluster See Chromosome Naming Scheme for a detailed description Genomic Position Genomic Start Stop position for this TC Public Gene IDs Public Gene IDs for this TC Group Whether this TC is coding non coding or other Gene Level Differential Expression Analysis 57 Sorting Columns Right Click Method 1 Select a column then right click on it The following window appears Figure 3 15 Figure 3 15 Right click Column Menu 2 Click to select either Sort By Ascending A Z or Sort By Descending Z A Double Click Method 1 Double click on a column header to sort its data in an ascending order Double click on the same
42. window Select Analysis Alternative Splicing Analysis Gene Level Differential Expression Analysis Exon Level Differential Expression Analysis The New Analysis window appears Figure 4 2 Figure 4 2 New Analysis window Transcriptome Analysts Console Open Existing Result Preferences 4m Exon Level Differential Expression Analysis Remove Selected Parse File Names Show Grouped Files Name Array Type File Type File Path Click Import Data to import files for analysis Condition1 Condition2 Each condition must have at least one file Analysis File C Users ppavic Documents TAC AnalysisResults Analysis_35 tac 2 Click Import Data The following window appears Figure 4 3 It displays the data path you set up earlier and its files Chapter 4 Exon Level Differential Expression Analysis 82 EE NOTE The first time you launch TAC it asks you to define a path to store your library and annotation files For your convenience TAC retains this path information Affymetrix recommends you use the Expression Console library path you already configured C Users ppavic Desktop Exon_Data HuEx 1_O st v2 colon cancer Up One Level Ps 10 5N ma exon all dabg chp 10 5N ma exon core dabg chp 11 amp T ma exon all dabg chp 11 amp T ma exon core dabg chp 12 amp N ma exon all dabg chp 12 amp N ma exon core dabg chp 13 7T ma exon all dabg chp
43. 03389 g 1 1320 511 272 94 649E 13 875E 10 CRP Homo nh ukli a a We oe TC04001274 hg 1 1164 3 61 260 30 2 59E 11 5 48E 09 GC Homo s T TW a I NW Y 1C12002283 hg 1 10 79 2 93 23131 8 30E 10 4925 08 L Se ETTI it TC02004095 hg 1 1153 371 224 82 974E 10 5 46E 08 L DOPE HE RES NI s E y TC10002643 hg 1 11 30 349 22419 2 09E 09 9 41E 08 Y TC04001662 hg 1 12 92 sai 224 04 2 67E 12 1 59E 09 FGA Homo s Zu u anf TM LT TCO1004981 hg 1 1232 451 223 58 474E 10 3 43E 08 Uu IEBSIS EEUU BEBn IEt 1C04002794 hg 1 13 56 578 219 16 7 63E 11 1 04E 08 apone 11 01 3 28 212 91 4 58E 11 iem oj EF at j Nhl Ih ME a TC01000708 hg 1 1057 287 20915 5 79E 11 873E 09 ANGPTL3 Homos ft Tah y oe The TC04002793 hg 1 13 20 5 50 208 88 238 1 524 09 1C04000777 hg 1 1228 4 57 208 57 321E 12 1 72E 09 FGB Homo s ONT a ed a EEE TC02004094 hg 1 12 60 434 202 23 7 24E 10 4 49E 08 nie he pA poo TC10000675 hg 1 1176 412 19845 444E 11 7 44E 09 CYP2C9 Homo s 1C16001249 hg 1 1244 4 83 19440 3 96E 13 7 04 10 TAT Homo s 12 hl Li D I L I mrt TC05003424 hg 1 11 86 4 26 193 46 721E11 100E 08 C9 Homo s ug A LI TC04001663 hg 1 1121 374 177 35 188E11 4 75E 09 FGG Homo s TC10001546 hg 1 1167 4 20 17655 321E 10 2 69E 08 CYP2C8 Homo s CNN IUSSIT P 1j TC04001265 hg 1 10 67 322 174 52 197E 11 4 81E 09 UGT2B4 Homos CV Va a TC01003535 hg 1 1249 5 06 172 39 7 56E 13 8 86E 10 SERPINCL Homo s TC17000542 hg 1
44. 1 4 94 7 24E 10 449E 08 1C03000191 hg 1 i 6 42 7 23E 10 4 49E 08 XYLB 110 1E 11 1C02004572 hg 1 7 20 746E 10 447E 08 1C17000165 hg 1 6 61 716E 10 447E 08 HS3ST3B1 Homos 1C04001090 hg 1 6 27 7 10E 10 444 08 SEPSECS Homo s 1C03002093 hg 1 j 6 10 j 07E 10 4 43E 08 MASP1 Homo s TCOY000342 hg 1 9 83 6 98E 10 4 40E 08 1C08001599 hg 1 8 65 6 98E 10 4 40E 08 TMEM6S Homo s TC15000442 hg 1 4 81 6 84E 10 4 38E 08 IPC Homo s 1C12001429 hg 1 7 56 6 90E 10 4 36E 08 SLC38A4 Homos 1C01001820 hg 1 6 30 l 6 89E 10 4 36E 08 MARC1 M Homo s 1C01001731 hg 1 l 8 65 6 88E 10 4 36E 08 PFKFB2 Homo s 1C10002092 hg 1 1219 7 6 88E 10 4 36E 08 1C04001132 hg 1 7 67 6 84E 10 4 35E 08 C4orf34 Homo s 1C11001465 hg 1 6 67 6 83E 10 4 35E 08 HPSS Homo s 1C03001414 hg 1 7 03 6 82E 10 4 35 08 MSTi AC Homo s 1C11000238 hg 1 8 23 6 76E 10 4 32E 08 SAA1 5S r Homo L 1C11000718 hg 1 7 04 6 76E 10 4 32E 08 CCND1 Homo s 1C10000347 hg 1 8 01 6 74E 10 4 32E 08 PRKG1 Homo s 1C22001430 hg 1 6 57 6 71E 10 4 31E 08 CYP2D6 Homo s 1C04000828 hg 1 6 58 72 6 70E 10 4 31 08 MSMOL S Homo s 1C08001282 hg 1 8 18 6 70E 10 4 31E 08 PDE7A Homo s 1C10002971 hg 1 370 6 63E 10 4 29E 08 CYP2C19 Homos TCOX001416 hg 1 941 6 61E 10 4 29E 08 ARHGEF6 Homos 1C05001742 hg 1 6 31 6 53E 10 4 24E 08 ALDH7A1 Homos 1C17001201 hg 1 743 6 527 10 4 24E 08 SHMT1 Homo s TC08001521 hg 1 740 6
45. 1228 4 98 156 84 6 73E 13 875E 10 G6PC Homo s tea Pe OE D TC01001733 h9 1 11 48 4 20 15543 6696 11 9 55E 09 CABPA AX Homo s M PL Pe a g g EF 8 TC15000391 ng 1 1211 4 85 153 97 2420 13 678E 10 SLC27A2 Homo s f I i TC01001354 hg 1 12 30 5 04 153 22 7 38E 10 4 56E 08 APCS Homo s i Eg DT _ 1C12003240 hg 1 10 29 3 03 152 79 amp 25E 11 9120 09 SLCO181 Homos NIE ny uM ERR 1C02001628 hg 1 13 08 5 88 147 58 2 08E 13 6 35E 10 APOB Homo s LV ES i A ff I 1C04000789 hg 1 10 63 345 145 55 9 64E 12 3 10E 09 TDO2 Homo s 20 WWE NN oT i I 212 NM NEN E 1C09002906 ng 1 13 04 5 86 14516 1 62E 12 121E 09 ORM2 Homo s 1C03001997 hg 1 11 02 3 89 139 82 229E 11 5 13E 09 SLC2A2 Homo s z ME ng TC19001670 hg 1 1089 3 78 137 65 247E 10 2 02E 08 SULT2A1 Homos 22 feat 4h TC04000403 hg 1 1227 519 13544 1 83E 12 1 27E 09 ALB Homo s TCA ctg9 hapi 10 79 32 134 37 829E 11 1 09E 08 UGT2810 Homos x be 4 TC09001419 hg 1 1172 4 69 131 03 1 09E 09 5 92E 08 BAAT Homo s 1C03001023 g 1 1103 4 01 129 86 4 93E 11 7 85E 09 HRG Homos Gene Rows 1185 Selected Rows 17 Selecte YT fse 1 Click the blue up or down arrows shown to navigate through each highlighted gene or right click in the table to get the gene symbols IDs of these lassoed genes Using the Table to Investigate Pre Lassoed Selections See Saving Table Information on page 61 to search
46. 38 370 6 63E 10 4 29E 08 CYP2C19 TCOX001416 hg 1 6 81 941 6 61E 10 4 29E 08 ARHGEF6 TC05001742 hg 1 8 51 631 6 53E 10 4 24E 08 ALDH7A1 TC17001201 hg 1 1020 743 6525 10 4 24 08 SHMT1 TCO8001521 hg 1 4 63 740 648E 10 4 22E 08 ANGPT1 TC22000166 hg 1 615 1036 646E 10 4 22 08 MYO18B TC19002460 hg 1 743 10 90 637E 10 4 17E 08 zi Gene Rows 1945 Selected Rows 18 Selected 2 Click the blue up or down arrows shown to navigate through each highlighted gene or right click in the table to get the gene symbols IDs of these lassoed genes Using the Table to Investigate Lassoed Selections See Saving Table Information on page 61 to search and copy your lassoed selection Gene Level Differential Expression Analysis 66 Copying Lassoed Selections 1 Right click on the graph The following menu appears Figure 3 31 Figure 3 31 Right click Graph Menu Copy lassoed probe sets Copy differentially expressed probe sets Print Save as PNG Copy lassoed probe sets 1 Click Copy lassoed probe sets to copy your currently lassoed probes to the Windows Clipboard for pasting Copy differentially expressed probe sets 1 Click Copy differentially expressed probe sets to the Windows Clipboard for pasting Print 1 Click Print to print the graph to a pre conf
47. 40 044 6 261 776 6 291 183 6 346 413 6 399 365 6 420 169 6 551 817 6 610 482 6 733 977 and EP 619 321 373 203 and other U S or foreign patents Copyright 2013 Affymetrix Inc All rights reserved Contents Chapter 1 Chapter 2 Introduction ee ee tee eee eee 7 Transcriptome Analysis Console TAC 2 0 2 0 00 ee ee eee 7 Software and Hardware Requirements 0 00 ccc es 7 Stal AUOMIASIMUCHONS aeee eea a a a e a dyad tae E a E E oes 7 Preferences TT L 8 Parts of the Preferences Window llle 8 Setting Up a Library Pali 20 ows ae xac eee Ne EREE RARER 8 Installed Array Types 2 0 ne ee eens 9 Download Array Type Files Internet Connection Required 0 0005 9 Internet Settings 2 ee ee eee eens 10 Algotthim OPOS zx sse dcs dh 9 oad ed Ree eee eR EORR XE eee 3C ROR de S eee ees 11 Annotation Files llle 11 Alternate Splicing Analysis naana anaana 11 Las E C 13 Default Button 2 6 eee 13 Open Existing Result Tab 2 eee 13 Browsing For Existing Analysis Result 0 0 ee 14 Alternative Splicing Analysis llle 16 Setting Up an Analysis Using Alt Splice CHP Files llle 16 Parsing Imported Data File Names Optional lille 18 Importing CHP Files into Different Condition Groups 0 0 0 000 eee eee 20 Importing Files using Drag and Dr
48. 485 10 4 22E 08 ANGPT1 Homo s 1C22000166 hg 1 615 10 36 6 46E 10 4 22E 08 MYO188 Homo s 1C19002460 hg 1 743 10 90 6 37E 10 4 17E 08 512 256 128 64 32 16 8 16 32 64 128 256 512 120 1E 12 100 1E 10 anjea d 91 607 0p aouesyiubis a Gene Rows 1945 Selected Rows 18 Select 2 Click the blue up or down arrows shown to navigate through each highlighted gene or right click in the table to get the gene symbols IDs of these lassoed genes Using the Table to Investigate Lassoed Selections See Saving Table Information on page 61 to search and copy your lassoed selection Copying Lassoed Selections 1 Right click on the graph The following menu appears Figure 3 36 Figure 3 36 Right click Graph Menu Copy lassoed probe sets Copy differentially expressed probe sets Print Save as PNG Copy lassoed probe sets 1 Click Copy lassoed probe sets to copy your currently lassoed probes to the Windows Clipboard for pasting Gene Level Differential Expression Analysis 70 Copy differentially expressed probe sets 1 Click Copy differentially expressed probe sets to the Windows Clipboard for pasting Print 1 Click Print to print the graph to a pre configured printer The Print window appears 2 Configure the printing options as you normally would
49. 8 hg 1 TC02002469 hg 1 ITGB6 TC21000570 hg 1 TC14002034 hg 1 TC05001732 hg 1 RP11 43D2 5 TC14000952 hg 1 U6 TC17002572 hg 1 TC14001151 hg 1 AKO96898 TC15001780 hg 1 DQ601279 TC15000804 hg 1 DQ601279 TC15000782 hg 1 DQ601279 TC15000769 hg 1 DQ601279 TC12002953 hg 1 ConditionzMuscle File HTA2 Muscle BetaSamplePool 2 v03 ID TC15000769 hg 1 DQ601279 Signal 6 05 Efi IMPORTANT The processing limit of the Hierarchical Clustering Graph is 5000 genes Selecting Interesting Probe Sets There are 3 methods to select interesting probe sets Gene Level Differential Expression Analysis 77 Method 1 Zoom Slider 1 Click on the Zoom slider bar Figure 3 47 then hold down the left mouse button and move the bar down to magnify point s of interest Figure 3 47 Zoom and Scroll Tool oom scroll Method 2 Scroll Slider 1 Click on the Scroll slider bar Figure 3 47 then hold down the left mouse button and move the bar up or down to scroll through the results Method 3 Mouse Zoom 1 To zoom in on a region of interest left click hold then move the mouse to frame your selection Figure 3 48 Figure 3 48 Mouse Zoom Tool BEEN b 2 Release the mouse button Your region of interest is now magnified revealing its Transcript Cluster IDs and their Gene Symbols left column Figure 3 49 You can also right click on a probe set of interest then export it
50. Bi weight Avg Tukey s Bi weight average of exon intensity of all the samples in a condition Bi weight average Signal log2 of sample 1 exon1 intensity sample 2 exon intensity sample N exon1 intensity Condition2 Bi weight Avg Signal log2 Condition1 Standard Deviation Standard Deviation of exon intensities from all samples in a condition Condition2 Standard Deviation STDEV of sample 1 exon1 intensity sample 2 exon1 intensity sample N exon1 intensity Fold Change linear Condition1 This shows the fold change in linear space of Condition1 vs Condition2 VS Condition2 2 Condition Bi weight Avg Signal log2 Condition2 Bi weight Avg Signal log2 ANOVA p value Condition1 vs One Way Between Subject ANOVA p value Condition 1 vs Condition2 Condition2 FDR p value Condition1 vs FDR adjusted p value based on Benjamini Hochberg Step Up FDR controlling Procedure Condition2 IMPORTANT All ANOVA p values from all exons are sent to FDR for correction Sorting Columns TAC uses a 2 level sorting process for the exon information right side of the Exon table First it sorts exon within each gene based on the exon data then it sorts each gene Figure 4 10 Figure 4 10 2 Level Sort Results Example Conditionl Bi weight Avg Signa Chapter 4 Exon Level Differential Expression Analysis 90 Right Click Method 1 Select a column then right click on it
51. Figure 4 8 Figure 4 8 Exon Table Analysis_34 tac Analysis Result Ta Table Comparison vs Search Show Hide Columns M Export 7 Clear Current Filter s Reset to Default w unl o en Lh un 3759335 gap junction protein gamma 9 i I 41 0 002324 0 749784 1 45kDa 9 0 002820 0 762914 9 i 0 003695 0 788452 3759342 T l 8 39 0 028490 0 925210 8 16 0 000245 0 590078 6 97 0 000888 0 662675 6 04 0 000331 0 609967 5 77 0 002177 0 741288 4 61 0 001483 0 699401 9 223 0 033223 0 932979 2 68 19 07 0 017762 0 898500 2345061 CLCA4 chloride channel accessory 4 2345085 60 190 17 58 0 028575 0 925210 2345078 1645 0 025509 0 918107 2345074 1291 0 040962 0 940868 2345086 863 496 123 0 027639 0 923545 w w N wi un o tu us B w m w lt un o e 2 N NI N N N NJ w w wj wj w a ajala a vj ut un nj un un o 0 o o0 o oO I O O OF w W W W w eS es PSR a eS Ol N wl i Ol u w uo mam 2 230 6M 0028 asso70 24507 2042189 _ 094227 transmembrane and 3751864 36 70 0 003471 0 782676 immunoglobulin domain F MEET 3751867 844 0 014050 0 879713 Ini 3751861 7 03 0 019950 0 905471 3751868 7 01 0 010763 0 862316 3751870 4 03 146 5 92 0 020530 0 908261 3751862 446 1 99 5 54 0 000642 0 646947 3751869 3 62 0 027429 0 922599 synaptopodin 2 2741181 29 96 0 000257 0 593406 2741192
52. File Names table now appears as shown Figure 2 6 Figure 2 6 Parse File Name table Separating Characters Parse Add Attributes Attribute 1 CQ b Attribute QQ IP Attribute QQ P Attributes P Attributes O P Attributes Q Attribute Q HTA2 Liver BetaSamplePool 1 v03 rma alt splice dabg HTA2 Liver BetaSamplePool 2 v03 rma alt splice dabg HTA2 Liver BetaSamplePool 3 v03 rma alt splice dabg HTA2 Liver BetaSamplePool v03 rma alt splice dabg HTA2 MAQUA PolyA EC1 1 w03 rma alt splice dabg HTA2 MAQUA PolyA EC1 2 w03 rma alt splice dabg HTA2 MAQCB PolyA EC1 1 w03 rma alt splice dabg HTAZ2 MAQCB PolyA EC1 2 w03 rma alt splice dabg HTA2 Muscle BetaSamplePool 1 v03 rma alt splice dabg 5 Do the following to clean up attributes parsed from the sample file names Click inside any of the Attribute text fields Attribute1 to type in a new Attribute name If desired click to change the default separating character for combing your parsed attributes The default separating character is a period These separating characters are useful if you ever want to combine multiple parsed attributes to a new attribute Click to join together a neighboring attribute column Click Q to remove an attribute column from the table Alternative Splicing Analysis 20 In the example above since Attribute 1 3 4 5 and 7 are redundant and not useful they are removed The table now appears as shown Figure 2 7 Figure 2
53. IDs Internet Connection Required By default the transcript isoforms are displayed using Affymetrix Transcript Isoform IDs 1 Click on the drop down arrow then click to select a different type of transcript isoform IDs Figure 2 47 Ensembl Transcripts External public Transcript ID source RefSeq Genes External public RefSeq Transcript ID source Other Other public Transcript IDs Figure 2 47 Isoform drop down menu Afix Transcript Isoforms Affx Transcript Isoforms Ensembl Transcripts RefSeq Genes Other Linking Isoforms to an External Source Internet Access Required 1 Right click on a Isoform of interest Alternative Splicing Analysis 45 The following menu appears Figure 2 48 Figure 2 48 Copy ID and Link to Menu fo Copy ID Link to Ensembl 2 Click on Link to selection Selecting RefSeq links to the NCBI website Selecting Ensembl ID links to the Ensembl website If the Isoform does not have a RefSeq or Ensembl ID associated with it then linking to an external website is not available If this 1s the case click Copy ID to copy the Isoform ID to Windows Clipboard for pasting to another source NOTE Available Link to selections are depend on the Isoform selected from the Isoform drop down menu Figure 2 47 Example If Ensembl Transcripts is selected then the Link to NCBI is grayed out unavailable as shown in Figure
54. KG Affymetrix User Manual Transcriptome Analysis Console TAC Software maaa ii ae For Research Use Only Not for use in diagnostic procedures a P N 703150 Rev 1 Trademarks Affymetrix Axiom Command Console DMET GeneAtlas GeneChip GeneChip compatible GeneTitan Genotyping Console NetAffx and Powered by Affymetrix are trademarks or registered trademarks of Affymetrix Inc All other trademarks are the property of their respective owners Limited License Subject to the Affymetrix terms and conditions that govern your use of Affymetrix products Affymetrix grants you a non exclusive non transferable non sublicensable license to use this Affymetrix product only in accordance with the manual and written instructions provided by Affymetrix You understand and agree that except as expressly set forth in the Affymetrix terms and conditions no right or license to any patent or other intellectual property owned or licensable by Affymetrix is conveyed or implied by this Affymetrix product In particular no right or license is conveyed or implied to use this Affymetrix product in combination with a product not provided licensed or specifically recommended by Affymetrix for such use Patents Arrays Products may be covered by one or more of the following patents and or sold under license from Oxford Gene Technology U S Patent Nos 5 445 934 5 700 637 5 744 305 5 945 334 6 054 270 6 1
55. LJ36877 Coding JUCO9006864 hg 1 1113 80 83 109 46 0 000010 AK094196 og su acae PSR09012529 hg 1 1041 37 01 5140 0 000004 I Tiipl PLIN2 transcript variant 1 JUCO9006867 hg 1 25 39 4 05 9 26 36 98 0 000763 25 45 0 54 9 72 Homo sapiens chromosome 10 open reading frame 71 C100rf71 transcript variant 1 mRNA Homo sapiens chromosome 10 open reading frame 71 C100rf71 transcript variant 1 mRNA Homo sapiens chromosome 10 open reading frame 71 C100rf71 transcript variant 2 mRNA m n JUC09006875 hg 1 5 29 414 2 22 6 13 7 79 3 16 0 030391 posna aaf s um 5 35s as oamse AX748175 fis clone TESTI2027238 PSRO1022404hg1 Homo sapiens pre B cell g 63 soj 13 leukemia homeobox 1 PBX2 nai uei te aa Homo sapiens pre B cell leukemia homeobox 1 PBX1 bebe aed oan PSROI022388ng1 728 728 100 009 245 580 0 000038 leukemia homeobox 1 PBX1 transcript variant 3 mRNA PSROI022406ng1 549 563 110 160 409 S61 0000085 transcript variant 3 mRNA g 532 560 22 TC01001435 hg 1 714 Ducwoigmi sm ta 3 aaa aaa 5 eamus pem 3 ase asa 3s sa 4 ames Psonn es 3 ase oa 2 4 ooer pswiwwmei soa 3s ae 2 aoe 3se oaoniso Psoas a sea ase aaa Au 3a5 amu PSR01022411 hg 1 5 38 6 15 1 71 1 84 3 62 343 0 003402 4 b Gene Rows 9452 Exon Rows 61935 Selected Rows 0 Parts of the Ta
56. Muscle MAQCA Liver Muscle MAQCA Liver Muscle MAQCA Liver Muscle MAQCB Liver Muscle MAQCA Liver Muscle Liver Muscle MAQCB Liver Muscle MAQCAB Liver Muscle MAQCB Liver Muscle MAQCB Liver Muscle Liver Muscle Liver Muscle Date Created 12 18 2012 1 06 19 PM 12 18 2012 11 14 02 AM 12 18 2012 10 42 03 AM 12 18 2012 10 32 15 AM 12 18 2012 10 00 08 AM 12 13 2012 9 57 57 AM 12 10 2012 2 52 29 PM 12 10 2012 10 40 45 AM 12 5 2012 11 22 52 AM 12 4 2012 4 30 54 PM 11 16 2012 12 48 50 PM 12 4 2012 10 46 21 AM 11 28 2012 2 02 22 PM 11 28 2012 1 23 34 PM 11 28 2012 1 18 48 PM 11 19 2012 10 04 21 AM 11 8 2012 1 03 33 PM 11 12 2012 3 25 28 PM Last Opened 12 18 2012 1 25 37 PM 12 18 2012 1 03 09 PM 12 18 2012 10 49 54 AM 12 18 2012 10 38 14 AM 12 18 2012 10 35 47 AM 12 17 2012 3 22 48 PM 12 17 2012 1 04 44 PM 12 10 2012 2 49 29 PM 12 10 2012 10 31 59 AM 12 5 2012 11 01 10 AM 12 4 2012 3 37 22 PM 12 4 2012 3 32 21 PM 12 4 2012 10 14 25 AM 11 28 2012 1 57 43 PM 11 28 2012 1 19 52 PM 11 20 2012 8 56 24 AM 11 16 2012 11 26 53 AM 11 16 2012 11 26 51 AM Browse For Existing Analysis Result 2 Double click on a recent study or single click on it then click Open Selected File After a few moments your recent study opens in the same state you last left it Browsing For Existing Analysis Result Do the following if you cannot locate a study 1 TAC stores twenty
57. PolyA EC1 1 v03 rma alt splice dabg AHTAMat HTA2 MAQCA PolyA EC1_2 v03 rma alt splice dabg AHTA dat HTA2_MAQCB_PolyA_EC1_1 v03 rma alt splice dabg AHTA dat HTA2_MAQCB_PolyA_EC1_2 v03 rma alt splice dabg HTA2_Muscle_BetaSamplePool_1 v03 rma alt splice dabg HTA 2 0 rma alt splice dabg LAHTA dat HTA2 Muscle BetaSamplePool 2 03 rma alt splice dabg HTA 2 0 rma alt splice dabg LAHTA dat HTA2 Muscle BetaSamplePool 3 v03 rma alt splice dabg HTA 2 0 rma alt splice dabg L HTAMdat HTA2 Muscle BetaSamplePool 4 v03 rma alt splice dabg HTA 2 0 rma alt splice dabg LAHTAMdat Condition1 Condition2 New Condition Each condition must have at least one file Analysis File C Users ppavic Documents TAC AnalysisResults Analysis_26 tac a Click Show Grouped Files checkbox Figure 2 4 to display sample file names and attributes even after they have been added to conditions Samples that have been added to different conditions are displayed in gray Click Remove Selected Figure 2 4 to remove a file s from the Sample File window Parsing Imported Data File Names Optional This option gives you the ability to parse attributes from the sample file names and helps you set up conditions 1 Click Parse File Names Alternative Splicing Analysis 19 The following window appears Figure 2 5 Figure 2 5 Parse File Name window Parse names to attributes CU uU Uk X a m Change files names i
58. Using the Table to Investigate Pre Lassoed Selections 00 0000 2 eee eee ES Copying Lassoed Selections 0 ee eee 43 Clearing Lassoed Selections llle 74 Changing Graph Colors llle 74 Hierarchical Clustering Graph uus ae atre mera Fe ao e doi ero acd e E OR eee 25 Selecting Interesting Probe Sets llli 76 Exporting Probe Sets of Interest lille 78 Chanding Glapi COM MT 78 Obtaining Information Related to Individual Probe Sets 00 0 000 79 woe MMM O 79 Signal Intensity Scale uuo ducere dead QI EAS ue SSG Eee eo Ss requies Eee Se ES 80 Saving a Hierarchical Cluster uuu uq 9 65 o deed Hed bay ede ee bac Bowne eas 80 PouWMMO QOUOM 2ees ence ote ededs eee dors uae eee ee ogc ee he aneeeu yes 4 80 Exon Level Differential Expression Analysis 00000eeeeee 81 Setting Up an Analysis Using Exon CHP Files 0 00 0 ee 81 Parsing Imported Data File Names Optional 0 0 0 00 000 0c ccc eee 83 Importing CHP Files into Different Condition Groups 000 00 83 Importing Files using Drag and Drop iuuenis dee ox occ eu dived sas 84 Exon Level Differential Expression Analysis Summary Window Overview 85 Exon Level Differential Expression Analysis Table Window Overview 87 Column Headers uo a patus acere dead aki uw o o ara edu Pe RR erae RJV 88 Sorting Columns e 89 eaedem P
59. a Gene Level Differential Expression Analysis 56 Parts of the Table Column Headers Table Options Column Headers The factory default columns and 2 preset filters kd are as shown Figure 3 14 See Table 3 1 for definitions of these columns Figure 3 14 Default Table Column Headers Fold Change ANOVA p fg linear Liver value Liver San b x ae PC g am vs Muscle se vs Muscle 2 Click outside the Show Hide Drop down menu to close it Table 3 1 Gene Table Columns and their Definitions Column entries in BOLD are factory defaults Available Columns Description Transcript Cluster ID ID of Transcript Cluster TC Condition1 Bi weight Avg Tukey s Bi weight average of gene intensity of all the samples in this condition Bi weight Signal log2 average of sample 1 gene1 intensity sample 2 gene1 intensity sample N gene1 intensity Condition2 Bi weight Avg Signal log2 Condition1 Standard Deviation Standard Deviation of gene intensities from all samples in this condition Condition2 Standard Deviation STDEV of sample 1 genet intensity sample 2 gene intensity sample N gene1 intensity Gene Fold Change linear This shows the fold change in linear space of Condition1 vs Condition2 Condition1 VS Condition2 2 Condition1 Gene Avg Signal log2 Condition2 Gene Avg Signal log2 ANOVA p value Condition1 vs One Way Between Subject ANOVA p value Condition 1
60. aas ODED Se Rosen E maps S A diem moa eo 59 Chapter 4 Contents 5 Table Options 2 0 RR RR sl 59 Rearranging Factory Set Columns 0 naana aeaa 59 Reset IO DGlAUIC 42 eoe sine oes nterra eee eee peed waded eee eee eas 59 Searching Keywords 0 0 0 e 59 Semis MP 60 Exporting the Current Table with 1st Gene Symbols 0 0 00 00 00000 60 Gene Symbols tO EXPO acne dia beoe gra a edo ae RC RE ud CEES Role dec A d n 61 zqsenmujeri Pro E C r w 61 Saving Table Information 0 0 eee 61 COO selected HOG PPP 62 Copy Selected Row s Gene Symbols 0 anaana aaaea 62 Accessing External Databases Internet Connection Required 63 Searching the Affymetrix NetAffx Website 0 ee 63 Gene Level Differential Expression Analysis Graphs 0 0 2 0 000 eee 64 Scatter Plot Graph Overview lee eee eae 64 Using the Table to Investigate Lassoed Selections 0 0 00 00 cee eee 65 Clearing Lassoed Selections 0 ee eee 66 Changing Graph Colors 0 ee 66 Volcano Plot Graph Overview 0 0 0 0 0 RR Rel 67 Lassoing Genes of Interest lese 68 Using the Table to Investigate Lassoed Selections 0 00 00 cee eee 69 Copying Lassoed Selections l l 69 Clearing Lassoed Selections lille 70 Changing Graph GOO c 70 Chromosome Summary Graph Overview 0 0 0 eee 71
61. airings Figure 4 18 You must choose 2 different conditions Identical condition pairings generates the error message Please Choose Two Different Conditions Figure 4 18 Condition Pairing Drop down Comparison Liver vs Muscle 5 Chapter 4 Exon Level Differential Expression Analysis 93 Ka IMPORTANT Table and graph results ONLY reflect your current Condition pairing Exporting Options If you want to export Save your analysis table click drop down The following Export options appear Figure 4 19 Figure 4 19 Export Menu Export Current Table with Ist gene symbol only Export Current Table Export All Data Exporting the Current Table with 1st Gene Symbols o NOTE This option shows the first gene symbol only if there is more than one gene symbol for the selected transcript cluster 1 Click Export Current Table with 1st gene symbol only The Save As window appears 2 Click on an existing folder or click New Folder to choose a new save location 3 Type a filename for the table then click Save The table is now saved as a txt file Exporting the Current Table 1 Click Export Current Table The following window appears Figure 4 20 Figure 4 20 Export Window Column to Export Gene level information only All information Gene Symbols to Export 1st gene symbol only D All gene symbols Column to Export 1 Click either Gene level info
62. all dabg 15 8T rma exon all dabg rma exon all dabg l lT rma exon all dabg rma exon all dabg 7 AT rma exon all dabg rma exon all dabg 9 5T rma exon all dabg 1i 1 1 1 Click to Create New Condition Repeat the steps 1 3 above for Condition2 Colon Cancer To create more than 2 conditions click Click to Create New Condition Figure 4 5 then repeat steps 1 3 above for your 3rd Condition 8 If needed edit your Analysis result file path and or name by clicking inside the Analysis File text field Figure 4 6 or click Browse to select a new file destination Figure 4 6 Editable Analysis Result File Paths Analysis File O Users ppavic Documents TAC AnalysisResults Analysis_2 tac Browse ys pp y 9 After the Conditions have been labeled and populated click Run Analysis ER TAC auto saves your studies At any time click on the Open Existing Result tab to view recent analysis results Chapter 4 Exon Level Differential Expression Analysis 85 Please Wait appears then the Analysis Result viewer appears By default the Summary tab appears Figure 4 7 Figure 4 7 Default view after running an analysis Analysis_34 tac Analysis Result Summay SE Normal vs Colon Cancer Analysis Type Exon Level Differential Expression Analysis Array Type HuEx 1_0 st v2 Genome Version hg19 Annotation File HuEx 1_0 st v2 na33 1 hg19 transcript csv Annotation File HuEx 1 0 st v2 na33 1 hg19
63. ample X A Cag orat poe s IDZTC08000353 hg 1 Signal 3 14 10 54 FC 169 21 pValz2 43E 11 chr8 49966870 49988649 gene symbol C8orf22 ER The best way to view individual probe sets is to make the graph full screen by un checking Volcano Plot Graph Overview a The Volcano Plot graph is a type of scatter plot that is used to quickly identify changes in large datasets It plots significance versus fold change on the y and x axes respectively a X axis is the linear fold change from current condition pair Y axis is 10log10 p value of the ANOVA p values In the example below Figure 3 34 The gray TCs are the ones filtered out by the table The green TCs are down regulated in Liver Condition vs Muscle Condition2 while the red TCs are represented as up regulated in Liver Condition vs Muscle Condition2 Gene Level Differential Expression Analysis 68 If you switch the condition pair or the filter criteria in the table the data in the graph will change accordingly Figure 3 34 Volcano Plot Graph Analysis 29 tac Analysis Result E Split View Summary Scatter Plot ol Chromosome Summary Hierarchical Clustering i Up in Liver vs Muscle B Down in Liver vs Muscle 7 Clear Selection ka Save PNG A Print 150 1E 15 140 1E 14 130 1E 13 120 1E 12 110 1E 11 100 1E 10 90 1E 09 80 1E 08 70 1E 07 o pw zh eo e a eo ae bal LI e ET eo io a EJ
64. and copy your lassoed selection Copying Lassoed Selections 1 Right click on the graph The following menu appears Figure 3 41 Figure 3 41 Right click Graph Menu Copy lassoed probe sets Copy differentially expressed probe sets Print Save as PNG Copy lassoed probe sets 1 Click Copy lassoed probe sets to copy your currently lassoed probe sets to the Windows Clipboard for pasting Copy differentially expressed probe sets 1 Click Copy differentially expressed probe sets to the Windows Clipboard for pasting Gene Level Differential Expression Analysis 74 Print 1 Click Print to print the graph to a pre configured printer The Print window appears 2 Configure the printing options as you normally would then click OK Save as PNG 1 Click Save PNG to save the graph as a PNG image file The Save As window appears 2 Click on an existing folder or click New Folder to choose a new save location 3 Type a filename for the graph then click Save The graph is now saved as a png file Clearing Lassoed Selections This option is enabled after genes have been lassoed 1 Click Clear Selection to clear lassoed genes from the graph and table En m ud ER You can also clear a lassoed selection by lassoing a white blank space within the graph Changing Graph Colors Use the drop down menus to select your up and down regulated probe set graph colors Figure 3 42 Figure 3 42 Graph Color Men
65. ble Alternative Splicing Analysis 25 Column Headers Table Options Column Headers The factory default columns and 2 preset filters bd are as shown Figure 2 14 See Table 2 1 for definitions of these columns Figure 2 14 Default Table Column Headers Showing or Hiding Table Columns 1 Click the drop down menu to show or hide columns in the splicing table 2 Click outside the Show Hide Drop down menu to close it Table 2 1 Available Splicing Table Columns BOLD columns represents columns that TAC displays by default Available Columns Description Transcript Cluster ID ID of Transcript Cluster TC Condition1 Expressed Condition2 Expressed True or False statement regarding whether this TC is expressed in a condition based on criteria specified in the Algorithm Options A condition has this gene expressed if greater than or equal to 50 36 of its samples have thes gene expressed As an example True means gt 50 samples are expressed in this condition False means 5096 samples are expressed in this condition Condition Expressed Detail Condition2 Expressed Detail Number of expressed samples total samples in a condition A gene is expressed in a sample if greater than or equal ta 50 56 of its eligible PSRs have DABG p value less than 005 As an example 4 4 means 4 out of 4 samples met this criteria gt 50 eligible PSRs have DABG lt 0 05 1 4 means only 1 out of 4 samples m
66. c LLL 9 Copy Selected ID s r JUC15013928 hg 1 7596 TOT 1 85 1 12 0 89 4 03 The selected PSR Junction IDs are now copied to the Windows Clipboard for pasting Accessing External Databases Internet Connection Required 1 To link out to various external databases right click on a TC of interest The following menu appears Figure 2 32 Figure 2 32 Search Database menu 6 75E 10 CRP an 6 49E 13 Search NCBI Entrez Databases 2 59E 11 5 48E 09 H Search NCBI Gene Database amp 3o0E 10 4 97F 08 EE Search Ensembl Databases O74E 10 5465 08 Fe TC100026434 View in UCSC Genome Browser 2 08E 09 9 41E 08 en TCO4001662 Search Affymetrix NetAffx 2 57E 12 1 59E 09 TCO1004981 44E 10 3 43E 08 a ene er 4746 10 343E 08 7 63E 11 1 04E 08 AE Copy Selected ID s laseri aene nal TC12002282 TCO1000 08 TCOADO2793 hg 1 Copy Selected Row s Gene Symbols All Gene Symbols 2 Click to select the external database you want to visit Your internet browser opens to the appropriate website Searching the Affymetrix NetAffx Website 1 Click to select Search Affymetrix NetAffx Alternative Splicing Analysis The internet browser opens to the Affymetrix Customer Login window 2 Enter your NetAffx Email ID and Password then click Submit Your internet browser opens to the Netaffx Query C
67. cle Gene Avg Signal log2 9 15 B B B Splicing Index Track L D D b Bl Liver vs Muscle Gene Fold Change linear 1 10 D Lb D UA 2 From the Splicing Table click on a PSR Junction of interest Figure 2 38 3 The selected entry in the splicing table is also highlighted in the Splicing Viewer Figure 2 38 ER When you click to select a Junction its detected PSRs are also highlighted Figure 2 38 Highlighted Table and Viewer entries sequence Homo sapiens piRNA piR 41365 complete sequence PSRO9014755 hg 1 4 07 PSR09014745 hg 1 0 26 1C10000779 9 01 873 1 21 SLK Homo sapiens STE20 like Coding JUC10005171 hg 1 8 30 5 14 0 68 3 61 7 60 Kinase SLK mRNA Homo PSR10009288 hg 1 9 19 6 72 5 54 0 16 2 02 4 56 sapiens STE20 like kinase SLK 1 t l mRNA JUC10005163 hg 1 3 35 1 93 2 67 5 66 6 86 231 PSR10009291g i 4 24 5 31 2 10 4 79 3 35 2 72 t Gene Rows 12538 Exon Rows 67064 Selected Rows 0 laa Save PNG s Print ID TC10000779 hg 1 Find in Table Gene Symbol SLK Location chr10 105 726 959 105 788 991 Strand Positive Splicing index Intensity Structure View e E liver Gene Avg Signal log2 9 01 Show Junction Combine Neighboring Psrs Enable Tooltip gt 3 gt 7 E
68. ct ANOVA p value Condition 1 vs Condition2 FDR p value Condition1 vs Condition2 FDR adjusted p value based on Benjamini Hochberg Step Up FDR controlling Procedure IMPORTANT Only ANOVA p values from PSR and Junctions that are expressed in at least one condition and its gene must be expressed in both conditions in order to be sent to FDR for correction Alternative Splicing Analysis 27 Sorting Columns TAC uses a 2 level sorting process for the PSR junction information right side of the splicing table First it sorts PSRs junctions within each TC based on the PSR Junction data then it sorts each gene For the gene level information left side of the splicing table it performs 1 level sort PSRs Junctions are auto sorted by PSR Junction ID column Figure 2 15 Figure 2 15 2 Level Sort Results Example Right Click Method 1 Select a column then right click on it The following window appears Figure 2 16 Figure 2 16 Right click Column Menu Liver Bi Muscle Bi 2 Click to select either Sort By Ascending A Z or Sort By Descending Z A Double Click Method 1 Double click on a column header to sort its data in an ascending order Double click on the same column header to sort its data in a descending order Filtering Column Data All table columns are filterable 1 Select a column then right click on it The following window appears Figure 2 16
69. d If the PSR has data and is expressed in at least one of the conditions then TAC checks whether it is presented in an isoform or not If the isoform has at least one exon that contains a specific PSR then it is considered Present The score for a specific PSR is calculated as follows If FDR p value lt p value cutoff set in Preferences page then ScoreMultiplier 1 5 otherwise the ScoreMultiplier 1 If Up Regulated and present then score Splicing Index ScoreMultiplier If Up Regulated and not present then score Splicing Index ScoreMultiplier If Down Regulated and not present then score Splicing Index ScoreMultiplier If Down Regulated and present then score Splicing Index ScoreMultiplier If Splicing Index 0 then score 0 Score of the Isoform the sum of the PSR s scores If the user selects Ensembl Transcripts or RefSeq Genes then the software performs 2 level sorting It sorts Isoforms with IDs first then it performs a sub sort within the first sorted result Parts of the Genomic View Figure 2 43 Genomic View Example Genomic View a 1 Up Regulated on Top lll Down Regulated on Top Show Junction Truncate Intronic Region EAC UCSC psr100092 P P p ef ps p PSR10009283 hg 1 L Isoform Affx Transcript Isoforms TR10005657 hg jefe feleexfefe exrooosozang e fein E E Edeel 1 11 189300029500 TR10001881 hg Ex10012906 hg fe
70. differential expression analysis Download Array Type Files Internet Connection Required 1 Click Download Array Type Files to download library and annotation files from Affymetrix com The NetAffx Account Information dialog window appears 2 Enter your Netaffx account email and password then click OK or click the Register Now link to obtain a new account Do the following to download library and or annotation files 1 Click Download Array Type Files The NetAffx Account Login window appears Figure 1 5 Figure 1 5 NetAffx Account Login MetAffx Account Information Enter your Affymetrix com email address and password Email EE Password Register Now Cancel 2 Enter your NetAffx email and password then click OK Chapter 1 Introduction 10 The following window appears Figure 1 6 Figure 1 6 NetAffx Library Files NetAffx Library Files Select the library files to download H U133A O HG U219 C HuEx 1_O st v2 J HuGene 2_Ost v1 3 Click the checkbox next to the library file s you want to download then click Download A progress bar appears then the downloaded library and annotation file s appear in the Installed Array Types panel Internet Settings To enable the Internet Settings option click on the down arrow button Figure 1 7 Figure 1 7 Internet Settings y Internet Settings If you are using a Proxy S
71. e Alternative Splicing Analysis Array Type HTA 2_0 Genome Version hg19 Map File HTA 2 0 MappingFile v0 3 1 revi map Annotation File HTA 2 0 MappingFile v0 3 1 transcript csv Summary 1 Total number of genes 67536 a Coding 44707 b NonCoding 22829 2 Liver vs Muscle a Coding 33723 genes expressed in both conditions 8270 expressed genes have at least one PSR with Splicing Index linear 2 or Splicing Index linear 2 to indicate alternative splicing 2059 genes expressed in Liver but not expressed in Muscle 2565 genes expressed in Muscle but not expressed in Liver 6360 genes not expressed in both conditions b NonCoding 14609 genes expressed in both conditions 1182 expressed genes have at least one PSR with Splicing Index linear 2 or Splicing Index linear 2 to indicate alternative splicing 1495 genes expressed in Liver but not expressed in Muscle 1D 1c10000779 hg 1 ao in Table Gene Symbol SLK Location chr10 105 726 959 105 788 991 Strand Positive Splicing Inde MY adSave PNG s Print 9 Intensity Structure View Show Junction Combine Neighboring Psrs Enable Tooltip 5 Ea B Liver Gene Avg Signal log2 9 01 lll MAQCA Gene Avg Signal log2 8 73 Bl Liver vs MAQCA Gene Fold Change linear 1 21 E i B Genomic View oe Show Junction Truncate Intronic Region lll Enable Tooltip P5100082 T e T Mfes ele esmiooszesng2 JJ Mif AALL Isoform Ensembl Tran
72. e Splicing Analysis 31 Changing Condition vs Condition Pairings 1 Use the Comparison drop down menus to change your condition pairings Figure 2 25 You must choose 2 different conditions Identical condition pairings generates the error message Please Choose Two Different Conditions Figure 2 25 Condition Pairing Drop down Comparison Liver vs Muscle rE m IMPORTANT Table and graph results ONLY reflect your current Condition pairing Exporting Options If you want to export Save your analysis table click drop down The following Export options appear Figure 2 26 Figure 2 26 Export Menu Export Current Table with 1st gene symbol only Export Current Table Export All Data Exporting the Current Table with 1st Gene Symbols m NOTE This option shows the first gene symbol only if there is more than one gene symbol for the selected transcript cluster 1 Click Export Current Table with 1st gene symbol only The Save As window appears 2 Click on an existing folder or click New Folder to choose a new save location 3 Type a filename for the table then click Save The table is now saved as a txt file Exporting the Current Table 1 Click Export Current Table Alternative Splicing Analysis 32 The following window appears Figure 2 27 Figure 2 27 Export Window Column to Export Gene level information only All information Gene Symbols to Export
73. e dabg LAHTA dA MAQC HTA2 Muscle BetaSamplePool 1 v03 rma alt splice dabg IHTA 2 0 rma alt splice dabg l AHTAda Irma alt splice dabg EJ HTA2 Muscle BetaSamplePool 2 v03 rma alt splice dabg LXHTA da scl Irma alt splice dabg EE HTA2 Muscle BetaSamolePool 3 v03 rma alt solice dabolHTA 2 0 rma alt solice daboll WHA ae umm Importing CHP Files into Different Condition Groups m IMPORTANT Customize your condition names first then add the CHP files into each condition uuu Hb a e Click on the Condition1 window header field to rename it to an appropriate Condition name Figure 2 9 Click to select and highlight the data you want to use for Conditionl Click in the Condition window to add your selected files to the Condition window If needed click to move selected files back to the Sample File window If needed click O to delete your current Condition and move all its files back to the Sample File window Repeat the steps 1 3 above for Condition2 To create more than 2 conditions click Click to Create New Condition Figure 2 9 then repeat steps 1 3 above for your 3rd Condition If needed edit your Analysis result file path and or name by clicking inside the Analysis File text field Figure 2 10 or click Browse to select a new file destination Alternative Splicing Analysis 21 Importing Files using Drag and Drop Click Shift or click Ctrl to select a group of files 2 Click and hold onto the
74. e transcript isoforms A gene is expressed in a sample if greater than or equal ta 50 76 of its eligible PSRs have DABG p value less than 0 05 A condition has this gene expressed if greater than or equal to 50 75 of its samples have this gene expressed 4 4 PSR lunction is expressed in a condition if greater than or equal to 50 7a of the samples have DABG p value less than 0 05 o9 Discovery Rate lt 0 05 Chapter 1 Introduction 12 The Splicing Index algorithm compares normalized signal estimates from one condition to another See the equation below Figure 1 13 Figure 1 13 Splicing Index Algorithm Exon 1 Condition 1 Intensity S t Gene 1 Condition 1 Intensit Splicing index Exon 1 Condition 2 Intensity Gene 1 Condition 2 Intensity In order for Transcriptome Analysis Console to perform a Splicing Index two key criteria must be met They are as follows 1 Criteria 1 A Transcript Cluster gene must be expressed in both conditions Therefore for each condition you need to determine whether a gene is expressed or not Configurable Parameter 3 A gene can be considered expressed in a condition if it meets the criteria specified in parameter 3 As an example if at least 50 of the samples are expressed then this gene is expressed in this condition Configurable Parameter 2 You must determine whether a gene is expressed by looking at the DABG p values for all the eligible exons PSRs As an exam
75. eful they are removed The table now appears as shown Figure 3 7 Figure 3 7 Parse Table with 2 Filenames Separating Characters _ Parse Add Attributes Tissud J M Attributes amp Liver rma gene full Liver rma gene full Liver rma gene full Liver rma gene full MAQTA v03 MAQTA v03 MAQCE v03 MAQCE v03 kA irela rnaa3 cumacf all 5 To save your parsed attributes to the Same File window click Add Attributes The parsed file name attributes Figure 3 7 are added to the Sample File window as additional attributes Figure 3 8 Figure 3 8 Parsed Results shown in the Sample File Window Open Existing Result Preferences 4m Gene Level Differential Expression Analysis Import Data Remove selected Parse File Names Show Grouped Files Name Array Type File Type File Path Tissue Attribute 6 HTA2 Liver BetaSamplePool 1 03 rma gene full HTA 2_0 rma gene fulllLAHTAd Liver rma gene full HTA2 Liver BetaSamplePool 2 v03 rma gene full Liver rma gene ful HTA2 Liver BetaSamplePool 3 v03 rma gene full Liver rma gene ful HTA2 Liver BetaSamplePool 4 03 rma gene full Liver rma gene ful da da HTA2 MAQCA PolyA_EC1_1 v03 rma gene full MAQCA v03 HTA2 MAQCA PolyA EC1 2 03 rma gene full MAQCA v03 Importing CHP Files into Different Condition Groups o IMPORTANT Customize your condition names first then add the CHP files into each condition Do t
76. enter and displays information about your gene of interest 35 Hu NOTE The NetAffx Query Center is compatible with Windows Internet Explorer and Firefox Chrome is not supported at this time EE NOTE If a Probe Set or Transcript Cluster is not available an appropriate message appears Alternative Splicing Analysis 36 Viewing Results in the Splicing Viewer m NOTE The Splicing Viewer lower panel only reflects the active TC in the table the TC with the TAC icon next to it Figure 2 33 Using the Splicing Table and Splicing Viewer 1 Double click on any TC row or single click on it then click View Selected Gene to see the results in the Splicing Viewer Figure 2 33 Splicing Table and Viewer Analysis 28 tac Analysis Result n Sn El Split View Summary Table S Comparison Liver vs MAQCA Search SLK Prev Next Show Hide Columns I Export gt Clear Current Filter s Reset to Default Show All Data re V Show Expressed Genes Only V Show Expressed PSR JUC Only V Have at least one PSR 0g2 Signal log2 L ers MAQ ame me men miim Signa log2 Signa log vs MAQCA Sig sequence Homo sapiens aiiis j S i piRNA piR 41365 complete PSR09014755 hg 1 248 3 53 2 07 2 56 sequence PSR09014745 hg 1 648 797 2 81 1 54 9 01 8 73 SGU UA SK WonemsesSsEDUe Coamo puao 630 sa 3M oe sa 74 mod rai ie PSRIDUEERSDQI ere O ane a 48 pk r ium si sm era
77. ere is an Exon Skipping Event in muscle Figure 2 42 Alternative Splicing Event Example P5R10009288 hg 1 Position chr10 105 770 574 105 770 666 Liver Bi weight Avg Signal log2 9 19 PSR10009288 hg 1 Position chr10 105 770 574 105 770 666 Muscle Bi weight Avg Signal log2 5 47 PSR10009288 hg 1 Position chr10 105 770 574 105 770 666 Splicing Index 14 44 Liver Normalized Avg Signal log2 0 16 Muscle Normalized Avg Signal log2 3 69 FOR p value 0 000046 Click to return the Information Header to its collapsed view Alternative Splicing Analysis 42 Genomic View The Genomic View displays gene structure and transcript isoforms that belong to a TC All PSRs and Junctions are represented as their true genomic sizes Example The larger the box the larger the exon Each PSR Junction color is based on the color used in the Splicing Index track Below each gene structure transcript isoforms are displayed in order The order starts with the transcript isoforms that most likely exist in Condition to transcript isoforms that most likely exist in Condition2 The transcript isoforms that fit Condition are auto sorted to the top Transcript isoforms that fit Condition2 are auto sorted to the bottom How Each Isoform Sorting Score is Calculated All Splicing Index equations use log2 scale data TAC sorts each selected PSR If no PSRs are selected then all available PSRs are use
78. erver click its checkbox then complete the required fields Figure 1 8 Figure 1 8 Internet Settings Proxy Server Information Internet Settings 4 Proxy Server Address Port User Password Chapter 1 Introduction 11 Algorithm Options To customize Algorithm Options click on the down arrow button Figure 1 9 Figure 1 9 Algorithm Options Drop down Algorithm Options If you have more than one array type installed and you want to change algorithm options for another array use the drop down menu to select a different array Figure 1 10 Figure 1 10 Algorithm Options Algorithm Options Array Type HTA 2_0 z Annotation Files Use the drop down menus to select different versions of annotation files for various types of analysis Figure 1 11 Figure 1 11 Annotation Files Annotation Files Gene HTA 2 0 MappingFile vO 3 transcript csv HTA 2_0_MappingFile v0 3 revl map Splice Alternate Splicing Analysis Here you can customize algorithm parameters by typing values in the text boxes Figure 1 12 A NOTE Alternate Splicing Analysis is only available for certain arrays Please contact Affymetrix support regarding which array is supported Figure 1 12 Configurable Parameters 1 5 Alternative Splicing Analysis Use an eligible PSR to determine gene expression if it is present in greater than or equal to 50 76 Of all th
79. et this criteria gt 50 eligible PSRs have DABG lt 0 05 Condition1 Gene Avg Signal log2 Condition2 Gene Avg Signal log2 Tukey s Bi weight average of gene level intensity of all the samples in a condition Bi weight average of sample 1 gene1 intensity sample 2 gene intensity sample N genet intensity Condition1 Standard Deviation Condition2 Standard Deviation Standard Deviation of gene intensities from all samples in a condition STDEV of sample 1 gene1 intensity sample 2 gene1 intensity sample N gene1 intensity Gene Fold Change linear Condition1 vs Condition2 This shows the fold change in linear space of Condition vs Condition2 2 Condition1 Gene Avg Signal log2 Condition2 Gene Avg Signal log2 Gene Symbol Description Gene symbol for this transcript cluster Note RefSeq gene symbol is listed as the first gene symbol if there are more than 1 gene symbol Note A TC with no gene symbol may be auto assigned a public gene ID Gene Description for this TC Chromosome Genomic Position Chromosome for this transcript cluster See Chromosome Naming Scheme for a detailed description Genomic Start Stop position for this TC Public Gene IDs Group Public Gene IDs for this TC Whether this TC is coding non coding or other PSR Junction ID ID of Probe Selection Region PSR and Junction Probe Sets Alternative Splicing Analysis 26
80. example below the signal from one of the Liver samples is shown as 11 68 as well as the ID Filename and Condition name Figure 3 52 Figure 3 52 Mouse Over Gene TC 10001546 hg 1 CYP2C8 TCO0500342 4 hgr 1 C8 ConditionzLiver Flez HTA2 Liver BetaSamplePool 1 03 IDZTC10001546 hg 1 CYP2C8 Signalz11 68 Conditions This header displays the Conditions Figure 3 53 Gene Level Differential Expression Analysis 80 1 Mouse over a probe set below the condition legend to show what number represents what condition Figure 3 53 Condition Legend Signal Intensity Scale Displays the signal intensity range of your current condition pair from minimum to maximum Figure 3 54 Figure 3 54 Signal Intensity Scale 2 07 14 00 Saving a Hierarchical Cluster 1 To save the cluster as a PNG image file click Save PNG The Save As window appears 2 Click on an existing folder or click New Folder to choose a new save location 3 Type a filename for the cluster then click Save The cluster is now saved as a png file Printing Option 1 To print the cluster to a pre configured printer click Print The Print window appears 2 Configure the printing options as you normally would then click OK Exon Level Differential Expression Analysis Setting Up an Analysis Using Exon CHP Files 1 At the main TAC window click Exon Level Differential Expression Analysis Figure 4 1 Figure 4 1 Main TAC
81. export Save your analysis table click drop down The following Export options appear Figure 3 23 Figure 3 23 Export Menu Export Current Table with 1st gene symbol only Export Current Table Export All Data Exporting the Current Table with 1st Gene Symbols m NOTE This option shows the first gene symbol only if there is more than one gene symbol for the selected transcript cluster 1 Click Export Current Table with 1st gene symbol only The Save As window appears 2 Click on an existing folder or click New Folder to choose a new save location 3 Type a filename for the table then click Save The table is now saved as a txt file Gene Level Differential Expression Analysis 61 Exporting the Current Table 1 Click Export Current Table The following window appears Figure 3 24 Figure 3 24 Export Window Column to Export Gene level information only All information Gene Symbols to Export 1st gene symbol only D All gene symbols Column to Export 1 Click either Gene level information only or All information Gene Symbols to Export 1 Click either 1st gene symbol or All gene symbols 2 Click OK The Save As window appears 3 Click on an existing folder or click New Folder to choose a new save location 4 Type a filename for the table then click Save The table is now saved as a txt file Exporting All Data 1 Click Export AII Data EE NOTE On
82. f needed for the parsing It will not change actual file names HTA2 Liver BetaSamplePool 1 403 rma alt splice dabg HTA2 Liver BetaSamplePool 2 403 rma alt splice dabg HTA2 Liver BetaSamplePool 3 403 rma alt splice dabg HTA2 Liver BetaSamplePool 4 403 rma alt splice dabg HTA2 MAQCA PolyA ECI 1 v03 rma alt splice dabg HTA2 MAQCA PolyA ECI 2 03 rma alt splice dabg UTAT AAAS OR Dash ED 15 s alten lies dake Separating Characters _ Parse Add Attributes Attribute 1 CQ b Attribute2 QQ IP Attribute QQ P Attributes QQ P Attributes O P Attributes Q Attribute Q HTA2 Liver BetaSamplePool v03 rma alt splice dabg HTA2 I BetaSamplePool v03 rma alt splice dabg 1 2 HTA2 BetaSamplePool 3 v03 rma alt splice dabg A HTA2 I BetaSamplePool v03 rma alt splice dabg HTA2 MAQCA PolyA EC1 1 v03 rma alt splice dabg HTA2 MAQTA PolyA EC1 2 vO3 rma alt splice dabg HTA2 MAQCB PolyA EC1 1 v03 rma alt splice dabg HTA2 MAQCE PolyA EC1 2 HTAZ2 Muscle BetaSamplePool 1 v03 rma alt splice dabg v03 rma alt splice dabg HTA2 Muscle BetaSamplePool Fd v03 rma alt splice dabg HT AD haere Eatatamnlale l 4 u rma alt enlira daha L 2 Determine what common separating characters reside within your file names The file name examples in Figure 2 5 are separated by an underscore and period 3 Typethe appropriate symbols in the Separating Characters field In this example an underscore and period 4 Click Parse The Parse
83. fferential Expression Analysis 49 Parsing Imported Data File Names Optional This option gives you the ability to parse attributes from the sample file names and helps you set up conditions 1 Click Parse File Names The following window appears Figure 3 5 Figure 3 5 Parse Filename Window Parse names to attributes Change files names if needed for the parsing It will not change actual file names HTA2 Liver BetaSamplePool 1 03 rma gene full HTA2 Liver BetaSamplePool 2 03 rma gene full HTA2 Liver BetasamplePool 3 03 rma gene full HTA2 Liver BetaSamplePool 4 03 rma gene full HTA2 MAQCA PolyA ECT 1 03 rma gene full HTA2 MAQCA PolyA ECT 2 v03 rma gene full UTAT MAAR Del EC 1 8602 a cuana fl Separating Characters y Parse Add Attributes Attribute1 Q Bb Attribute 2 QQ IP Attribute Q IP Attributes OH IP Attributes OM P Attributes Q Attribute HTA2 Liver BetaSamplePool 1 v 3 rma gene full HTA2 Liver BetaSamplePool z v 3 rma gene full HTA2 Liver BetaSamplePool 3 v 3 rma gene full HTA2 Liver BetaSamplePool 4 vO3 rma gene tull HTA2 MAQCA PolyA ECT 1 v03 rma gene full HTA2 MAQCA PaolyA ECT 2 v03 rma gene full HTA2 MAQCE PalyA ECT 1 v03 rma gene full HTA2 MAQCE PalyA ECT 2 v03 rma gene full HTA2 Muscle BetaSamplePool 1 v 3 rma gene full 2 Determine what common separating characters reside within your file names The file name examples in Figure 3 5 are separated by a
84. files in it Note Scroll the Summary window downward to reveal the other Conditions in your analysis Gene Level Differential Expression Analysis 55 Gene Level Differential Expression Analysis Table Window Overview 1 After reviewing the Summary click the Table tab or click to uncheck the checkbox to display the Table in full screen Figure 3 13 Analysis_23 tac Analysis Result TC17001810 hg 1 TC02004093 hg 1 TC01006295 hg 1 TC13000641 hg 1 TC01003389 hg 1 TC04001274 hg 1 TC12002283 hg 1 TC02004095 hg 1 TC10002643 hg 1 TC04001662 hg 1 TC01004981 hg i TC04002794 hg 1 TC12002282 hg 1 TC01000708 hg 1 TC04002793 hg 1 TC04000777 hg 1 TC02004094 hg 1 Comparison Liver vs Muscle Search 3 94 TCOX000676 hg 1 11 67 311 97 123E 11 3656 09 F9 Homo sapiens coagulation Show Hide Columns Export Clear Current Filter s prev Nec 12 93 509 21 2 86E 11 5 79E 09 APOH Homo sapiens apolipoprote Coding Coding NonCoding a m seo are Ces on me smempsem OOOO x e m zmew saem Coding Coding NonCoding NonCoding NonCoding Homo sapiens angiopoieti Coding aera 195 8 amp s zaen sae 4 94 NonCoding Homo sapiens fibrinogen be Coding NonCoding TC10000675 hg 1 412 7 44E 09 CYP2C9 Homo sapiens cytochro TC16001249 hg 1 4 83 7 04E 10 TAT Homo sapiens tyrosi
85. gulated Coding 1840 NonCoding 655 4 Liver vs MAQCA a 3167 genes are up regulated Coding 2436 NonCoding 731 b 3483 genes are down regulated Coding 2420 NonCoding 1063 5 Muscle vs MAQCA a 2420 genes are up regulated Coding 1834 NonCoding 586 b 3343 genes are down regulated Coding 2314 NonCoding 1029 Algorithm Options 1 One Way Between Subject ANOVA Unpaired Default Filter Criteria 1 Fold Change linear 2 or Fold Change linear 2 2 ANOVA p value Condition pair 0 05 Conditions Liver 4 1 HTA2 Liver BetaSamplePool 1 v03 rma gene full chp 2 HTA2 Liver BetaSamplePool 2 v03 rma gene full chp 3 HTA2 Liver BetaSamplePool 3 v03 rma gene full chp AS UTA livar DrtaCamnmlanan ANI rema cm mme fill chen Analysis_29 tac Analysis Result Volcano Plot Chromosome Summary m Up in Liver vs Muscle m Down in Liver vs Muscle A e YS QE n v CSAR So s ANN ic Be ox v i e tee p a od wet Split View Hierarchical Clustering Clear Selection ka Save PNG s Print Gene Level Differential Expression Analysis 53 Gene Level Differential Expression Analysis Summary Window Overview The Summary information is static and based on the algorithm parameters applied during the analysis Below is an example of the information collected in the Summary window Figure 3 12 Figure 3 12 Summary window Liver vs Muscle vs MAQCA Anal
86. gure 2 44 Up Regulated on Top Sort Order Example PSK Isoform Affx Transcript Isoforms a TR10005657 n D Ao E CUTE 7100018879 o E CUTE CONE o E TR10004124 hg LUILIDEMS 0 E CUTE PSRIODOSZ33 hg 1 P5R10009295 hg 1 x10009196 hg x10009196 hg EX LUCLTEES 7 0099 EA E CO a eo ET 2 Click to highlight a PSR then click button button to bring the isoforms with negative SI value PSR green to the top based on the Splicing Index and p value value of your highlighted PSR Note the sorting order of the PSR and Isoforms Figure 2 45 Figure 2 45 Down Regulated on Top Sort Order Example s Bsmagel psr10 PSR10009296 hg 1 BPSRIODOSZ33 hg 1 TR10008792 hg EX1000303B hg TR10004184 hg TR10000675 hg EX1000303B hg TR10001295 hg EX1000303B hg TR10001881 hg EX100Q0303B hg TR10005657 hg EX100Q0303B hg EX1lO0DQ08135 hg EX1lO0DQ08135 hg IMPORTANT If Other than Affx Transcript Isoforms IDs are used the software will do 2 level sorting It first sorts Isoforms with IDs for the selected source then it performs a sub sort within the sorted result Using the Show Junction Option The option 1s unchecked by default Check it to produce dotted lines to show the relationship of the detected Junctions and PSRs Uncheck this box to hide these junction lines Alternative Splicing Analysis 44 Using the Truncate Intronic Region Option
87. he Windows Clipboard for pasting Copy Selected ID s 1 Click to highlight light blue a ID or Ctrl left click to highlight multiple rows 2 Right click then click Copy Selected ID s to copy Transcript Cluster IDs Figure 3 26 Figure 3 26 Copy Selected ID s option TCO1003388 h TCO4001274 TC12002283 TCO2004095 TC10002643 TCOADO1662 TCO1004981 TCO4002794 TC12002282 TCO1000708 Search NCBI Entrez Databases Search NCBI Gene Database Search Ensembl Databases smEm amEm sms sam amem sabm meg sabm praca impm All Gene Symbols rm m 247 m 1 JN View in UCSC Genome Browser Search Affymetrix NetAtix Copy Selected Row s Ctrl C Copy Selected ID s ains Copy Selected Row s Gene Symbols k The selected TC IDs are now copied to the Windows Clipboard for pasting Copy Selected Row s Gene Symbols 1 Click to highlight light blue a row or Ctrl left click to highlight multiple rows 2 Right click on the selection then click to select Copy Selected Row s Gene Symbols Gene Level Differential Expression Analysis 63 3 Click to select either All Gene Symbols all possible gene symbols for a Transcript Cluster or First Gene Symbol Only the first gene symbol that belongs to the Transcript Cluster Figure 3 27 Figure 3 27 Copy Selected Row s gene Symbols options
88. he following to populate the Condition1 window 1 Click on the Condition1 window header field to rename it to an appropriate Condition name Click to select and highlight the data you want to use for Conditionl Click in the Condition window to add your selected files to the Condition window If needed click to move selected files back to the Sample File window aoe D M If needed click O to delete your current Condition and move all its files back to the Sample File window Gene Level Differential Expression Analysis 51 Importing Files using Drag and Drop Do the following to drag and drop files into a Condition window 1 Click Shift or click Ctrl then click to select a group of files 2 Click and hold onto the last file in the group then drag them into the appropriately labeled Condition window 3 Release the mouse button The Condition Liver window now contains your files 4 If needed click Q to move the file s back to the Sample File window Figure 3 9 Newly Labeled Condition with Samples Liver t 0 Condition2 HTA2 Liver BetaSamplePool 1 v03 rm HTA2 Liver BetaSamplePool 2 v03 rm HTA2 Liver BetaSamplePool 3 v03 rm HTA2 Liver BetaSamplePool 4 v03 rm p rcm fF tn a La Click to Create New Condition I LO Ko Pa AL 5 Repeat the steps 1 4 above for Condition2 To create more than 2 conditions recommended click Click to Create New Condition Figure 3 9 then re
89. igured printer The Print window appears 2 Configure the printing options as you normally would then click OK Save as PNG 1 Click Save PNG to save the graph as a PNG image file The Save As window appears 2 Click on an existing folder or click New Folder to choose a new save location 3 Type a filename for the graph then click Save The graph is now saved as a png file Clearing Lassoed Selections This option is enabled after genes have been lassoed as points of interest Fag 1 Click Clear Selection to clear lassoed genes from the graph and table ER You can also clear a lassoed selection by lassoing a white blank space within the graph Changing Graph Colors Use the drop down menus to select your up and down regulated probe set graph colors Figure 3 31 Figure 3 32 Graph Color Menus Red and Green are default 1 Up in Liver vs Muscle C Gene Level Differential Expression Analysis 67 Obtaining Information Related to Individual Probe Sets 1 Mouse over position the cursor over a probe set to show its details In the example below Figure 3 33 signals from both conditions 3 14 is the signal in Liver Conditionl and 10 54 is the signal in Muscle Condition2 are shown Fold change FC in linear scale ANOVA p value chromosome positions and gene symbol are also identified in the line below Figure 3 33 OK aem Xx x MER Ae am E d a eS Figure 3 33 Mouse Over Ex
90. ilters from the table headers Table Options Use the Table Options Menu Figure 3 19 to customize your table view Figure 3 20 Table options menu Comparison Liver WS Muscle Search Show Hide Columns N Export w Clear Current Filter s Reset to Default Rearranging Factory Set Columns 1 Click on a column you want to move 2 Drag it left or right to its new location 3 Release the mouse button 4 The column is now in its new position Reset to Default 1 Click Reset to Default to return the table to its factory setting Searching Keywords m NOTE The Search Tool is limited to finding matching strings It is not a full search engine 1 To search for a keyword within your table click inside the Search field then type your keyword 2 Click the Prev or INext buttons to search Figure 3 21 Figure 3 21 Search Tool Search SLK Prev Next Gene Level Differential Expression Analysis 60 Changing Condition vs Condition Pairings 1 Use the Comparison drop down menus to change your condition pairings Figure 3 22 You must choose 2 different conditions Identical condition pairings generates the error message Please Choose Two Different Conditions Figure 3 22 Condition Pairing Drop down Comparison Liver vs Muscle rE m IMPORTANT Table and graph results ONLY reflect your current Condition pairing Exporting Options If you want to
91. in a condition if it meets the criteria specified in parameter 3 As an example if at least 50 of the samples are expressed then this gene is expressed in this condition Configurable Parameter 2 You must determine whether a gene is expressed by looking at the DABG p values for all the eligible exons PSRs As an example at least 5096 of eligible PSRs must be expressed DABG p 0 05 for the gene to be considered expressed Appendix A Algorithms 99 Configurable Parameter 1 The way to decide whether a PSR is eligible is to see 1f it presents at least 50 of all the transcript isoforms for that gene Only PSRs 1 2 3 4 8 are considered as eligible PSRs in this gene as shown in the example below Figure A 3 Figure A 3 Configurable Parameters Exon Number 1 2 3 4 X X X amp PSR12 HPSR120011979 IPSR PSR120011982 P HPSR1 Isoform x120 EX120002723 a 0eu3sj EX120 Saz L X120016873 x1 L X120016872 HE 2 Criteria 2 A PSR or Junction can only be analyzed by Splicing Index 1f it expresses in at least one condition Configurable Parameter 4 To decide whether a PSR or junction is expressed in a condition you need as an example to check the DABG p values from all samples in that condition to see whether gt 50 of samples have DABG p lt 0 05 After a gene and PSRs junctions meet the criteria above normalization and comparison can begin In order to
92. ken into account when calculating the necessary available disk space requirement Larger data file sizes associated with Whole Transcriptome arrays should be taken into account when calculating the necessary available disk space requirement Table 1 1 System Requirements 64 bit Operating System Speed Memory Available Disk Web Browser RAM Space Microsoft Windows 7 professional 8 GHz Intel Pentium 16 GBRAM 150GBHD IE 7 0 and above operating system with Service Pack 1 Quad Core Processor data storage Microsoft Windows XP operating 4 GHz Intel Pentium 8 GB RAM 150 GB HD IE 7 0 and above system with Service Pack 2 0 Quad Core Processor data storage Recommended 16 GB of RAM Installation Instructions To install the Transcriptome Analysis Console software 1 Go to www affymetrix com and navigate to the following location Home gt Products gt Microarray Solutions gt Instruments and Software gt Software gt 2 Locate and download the zipped TAC software package 3 Unzip the file then double click TAC64 exe to install it 4 Follow the directions provided by the installer The setup process includes and installs the required Microsoft component NET Framework 4 Client Profile Chapter 1 Introduction 8 Preferences Tab Use the Preferences tab to setup or change a library path download library and annotation files and modify algorithm options Figure 1 1 Figure 1 1 Main Preferences window
93. le options menu Comparison Liver vs Muscle Search SLk Prev INext View selected Gene Show Hide Columns M Clear Current Filter s Reset to Default Show All Data 4 Show Expressed Genes Only x Show Expressed PSR JUC Only Have at least one PSR Show Data Options By default all 3 Show Data options are checked Figure 2 22 As these options are unchecked the gene count changes and the summary below the splicing table automatically updates the counts Figure 2 22 Show Data Options iV Shaw Expressed Genes Only 4 Show Expressed PSR JUC Only Have at least one PSR Show Expressed Genes Only Check this box to show ONLY the genes that are expressed in both selected Conditions Uncheck this box to show genes that are NOT expressed in both of your Conditions Transcript clusters that are expressed in only 1 condition False True True False or not expressed in either condition False False are highlighted in italic red a All currently set filters are cleared Show Expressed PSR JUC Only Check this box to show PSRs and Junctions that are expressed in at least one of your set Conditions Uncheck this box to show PSRs and Junctions that are NOT expressed in both of your Conditions The PSR and Junctions that are not expressed in both conditions False False are highlighted in italic red Alternative Splicing Analysis 30 a All currently set filters are cleared Have at Least One PSR
94. le rows 2 Right click on the selection then click to select Copy Selected Row s Gene Symbols Alternative Splicing Analysis 34 3 Click to select either All Gene Symbols all possible gene symbols for a Transcript Cluster or First Gene Symbol Only the first gene symbol that belongs to the Transcript Cluster Figure 2 30 Figure 2 30 Copy Selected Row s gene Symbols options TCOADO1662 Search Affymetrix NetAtix TC01003389 h 5 8 75E 10 CRP TCO4201274 Search NCBI Entrez Databases TCi2002283 Search NCBI Gene Database 830E10 492E08 TCO2004095 Search Ensembl Databases 974E 10 546E08 TC10002643 View in UCSC Genome Browser 209E 09 9416 08 TCO1004981 ft Copy Selected Row s 7 63E 11 1 04E 08 EE TCiz002282 Copy Selected ID s laseri z amp nena Tcoigoo7oa y Copy Selected Row s Gene Symbols All Gene Symbols T TCOADO2793 hg 1 Your selected rows with gene symbols are now copied to the Windows Clipboard Copy PSR Junction Information 1 Click to highlight light blue a PSR Junction ID or Ctrl left click to highlight multiple rows 2 Right click then click either Copy Selected ID s or Copy Selected Row s to copy PSR Junction IDs Figure 2 31 Figure 2 31 Copy Selected PSR JUC option NE Ec ENNIO a 2 aan saa 884 s MGUOSUUSI conyScuceanont per
95. lgorithm Options 1 One Way Between Subject ANOVA Unpaired Default Filter Criteria 1 Fold Change linear 2 or Fold Change linear gt 2 2 ANOVA p value Condition pair 0 05 Conditions Normal 4 1 10 5N rma exon all dabg chp 2 12 6N rma exon all dabg chp 3 14 7N rma exon all dabg chp 4 16 8N rma exon all dabg chp Colon Cancer 4 1 15 8T rma exon all dabg chp 2 1 1T rma exon all dabg chp 3 7 AT rma exon all dabg chp 4 9 5T rma exon all dabg chp 1 Summary information per this analysis NOTE Summaries vary between Gene Exon and Splicing analysis 2 Array summary Total number of Exons number of Exons that are differentially expressed and the number of Exons that are up and down regulated 3 Shows the algorithm used to perform the Exon Level Differential Expression Analysis This section displays the factory default filtering criteria results NOTE Only Exons that pass in the Exon table criteria are summarized 5 Displays each Condition name and the total number of CHP files in it Chapter 4 Exon Level Differential Expression Analysis 87 Exon Level Differential Expression Analysis Table Window Overview 1 After reviewing the Summary click the Table tab to display the Table in full screen Figure 4 8 m NOTE The left side of the table provides gene level information The right side of the table provides exon information and is organized by each transcript cluster
96. lice dabg chp LI HTA2 MAGCB PolyA ECT 1 v 3 ma alt splice dabg chp LI HTA2 MAGCB PolyA EC1 2 v3 ma alt splice dabg chp m HTA2 Muscle BetaSamplePool 1 v 3 rma alt splice dabg chp kl HTA2 Muscle BetaSamplePool 2 v 03 rma alt splice dabg chp kl HTA2 Muscle BetaSamplePool 3 v 3 rma alt splice daba chp m HTA2 Muscle BetaSamplePool 4 v 3 rma alt splice dabg chp m HTA2 Spleen PolyA EC1 1 v 3 ma alt splice dabg chp ki HTA2 Spleen PolyA EC1 2 v 3rma alt splice dabg chp D HTA2_Testes_PolyA_EC1_1 v03 ma alt splice dabg chp Deme 0 Cw Files of type Ateratwe saiang CHP Fis s Coreas 3 Single click Ctrl click or Shift click to select multiple files Figure 2 3 o NOTE To optimize the analysis Affymetrix recommends importing more than 1 sample per condition 4 Click Alternative Splicing Analysis 18 The selected files are now populated in the Sample File Window Figure 2 4 Figure 2 4 Import Data into Sample File Window Transcriptome Analysis Console Open Existing Result Preferences 4m Alternative Splicing Analysis Remove Selected Parse File Names Show Grouped Files Name Array Type File Type File Path HTA2 Liver BetaSamplePool 1 v03 rma alt splice dabg HTA 2 0 rma alt splice dabg LAHTAMdat HTA2 Liver BetaSamplePool 2 v03 rma alt splice dabg AHTA dat HTA2 Liver BetaSamplePool 3 v03 rma alt splice dabg AHTA dat HTA2 Liver BetaSamplePool 4 v03 rma alt splice dabg HTA dat HTA2 MAQCA
97. ls TCO1000 08 TCOADOZ2793 hg 1 2 Click to select the external database you want to visit Your internet browser opens to the appropriate website Searching the Affymetrix NetAffx Website 1 Click to select Search Affymetrix NetAffx The internet browser opens to the Affymetrix Customer Login window 2 Enter your NetAffx Email ID and Password then click Submit Your internet browser opens to the Netaffx Query Center and displays information about your gene of interest m NOTE The NetAffx Query Center is compatible with Windows Internet Explorer and Firefox Chrome is not supported at this time Gene Level Differential Expression Analysis 64 m NOTE If a Probe Set or Transcript Cluster is not available an appropriate message appears Gene Level Differential Expression Analysis Graphs El NOTE Scatter Plot Volcano Plot Chromosome Summary Graphs Gene Tables and your selections stay in sync with each other The data displayed is always based on your current Condition pairings Scatter Plot Graph Overview The Scatter Plot is a standard scatter plot graph of your current condition paring Figure 3 29 The size of the X is based on p value the smaller p value the bigger the X The signal is log2 based data The scale is also log2 based scale for signals X axis is second condition The Y axis is first condition on the top left of the table The gray TCs are the ones filtered out by the table
98. ltered column you want to clear The following window appears Figure 4 15 Figure 4 15 Right click Column Menu NE mE Ar Sort By Ascending ij Sort By Descending Y Filter Clear Column Filters x Hide Column Chapter 4 Exon Level Differential Expression Analysis 92 2 Click Clear Column Filters The filter is removed All Filters 1 Click Clear Current Filters remove all currently active filters from the Splicing Table Table Options Use the Table Options Menu Figure 4 16 to customize your table view Figure 4 16 Table options menu Comparison Normal Vs Colon Cancer Search Show Hide Columns w Clear Current Filter s Reset to Default Rearranging Column Orders 1 Click on a column you want to move 2 Drag it left or right to its new location 3 Release the mouse button The column is now in its new position Resetting Table Defaults 1 Click Reset to Default to return the table to its factory setting Searching Keywords nm NOTE The Search Tool is limited to finding matching strings It is not a full search engine 1 To search for a keyword within your table click inside the Search field then type your keyword 2 Click the Prev or Next buttons to search Figure 4 17 Figure 4 17 Search Tool JF Search SLK Prev Next Changing Condition vs Condition Pairings 1 Use the Comparison drop down menus to change your condition p
99. ly currently paired data is exported including data in the hidden columns and the paired data s gene level information The Save As window appears 2 Click on an existing folder or click New Folder to choose a new save location 3 Type a filename for the table then click Save The table is now saved as a txt file Saving Table Information Use this copy feature to save table information to the Windows Clipboard then use this buffered information for pasting into other applications or websites Gene Level Differential Expression Analysis 62 Copy Selected Row s 1 Click to highlight light blue a row or Ctrl left click to highlight multiple rows 2 Right click then click to select Copy Selected Row s Figure 3 25 Figure 3 25 Copy Selected Row s option TOOL00336S h TC04001274 TC12002283 TCO2004095 TC10002643 TCOADO 662 TCO1004981 Search NCBI Entrez Databases Search NCBI Gene Database Search Ensembl Databases smEm amEm suem sam amem sabm mug 343606 praca aoa All Gene Symbols First Gene Symbol Only View in UCSC Genome Browser Search Affymetrix NetAtix Copy Selected Row s m Cirl TCiz00z2g Copy Selected ID s Too1o0e708 Copy Selected Row s Gene Symbols d TCO4002793 hg 1 The selected gene level information shown on the left side of the splicing table are now copied to t
100. n screen regardless if your parameter setting applies to your analysis or not Default Button Click Default bottom right to RESET Algorithm parameters to their factory settings Open Existing Result Tab TAC always auto saves your studies At any time you can view recent analysis results 1 Click on the Open Existing Result tab m TIP Click any of the window s header columns to sort your recent studies by Ascending A Z or Sort By Descending Z A Chapter 1 Introduction 14 The Open Existing Result window appears Figure 1 16 Figure 1 16 Open Existing Result window Transcriptome Analysis Console New Analysis Preferences Recent Analysis Results File Name Analysis_34 tac Analysis_33 tac Analysis_32 tac Analysis_31 tac Analysis_30 tac Analysis_29 tac Analysis_28 tac Analysis_27 tac Analysis_26 tac Analysis_25 tac Analysis_19 tac Analysis_24 tac Analysis_23 tac Analysis_22 tac Analysis_21 tac Analysis_20 tac Analysis_14 tac Analysis 15 tac Array Type HuEx 1_0 st v2 HuEx 1_0 st v2 HuEx 1_0 st v2 HuEx 1_0 st v2 HuEx 1_0 st v2 HTA 2_0 HTA 2_0 HTA 2_0 HTA 2_0 HTA 2_0 HTA 2_0 HTA 2_0 HTA 2_0 HTA 2_0 HTA 2_0 HTA 2_0 Analysis Type Exon Exon Exon Gene Exon Gene Splicing Splicing Splicing Splicing Gene Splicing Gene Gene Gene Splicing Conditions Normal Colon Cancer Condition1 Condition2 Normal Colon Cancer Normal Colon Cancer Normal Colon Cancer Liver
101. n the Information Header to its collapsed view a Click either to view the Splicing Table and Splicing Viewer side by side or top and bottom Alternative Splicing Analysis 38 Structure View Structure View displays gene structure All PSRs and Junctions are represented in the structure view with boxes that have same size a An Inclusion junction detects 2 neighboring PSRs The PSRs detected by an inclusion junction are linked and graphically represented as dotted lines when you mouse over or click that junction a An Exclusion junction detects PSRs that are apart from each other The PSRs detected by an exclusion junction are linked and graphically represented as dotted lines when you mouse over or click that junction 5n A crossed out box represents a PSR Junction that does not contain data Ayoz A diagonally crossed out box represents a PSR Junction that is not expressed in at least one condition bold top and bottom borders represents a PSR Junction that has passed through the current table s filtering criteria This only applies to PSRs Junctions currently filtered in the table A Spacer represents a transcript cluster TC probe selection region PSR where a selection of probes is not possible Note Spacer is typically a region with less than 25 bases Occasionally some of them can exceed 25 bases Parts of the Structure View Figure 2 36 Structure View M ll Liver Gene Avg Signal log2 9 01 E B Muscle Ge
102. n the text boxes Figure A 1 m NOTE Alternate Splicing Analysis is only available for certain arrays Please contact Affymetrix support regarding which array is supported Figure A 1 Configurable Parameters 1 5 Alternative Splicing Analysis Use an eligible PSR to determine gene expression if it is present in greater than or equal to 50 76 of all the transcript isoforms A gene is expressed in a sample if greater than or equal to 50 76 of its eligible PSRs have DABG p value less than 0 05 A condition has this gene expressed if greater than or equal to 50 75 of its samples have this gene expressed 4 PSR lunction is expressed in a condition if greater than or equal to 50 75 of the samples have DABG p value less than 0 05 Discovery Rate lt 0 05 The Splicing Index algorithm compares normalized signal estimates from one condition to another See the equation below Figure A 2 Figure A 2 Splicing Index Algorithm Exon 1 Condition 1 Intensity T Gene 1 Condition 1 Intensit Splicing l index Exon 1 Condition 2 Intensity Gene 1 Condition 2 Intensity In order for Transcriptome Analysis Console to perform a Splicing Index two key criteria must be met They are as follows 1 Criteria 1 A Transcript Cluster gene must be expressed in both conditions Also for each condition you need to determine whether a gene is expressed or not Configurable Parameter 3 A gene can be considered expressed
103. n underscore and period 3 Typethe appropriate symbols in the Separating Characters field In this example an underscore and period 4 Click Parse The Parse File Names table now appears as shown Figure 3 6 Figure 3 6 Parse Filenmae Table Separating Characters _ Parse Add Attributes Attribute 1 CQ b Attribute QQ IP Attribute QQ IP Attributes P Attributes O P Attributes Q Attribute HTA2 Liver BetaSamplePool 1 v03 rma gene full HTA2 Liver BetaSamplePool 2 v03 rma gene full HTA2 Liver BetaSamplePool 3 v03 rma gene full HTA2 Liver BetaSamplePool v03 rma gene full HTA2 MAQUA PolyA EC1 1 w03 rma gene full HTA2 MAQUA PolyA ECT E v03 rma gene full HTA2 MAQCB PolyA EC1 1 w03 rma gene full HTA2 MAQCB PolyA EC1 2 w03 rma gene full HTA2 Muscle BetaSamplePool 1 v03 rma gene full Do the following to clean up attributes parsed from the sample file names Click inside any of the Attribute text fields Attibute1 to type in a new Attribute name Gene Level Differential Expression Analysis 50 Click to enter a unique separating character for your parsed filename The default separating character is a period These characters are useful if you ever want to return a file to its original name Click to join together a neighboring attribute column Click to remove an attribute column from the table In the example above since Attribute 1 3 4 5 and 7 are redundant and not us
104. ne Avg Signal log2 9 15 z PSR1 PSR1 PS O08 uH ud atu BG D B Intensity Track 1 or Condition Gene Avg Signal log2 9 01 Figure 2 36 Intensity Track 2 or Condition2 Gene Avg Signal log2 9 15 Splicing Index Track or Conditionl vs Condition2 Gene Fold Change linear 1 10 a Show Junction checkbox a Combine Neighboring Psrs checkbox Enable Tooltip checkbox Zoom Fit button Zoom Tool Slider bar Save as PNG a Print Using the Intensity and Splicing Index Tracks with the Table The Structure view displays 3 tracks Intensity Track 1 top shows the Bi weight Avg Signal log2 from each PSR and Junction for Condition 1 Intensity Track 1 middle shows the Bi weight Avg Signal log2 from each PSR and Junction for Condition2 The Splicing Index Track bottom shows the Splicing Index values linear from the Condition and Condition2 comparison Alternative Splicing Analysis 39 1 Click each Intensity Track M far left to expand show them Figure 2 37 Figure 2 37 Intensity and Splicing Index Tracks 7 1D TC10000779 hg 1 Find in Table Gene Symbol SLK Location chr10 105 726 959 105 788 991 Strand Positive Splicing Index E Intensity Structure View j __ _ E ll Liver Gene Avg Signal log2 9 01 Show Junction Combine Neighboring Psrs Enable Tooltip B D n E B Mus
105. ne ami TC05003424 ng 1 4 26 Homo sapiens compleme TC04001663 hg 1 34 4 75E 09 FGG Homo sapiens fibrinogen ga TC10001546 hg 1 4 20 2 69E 08 CYP2C8 Homo sapiens cytochrom TC04001265 hg 1 Homo sapiens UDP giu TC01003535 hg 1 1C17000542 hg 1 1C01001733 hg 1 TC15000391 hg 1 TC01001354 hg i TC12003240 hg 1 TC02001628 hg 1 TC04000789 hg 1 TC09002906 hg 1 TC03001997 hg 1 TC19001670 hg 1 TC04000403 hg 1 TC4 ctg9 hapi TC09001419 hg 1 TC03001023 ha 1 4 Gene Rows 5827 Selected Rows 0 Selected 4r 8 86E 10 Homo sapiens serpin pep 12 28 156 84 6 73E 13 875E 10 Homo sapiens glucose 6 ph 1148 15543 6 69E 11 9 55E 09 C4BPA AX Homo sapiens complement Coding 1211 485 153 97 2727 13 6 78E 10 SLC27A2 Coding 10 29 308 152 79 6 25E 11 9 12E 09 SLCO181 Homo sapiens solute cari Coding 13 08 147 58 2087 13 6 35 10 Homo sapiens apolipoprote Coding 4 81E 09 UGT2B4 SERPINC1 Coding Coding 10 63 145 55 9 64E 12 3 10E 09 Homo sapiens tryptophan 2 Coding 10 89 137 65 247E 10 2 02E 08 SULT2A1 Homo sapiens sulfotransfera 1227 13544 1 83E 12 1 27E 09 Homo sapiens albumin AL 10 79 134 37 8 29E 11 1 09E 08 UGT2810 _ Homo sapiens UDP glucuron 1172 469 131 03 1 09 09 5 92E 08 Homo sapiens bile acid CoA 11 03 4 01 129 86 4 93F 11 7 85F 09 HRG Homo saniens histidine ri Cadin
106. nks E e MN WEOOBTRBT con traction RS NIE m Copy Selected ID s 7 96 707 1 85 112 0 89 4 26 JUC15013328 hg 1 03 The selected exons are now copied to the Windows Clipboard for pasting Accessing External Databases Internet Connection Required 1 To link out to various external databases right click on a TC of interest The following menu appears Figure 4 25 Figure 4 25 Search Database menu B 5E 10 CRP 7 H 6 49E 13 Search NCBI Entrez Databases Search NCBI Gene Database Search Ensembl Databases C02004035 TC10002643 TCOADO1662 TCO1004981 TC04002794 TC12002282 TCO1000708 TCOADOZ2793 hg 1 smEm emEm smi sam m View in UCSC Genome Browser Search Affymetrix NetAtix 2 67E 12 1 59E 09 474E 10 3 43E 08 EL 7 63E 11 1 04E 08 E 74 O Copy Selected Row s Gene Symbols All Gene Symbols Copy Selected Row s Copy Selected ID s 2 Click to select the external database you want to visit Your internet browser opens to the appropriate website Searching the Affymetrix NetAffx Website 1 Click to select Search Affymetrix NetAffx The internet browser opens to the Affymetrix Customer Login window 2 Enter your NetAffx Email ID and Password then click Submit Your internet browser opens to the Netaffx Query Center and displays information about your gene of
107. op ius sek xar tke dex RR REOR sen See edad 21 Alternative Splicing Analysis Table Window Overview 00 000 e eee eee eee 24 Column Headers 2 douce eau rode eon ain e SUE OC eae Ro eroe eee ee e E ated eas 25 Showing or Hiding Table Columns xu seei bw X x EERRE RP Rn RR hs SOUNO COMMING d F 27 weed ccc 27 Double Click Method suus ove deve Di Goes wine bie ORG RO A E eens sd 27 Editing Filtering Properties idi osucess o oe e oka eye Pwd Ed Pob Oe deena ees 28 Clear In TINGS seireun 134523 9 099 99999295 93 PSY I4 4ou dee Goose Es 29 hende T 29 ZU FUESIS 3p bie ua pace eek oe ee Oe SAG Rew ed pee E ee ee SR SNE M 29 Table OOUONS 244754254 he boda eee eae S SIX V rd add eR RE Iv d d VE EE S 29 SHOW Data OOUONS MERERI 29 Show Expressed Genes Only 000 0c eee 29 Show Expressed PSR JUC Only 2 0 eee 29 Have at Least One PSR eee ees 30 vue sab P c Pc 30 Rearrangimg Column Orders s ua toic d he wae Ree GR iE ode bod 30 Resetting Table Defaults llle 30 Searching KeywordS cosas saan murmur Re Mw ood ge geb oc e Eee eed Redd eee ob d 30 PROOFING ODIOS aeea doneaetaaeee hd deen ees ome concn eed ba4b hen waynes 31 Exporting the Current Table with 1st Gene Symbols 20 0000 0 00000 31 Contents 4 Exporting the Current Table 2 0 eee 3 Gene Symbols to Export 2 er 32 Exporting All Data 0 lis
108. peat steps 1 4 above for your 3rd Condition 7 If needed edit your Analysis result file path and or name by clicking inside the Analysis File text field Figure 3 10 or click Browse to select a new file destination Figure 3 10 Analysis File Field Analysis File O Users ppavic Documents TAC AnalysisResults Analysis_2 tac Browse y pp y y 8 After the Conditions have been labeled and populated click Run Analysis ER TAC auto saves your studies At any time click on the Open Existing Result tab to view recent analysis results Please Wait appears then the Analysis Result viewer appears By default this new viewer opens in a split screen configuration Figure 3 11 By default the Summary tab and Scatter Plot graph tabs appear side by side nm NOTE Click to uncheck the checkbox to display each window as a full screen Gene Level Differential Expression Analysis 52 Figure 3 11 Default View after running an analysis Liver vs Muscle vs MAQCA Analysis Type Gene Level Differential Expression Analysis Array Type HTA 2_0 Genome Version hg19 Annotation File HTA 2 0 MappingFile v0 3 1 transcript csv Summary 1 Total number of genes 67525 Coding 44696 NonCoding 22829 2 11047 genes are differentially expressed Coding 8129 NonCoding 2918 3 Liver vs Muscle a 3332 genes are up regulated Coding 2534 NonCoding 798 b 2495 genes are down re
109. perform normalization gene intensity needs to be calculated For a particular gene the gene intensity for each sample is calculated using the Tukey s Biweight average for all the eligible exons PSRs intensities in that gene Next normalize each PSR or junction intensity using the gene intensity of that sample Figure A 2 Normalized intensities from Condition is compared to normalized intensities from condition 2 using One way Between Subject ANOVA for the PSRs and junctions within a gene Configurable Parameter 5 After running ANOVA multi testing correction is performed using Benjamini Hochberg Step Up FDR controlling procedure for all the expressed genes and expressed PSRs Junctions expressed in at least one condition Pm NOTE By default the Alpha level is set as 0 05 in Parameter 5 False Discovery Rate field For more information go to http media affymetrix com support technical whitepapers exon_alt_transcript_analysis_whitepaper pdf 5 Benjamini Hochberg Step Up FDR controlling Procedure FDR control is a statistical method used in multiple hypothesis testing to correct for multiple comparisons FDR is controlled at certain alpha level default 0 05 in all result tables This means that the expected proportion of rejections that are in error is less than alpha Alpha level can be customized in the result tables by changing the default filtering criteria in the FDR p value column 0 05 to something else
110. ple at least 5096 of eligible PSRs must be expressed DABG p 0 05 for the gene to be considered expressed Configurable Parameter 1 The way to decide whether a PSR is eligible is to see 1f it presents at least 50 of all the transcript isoforms for that gene Only PSRs 1 2 3 4 8 are considered as eligible PSRs in this gene as shown in the example below Figure 1 14 Figure 1 14 Configurable Parameters Exon Number 1 2 3 4 X X X amp PSR12 HPSR120011979 PSR PSR120011982 P HPSRI Isoform EX120002721 H6120 _H 20002723 EX120002724 Ex120002721 HEx120 x120002723 H EX120002724 1 Ee eas HE 20 829002723 H 8120002724 EX120002721 Ex120 x120002723 H EX120002724 HEX12 EX120016873 x1 EX120016872 x1 2 Criteria 2 A PSR or Junction can only be analyzed by Splicing Index if it expresses in at least one condition Configurable Parameter 4 To decide whether a PSR or junction is expressed in a condition you need as an example to check the DABG p values from all samples in that condition to see whether gt 50 of samples have DABG p lt 0 05 After a gene and PSRs junctions meet the criteria above normalization and comparison can begin In order to perform normalization gene intensity needs to be calculated For a particular gene the gene intensity for each sample is calculated using the Tukey s Biweight average for all the eligible exons PSRs intensities in that gene
111. probeset csv Summary 1 Total number of exons 1432143 2 21795 exons are differentially expressed 3 Normal vs Colon Cancer a 13556 exons are up regulated b 8239 exons are down regulated Algorithm Options 1 One Way Between Subject ANOVA Unpaired Default Filter Criteria 1 Fold Change linear 2 or Fold Change linear 2 2 ANOVA p value Condition pair 0 05 Conditions Normal 4 1 10 5N rma exon all dabg chp 2 12 6N rma exon all dabg chp 3 14 7N rma exon all dabg chp 4 16 8N rma exon all dabg chp Colon Cancer 4 1 15 8T rma exon all dabg chp 2 1 1T rma exon all dabg chp 3 7 AT rma exon all dabg chp 4 9 5T rma exon all dabg chp Exon Level Differential Expression Analysis Summary Window Overview The Summary information is static and based on the algorithm parameters applied during the analysis Below is an example of the information collected in the Summary window O00 Chapter 4 Exon Level Differential Expression Analysis 86 Normal vs Colon Cancer Analysis Type Exon Level Diferential Expression Analysis Array Type HuEx 1 0 st v2 Genome Version hg18 Annotation File HuEx 1 0 st v2 na33 1 hg18 transcript csv Annotation File HuEx 1_0 st v2 na33 1 hg19 probeset csv Summary 1 Total number of exons 1432143 2 21795 exons are differentially expressed 3 Normal vs Colon Cancer a 13556 exons are up regulated b 8239 exons are down regulated A
112. recent results in the Open Existing Result window If you still cannot locate your study click Browse For Existing Analysis Result l Chapter 1 Introduction 15 The following window appears Figure 1 17 LE M ce TAC AnalysisResults Search AnalysisResults P Organize New folder Name Date modified ir Faworites BE Desktop EJ Analysis 1 10 25 2012 9 04 AM Jg Downloads 3 Analysis 2 10 24 2012 3 01 PM S Recent Places 3 Analysis 3 10 25 2012 9 01 AM amp Analysis 4 10 26 2012 10 33 Libraries 3 Analysis 5 10 26 2012 10 33 Documents 3 Analysis 6 11 1 2012 341 PM a Music 3 Analysis 7 11 2 2012 9 03 AM Pictures E Analysis 8 11 2 2012 9 26 AM BE Videos EJ Analysis 9 11 2 2012 1 46 PM EJ Analysis 10 11 5 2012 9 13 AM E Computer 3 Analysis 11 11 2 2012 312 PM EL Local Disk C EJ Analysis 12 11 5 2012 9 14 AM cx DATAPARTI D EJ Analysis 13 11 8 2012 12 58 PM DVD RW Drive E R Anabarin 1A 11 n amp rn 11 4 n n me me File name TAC Analysis Result 2 Click on a recent study from the TAC Analysis Results window then click Open After a few moments your recent study opens in the same state you last left it Alternative Splicing Analysis Setting Up an Analysis Using Alt Splice CHP Files 1 At the main TAC window click Alternate Splicing Analysis Figure 2 1 Figure 2 1 Main TAC window Select Analysis Alternative Splicing Analysi
113. rmation only or All information Chapter 4 Exon Level Differential Expression Analysis 94 Gene Symbols to Export 1 Click either 1st gene symbol or All gene symbols 2 Click OK The Save As window appears 3 Click on an existing folder or click New Folder to choose a new save location 4 Type a filename for the table then click Save The table is now saved as a txt file Exporting All Data 1 Click Export All Data EE NOTE Only currently paired data is exported including data in the hidden columns and the paired data s gene level information The Save As window appears 2 Click on an existing folder or click New Folder to choose a new save location 3 Type a filename for the table then click Save The table is now saved as a txt file Saving Table Information Use this copy feature to save table information to the Windows Clipboard then use this buffered information for pasting into other applications or websites Copy Selected Row s 1 Click to highlight light blue a row or Ctrl left click to highlight multiple rows 2 Right click then click to select Copy Selected Row s Figure 4 21 Figure 4 21 Copy Selected Row s option TCO1003388 7 TC04001274 TC12002283 fh TCO2004095 TC10002643 TCOADO1662 TCO1004981 TC04202794 TC12002282 TCO1000708 TCOADO2793 hg 1 Search NCBI Entrez Databases Search NCBI Gene Database Search Ensembl Databases pee sew ie ptu own
114. rms are Sorted on page 45 Figure 2 50 Get Score Notepad File Example Untitled Notepad File Edit Format View Help TR10005657 hg TR10005657 hg 4 86306598782539 Eeee 1R10001881 hg 3 44722348451614 es 1R10004184 hg 2 8886516392231 TR10004184 hg TR10000675 hg 5 69265922904015 TR10000675 hg TR10008792 hg 6 69293043017387 TR10001295 hg 8 10877293348312 TR10008792 hg TR10001295 hg Gene Level Differential Expression Analysis Setting Up an Analysis Using Gene CHP Files 1 At the main TAC window click Gene Level Differential Expression Analysis Figure 3 1 Figure 3 1 Main TAC Window Select Analysis Alternative Splicing Analysis Gene Level Differential Expression Analysis Exon Level Differential Expression Analysis 2 The New Analysis window appears Figure 3 2 Figure 3 2 New Analysis Window Transcriptome Analysis Console Open Existing Result Preferences 4m Gene Level Differential Expression Analysis Remove Selected Parse File Names Show Grouped Files Name Array Type File Type File Path Click Import Data to import files for analysis Condition1 Condition2 Each condition must have at least one file Bronse C Users ppavic Documents TAC AnalysisResults Analysis_12 tac Analysis File 3 Click Import Data The following window appears Figure 3 3 It displays the data path yo
115. s Gene Level Differential Expression Analysis Exon Level Differential Expression Analysis The New Analysis window appears Figure 2 2 Figure 2 2 New Analysis window Transcriptome Analysis Console Alternative Splicing Analysis Remove Selected Parse File Names Show Grouped Files Name Array Type File Type File Path Click Import Data to import files for analysis Condition1 Condition2 Each condition must have at least one file Analysis File C Users ppavic Documents TAC AnalysisResults Analysis_36 tac 2 Click Import Data The following window appears Figure 2 3 It displays the data path you set up earlier and its files o NOTE The first time you launch TAC it asks you to define a path to store your library and annotation files For your convenience TAC retains this path information Affymetrix recommends you use the Expression Console library path you already configured Alternative Splicing Analysis 17 IMPORTANT To perform an alternative splicing analysis you must import alt splice chp files Figure 2 3 HTA2 liver BetaSamplaePool 1 v 3 ma alt splice dabg chp HTA2 Liver BetaSamplePool 2 v 3 ma alt splice dabg chp HTA2 liver BetaSamplePool 3 v 3 ma alt splice dabg chp ss HTA Liver BetaSamplePool 4 v 3 ma alt splice dabq chp m HTA2 MAQCA PolyA EC1 1 v03 ma alt splice dabg chp HTA2 MAQCA PolyA EC1 2 v03 ma alt sp
116. scripts x Alternative Splicing Analysis 23 Alternative Splicing Analysis Summary Window Overview The information shown in the summary is based on the algorithm parameters applied during the analysis See the example below Figure 2 12 Figure 2 12 Summary Window Liver vs Muscle vs MAQCB Analysis Type Alternative Splicing Analysis Array Type HTA 2_0 Genome Version hg19 Map File HTA 2 0 MappingFile v0 3 1 rev1 map Annotation File HTA 2 0 MappingFile vO 3 1 transcript csv Summary 1 Total number of genes 67536 a Coding 44707 b NonCoding 22829 2 Liver vs Muscle a Coding 33723 genes expressed in both conditions 8270 expressed genes have at least one PSR with Splicing Index linear 2 or Splicing Index linear 2 to indicate alternative splicing 2059 genes expressed in Liver but not expressed in Muscle 2565 genes expressed in Muscle but not expressed in Liver 6360 genes not expressed in both conditions b NonCoding 14609 genes expressed in both conditions 1182 expressed genes have at least one PSR with Splicing Index linear 2 or Splicing Index linear 2 to indicate alternative splicing 1495 genes expressed in Liver but not expressed in Muscle Algorithm Options 1 Splicing Index 2 One Way Between Subject ANOVA Unpaired 3 False Discovery Rate 0 05 4 Use an eligible PSR to determine gene expression if it presents in gt 50 of all the transcript i
117. se 32 Saving Table Information eee een 22 Copy Selected Row s oonan auaa naaa eee 32 Copy Selected ID 5 usa auecacecere qaaa d adea ideni PERPE Bae hed SESS 33 Copy Selected Row s Gene Symbols 0 000 0000 eee 33 Copy PsR Iunctuon Information su saezsmeckh rcu RR PEERS ERERSRRSEXSS 34 Accessing External Databases Internet Connection Required 34 Searching the Affymetrix NetAffx Website llle 35 Viewing Results in the Splicing Viewer lel RI 36 Using the Splicing Table and Splicing Viewer llle 36 Parts of the Visualization Tab illleeee e 37 Changing the Factory Set Scale Limits 37 Setting New Scale Ranges llle 37 Changing Scale COlOlS s 44 ducawt seek goede es Sb ai E a eee eo 37 ZEN 4 oa aoa seta gs ae babe ee eons ou oe oS 44 E eee eee s os 37 Parts of the Structure VIEW scabs tase y druide Xd we xg ina TEE eee Oe 38 Using the Intensity and Splicing Index Tracks with the Table 38 Using the zoom Feature Losses ve xu Edu ua ed 296 2 9 dE AORTA e ERE ROUES 39 Using the Show Junction ODLOIY acxacgenssx um uU E bees Bo0a Pew mx e ass 40 Using the Combine Neighboring Psrs Option 0 0 00 ee 40 Using the Enable Tooltip Option llille eee 40 Identifying an Alternative Splicing Event using Structure View 00005 41 How Each Isoform Sorting Score is Calculated llle 42 Parts of the Genomic View
118. soforms 5 A gene is expressed in a sample if gt 50 36 of its eligible PSRs have DABG p value lt 0 05 6 A condition has this gene expressed if 250 of its samples have this gene expressed 7 A PSR Junction is expressed in a condition if 250 96 of samples have DABG p value 0 05 Default Filter Criteria 1 Splicing Index linear 2 or Splicing Index linear gt 2 2 FDR p value 0 05 3 A gene is expressed in both conditions 4 A PSR Junction must be expressed in at least one condition 5 A gene must contain at least one PSR Conditions Liver 4 1 HTA2 Liver BetasamplePool 1 v03 rma alt splice dabg chp 2 HTA2 Liver BetasamplePool 2 v03 rma alt splice dabg chp 3 HTA2 Liver BetaSamplePool 3 v03 rma alt splice dabg chp 4 HTA2 Liver BetasamplePool 4 v03 rma alt splice dabg chp Muscle 4 1 HTA2 Muscle BetaSamplePool 1 v03 rma alt splice dabg chp Fi UTAJ Adcorela Datatomnlsanaal 3142 ma 8 alt nonlic calemcsn 1 Summary information per this Alternative Splicing Analysis NOTE Summaries vary between Gene Exon and Splicing analysis 2 Array summary Total number of transcript clusters genes and numbers of coding and noncoding transcript clusters on this array 3 Summary of the Coding transcript clusters In the example above 33723 transcript clusters passed the Splicing Index criteria of Genes need to be expressed in both conditions 8270 transcript clusters have at least one Probe Selection
119. then click OK Save as PNG 1 Click Save PNG to save the graph as a PNG image file The Save As window appears 2 Click on an existing folder or click New Folder to choose a new save location 3 Type a filename for the graph then click Save The graph is now saved as a png file Clearing Lassoed Selections This option is enabled after genes have been lassoed as points of interest 1 Click Clear Selection to clear lassoed genes from the graph and table En m ud ER You can also clear a lassoed selection by lassoing a white blank space within the graph Changing Graph Colors Use the drop down menus to select your up and down regulated probe set graph colors Figure 3 37 Figure 3 37 Graph Color Menus Red and Green are default B Up in Liver vs Muscle a i Gene Level Differential Expression Analysis 71 Obtaining Information Related to Individual Probe Sets 1 Mouse over position the cursor over a probe set to show its details In the example below Figure 3 38 signals from both conditions 12 45 is the signal in Liver Condition1 and 4 22 is the signal in Muscle Condition2 are shown Fold change FC in linear scale ANOVA p value chromosome positions and gene symbol are also identified in the line below Figure 3 38 Figure 3 38 Mouse Over Example E 15 E 14 E 13 E 12 E 11 E 10 E 08 E 08 E O7 en i zh ce a e m ami L e rr a Lm e a i
120. u set up earlier and its files NOTE The first time you launch TAC it asks you to define a path to store your library and Dn files For your convenience TAC retains this path information Affymetrix recommends you use the Expression Console library path you already configured Gene Level Differential Expression Analysis 48 Figure 3 3 Import Data Window s Oo lll Current Directory C Users ppavic Desktop TAC_Samples Up One Level Li E HTA2 Liver BetaSamplePool 1 v03 rma gene4ull chp ma gene 4ull chp HTA2 Liver BetaSamplePool 3 v03 rma gene4ull chp EB HTA2 Liver BetaSamplePool 4 v03 rma gene4ull chp HTA2 MAQCA PolyA EC1 1 v03 1ma gene4ull chp HTA2 MAQCA PolyA EC1 2 v03 ma gene4ull chp HTA2 MAQCB PolyA EC1 1 v03 ma geneull chp HTA2 MAQCB PolyA EC1 2 v03 ma gene4ull chp M HTA2 Muscle BetaSamplePool 1 v03 rma gene4ull chp O HTA2_Muscle_BetaSamplePool_2 v03 ma gene ull chp HTA2 Muscle BetaSamplePool 3 v03 rma gene ull chp M HTA2_Muscle_BetaSamplePool_4 v03 ma gene ull chp HTA2_Spleen_PolyA_EC1_1 v03 ma gene ull chp Lj HTA2_Spleen_PolyA_EC1_2 v03 ma gene ull chp O HTA2_Testes_PolyA_EC1_1 v03 ma gene full chp m Files of type CHP Fies Cancel 4 Single click Ctrl click or Shift click to select multiple files as shown above nm NOTE To optimize the analysis Affymetrix recommends importing more than 1 sample per condition 5
121. us Red and Green are default B Up in Liver vs Muscle B I Gene Level Differential Expression Analysis 75 Obtaining Information Related to Individual Probe Sets 1 Mouse over position the cursor over a probe set to show its details In the example below Figure 3 43 signals from both conditions 11 81 is the signal in Liver and 9 16 is the signal in Muscle Condition2 are shown Fold change FC in linear scale ANOVA p value chromosome positions and gene symbol are also identified in the line below Figure 3 43 Figure 3 43 Mouse Over Example telpu t ee TEENIT NH wht 4 re 5 5548 a0 1 TIN Pe LL og I 91 Al TEER anl z VW W T nui E 21 1E LE d 22 rn 7 x Ht NL I NM m iM rg vit 1 ad IDZTCOXO000580 hg 1 Signal 11 81 9 16 FC 6 31 pValz6 90E 11 chrX 118370211 1183 78429 gene symbol PGRMCT ER The best way to view individual probe sets is to make the chromosome summary full screen by un checking E RIAT Hierarchical Clustering Graph Hierarchical Clustering is a method of cluster analysis which seeks to build a hierarchy of clusters for use as a data mining tool 1 Click on the Hierarchical Clustering tab If the message Figure 3 44 appears click on the Table tab to apply a stricter filter Figure 3 44 Clustering Filter Alert Too much data to cluster Apply a stricter filter to reduce the number of genes For instructions on how to modif
122. view Condition1 PSR JUC Expressed Detail Condition2 PSR JUC Expressed Detail Number of samples met the DABG cutoff total samples in a condition As an example 4 4 means 4 out of 4 samples met the DABG criteria DABG 0 05 0 4 means no sample met the DABG criteria DABG 0 05 Condition1 Bi weight Avg Signal log2 Condition2 Bi weight Avg Signal log2 Tukey s Bi weight average of PSR or junction intensity of all the samples in a condition Bi weight average of sample 1 PSR1 intensity sample 2 PSR1 intensity sample N PSR1 intensity Fold Change linear Condition1 vs Condition2 This shows the fold change in linear space of Condition vs Condition2 2 Condition1 Bi weight Avg Signal log2 Condition2 Bi weight Avg Signal log2 Condition1 Normalized Avg Signal log2 Condition2 Normalized Avg Signal log2 Tukey s Bi weight average of normalized PSR or junction intensity of all the samples in this condition Bi weight average of sample 1 PSR1 intensity sample 1 gene1 intensity sample 2 PSR1 intensity sample 2 gene1 intensity sample N PSR1 intensity sample N gene1 intensity Splicing Index linear Condition1 vs Condition2 This shows the normalized fold change in linear space of Condition1 vs Condition2 2 Condition1 Normalized Avg Signal log2 Condition2 Normalized Avg Signal log2 ANOVA p value Condition1 vs Condition2 One Way Between Subje
123. y filters See Filtering Column Data on page 57 The following message and button appears Figure 3 45 Figure 3 45 Hierarchical Clustering Analysis Button This might take a long time to perform hierarchical clustering Click to Perform Hierarchical Clustering Analysis 2 Click Click to Perform Hierarchical Clustering Analysis Gene Level Differential Expression Analysis 76 After a few moments the Hierarchical Clustering Analysis results appear Figure 3 46 EE NOTE The probe sets from your current condition pair that pass the filter criteria are sent to clustering The results shown reflect your current condition pairings only To confirm what pairing is active refer to the TAC Table s Comparison drop down selections Figure 3 22 Figure 3 46 Hierarchical Cluster Full screen een Analysis_15 tac Analysis Result a mm S ll Split View Summary Scatter Plot Volcano Plot Chromosome Summary laa Save PNG s Print TC05001422 hg 1 AC079467 1 TC06003665 hg 1 TC08001989 hg 1 TC03003169 hg 1 TC07001770 hg 1 PPP1R3A TC10002682 hg 1 TC06003061 hg 1 TC04001242 hg 1 TECRL TC01004414 hg 1 TC03000668 hg 1 KBTBD12 TC12001712 hg 1 BEST3 TC07002202 hg 1 TC10001316 hg 1 M1 TC02003720 hg 1 TCOX002125 hg 1 TC13000045 hg 1 SGCG TC06000999 hg 1 EYA4 TCO0X001155 hg 1 XIST TC04000568 hg 1 EGF TC15000672 hg 1 DQ587888 TC06002840 hg 1 TC02001077 hg 1 AC010680 1 TC1500273
124. ysis Type Gene Level Differential Expression Analysis Array Type HTA 2_0 Genome Version hg19 Annotation File HTA 2 0 MappingFile vO 3 1 transcript csv Summary 1 Total number of genes 67525 Coding 44696 NonCoding 22829 2 11047 genes are differentially expressed Coding 8129 NonCodina 2918 3 Liver vs Muscle a 3332 genes are up regulated Coding 2534 NonCoding 798 b 2495 genes are down regulated Coding 1840 NonCoding 655 4 Liver vs MAQCA a 3167 genes are up regulated Coding 2436 NonCoding 731 b 3483 genes are down regulated Coding 2420 NonCoding 1063 5 Muscle vs MAQCA a 2420 genes are up regulated Coding 1834 NonCoding 586 b 3343 genes are down regulated Coding 2314 NonCoding 1029 OOO Algorithm Options 1 One Way Between Subject ANOVA Unpaired Default Filter Criteria 1 Fold Change linear 2 or Fold Change linear gt 2 2 ANOVA p value Condition pair lt 0 05 Conditions Liver 4 1 HTA2 Liver BetasamplePool 1 v03 rma gene full chp 2 HTA2 Liver BetasamplePool 2 v03 rma gene full chp 3 HTA2 Liver BetasamplePool 3 v03 rma gene full chp 4 HTA2 Liver BetasamplePool 4 v03 rma gene full chp TT Muscle 4 1 HTA2 Muscle BetaSamplePool 1 v03 rma gene full chp 2 HTA2 Muscle BetaSamplePool 2 v03 rma gene full chp 3 HTA2 Muscle BetaSamplePool 3 v03 rma gene full chp 4 HTA2 Muscle BetaSamplePool 4 v03 rma gene full

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