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GeneSpring 7.2 Addendum

1. HAMPRO RMA in GS MPRO_Od vpxgl 145981 Preprocessor Thon IMPRO_Oday_C i 25 P TRANSCRIPTOME ANALYSIS STUDY MPRO_1d vpxgl 145952 Preprocessor Thon IMPRO_1day_ MPRO_1d vpxgl 145983 Preprocessor Thon IMPRO_1day_B MPRO_1d pxgh145984 Preprocessor Thon IMPRO_1day_C MPRO_2d vpxgl 145985 Preprocessor Thon IMPRO_2day_ MPRO_2d vpxgl 145986 Preprocessor Thon IMPRO_2day_E a MDRM A vee 4 ASORF Selected tems 0 Name ID Than MORM dau C Configure Columns Organization Folder Attachments Page 21 of 40 Choose the samples that you want to include in the analysis by selecting the samples in the top right window and clicking the Add button or press the Add all button to include all the samples To select samples from a different experiment select the experiment from the navigation tree on the left side of the window The samples associated with that experiment will be shown in the top right corner as before and you can make a different selection Select Samples BK Show All Filter On Experiment Fitter on Attribute Filter Results 21 items 15 selected 24 All Objects Nemo ID Notes _ 9 Experiments MPRO 2 pxal145985 Prenroci Eaa LI Hematopoiesis Study MPRO_2d vpxgl 1459 Prenrocessc Thon
2. Attributes and Parameters Sample Correlations Associated Files Graph To add an attachment drag a file into the table below or click on the Add File button MPRO_Oday_A CEL Attachment Sample Image MPRO_Oday_A tet Data File al Add File Extract File Delete File View File View Data File Format Export as Zip OK Cance Help 10f21 lt lt gt gt 2 6 4 Workflow The import of CEL files into GeneSpring follows the standard workflow with only two possible new steps as outlined below 1 Select the CEL files you want to load and drag them onto an open window of GeneSpring or use the File gt Import Data menu item 2 GeneSpring analyzes the files Please w A m x Analyzing files please 3 The Define File Format and Genome window appears with all the possible import format options for this file File Format Choose File Format Genome Select the genome set of genes on the array for this data If your genome does not appear on the list you can create a new one by selecting Create a New Genome Select Genome EHO Genomes or Arrays Attimetrix HG_U133 PLUS2_ Target HG_U95v2 HG U133 Dema Chips Te Demo Human C Create a New Genome a Nert Cancel Help 4 Affymetrix CEL file are identified along with the Array name that the CEL file relates to in the top drop down menu e If a file
3. Data Transformation Transform from log to linear valu Data Transformation Dye swap Per Spot Divide by control channel Data Transformation Reserve control channel Per Spot and Per Chip Intensity dependent Lowess t Per Chip Normalize to a median or percentile Per Chip Normalize to positive control genes Per Chip Normalize to a constant value Use Recommended Order Get Text Description Per Gene Normalize to specific samples Per Gene Normalize to median Use a Saved Scenario Save As Scenario Per Chip and Per Gene Median polishing Warnings No warnings El OK Cancel Help Although the normalization steps are not harmful some of them are not required and can be removed The first normalization step Data Transformation Set measurement less than 0 01 to 0 01 is a step that ensures that any value less then 0 01 is set to 0 01 This step is added to ensure no negative values are loaded since these values could cause problems for some of the analysis in GeneSpring The RMA and GC RMA algorithm will a ways return values that are positive so this step is not required and could be removed The second normalization step is the GeneSpring normalization step that ensures the expression values for each chip can be compared by dividing the expression values by the median value of all the expression values Per Chip Normalize to the 50 percentile Since the RMA and GC RMA algorithms perform th
4. 0 270 0 356 3108 992 12 819 2 655 0 693 0 647 0 062 0 062 0 062 0 065 3072 179 3 714 Ideal 1 000 1 000 1 000 1 000 1 000 1 000 0 000 16 000 0 000 1 000 1 000 1 000 1 000 1 000 1 000 0 000 16 000 Table 6 The results from the affycomp GC RMA and MASS original assessment of HG U95A spike in data Results from TableAll function in the BioConductor affycomp package Ideal indicates the number that the assessment would be if the algorithm and hybridizations were perfect HGU_95A New Assessment null log fc IQR null log fc 99 null log fc 99 9 low AUC med AUC high AUC weighted avg AUC 25 SD Median SD 75 SD 99 SD low slope med slope high slope low R2 med R2 high R2 0 25 0 0 5 0 25 GeneSpring RMA BioConductor RMA 0 194 0 412 0 574 0 313 0 851 0 458 0 443 0 096 0 114 0 135 0 218 0 293 0 733 0 473 0 032 0 457 0 332 0 238 0 291 0 194 0 412 0 575 0 314 0 851 0 459 0 444 0 096 0 114 0 135 0 218 0 293 0 734 0 473 0 032 0 457 0 332 0 236 0 291 1 0 5 0 295 0 295 2 1 0 479 0 479 4 2 0 641 0 641 8 4 0 712 0 713 16 8 0 780 0 780 32 16 0 788 0 788 64 32 0 753 0 753 128 64 0 629 0 630 256 128 0 559 0 559 512 256 0 406 0 407 1024 512 0 285 0 285 Table 7 The results from the affycomp GC RMA new assessment of HG U954A spike in data Results from TableAll function in the BioConductor affycomp package HGU_95A New Assessment GeneSpring GC RMA BioConduct
5. Add All gt gt gt MPRO_Oday_A v Ohr_B CHP 7 14 02 Affy WebC MPRO_Oday_A vOhr_C CHP 7 14 02 MPRO_Oday_A_ _Ohr_D CHP 111 4 02 Amersham C lt lt lt Remove All Mouse brain MPRO_Oday_B_v_Ohr_ACHP 7 14 02 PATHWAY exam MPRO_Oday_B_v Ohr B CHP 7 14 02 i EME gt PamGene data MPRO Oday B v Ohr C CHP 7 1 an2 z i gt 4 b Samples have multiple files subchips Previous Next Cancel Help The Preprocessing Data Files appears and indicates that GeneSpring is loading the files Preprocessing Data Files Seles Preprocessing Files CHP File Importer After the CHP files are processed you are given the opportunity to enter some additional Sample Attributes in the Import Data Sample Attributes window The attributes that are automatically loaded as described in the section above are not shown in this window but will be loaded Click Next to continue e The Import Data Sample Attributes window will possibly not appear or may contain different fields A different set of Standard Attributes may be the cause See the manual on Standard Attributes for more information Import Data Sample Attributes Seles Please select values for sample attributes Samples J S d d New Attribute Attribute Name Array Design Author Experiment Type Labe tribute Units Aes Numeric no no no toute PRO Oday Att 2 MPRO_Oday_B txt Replace Text a EN MPRo_oday_D te
6. decision will be guided by the two goals of preserving the free status of all derivatives of our free software and of promoting the sharing and reuse of software generally NO WARRANTY 15 BECAUSE THE LIBRARY IS LICENSED FREE OF CHARGE THERE IS NO WARRANTY FOR THE LIBRARY TO THE EXTENT PERMITTED BY APPLICABLE LAW EXCEPT WHEN OTHERWISE STATED IN WRITING THE COPYRIGHT HOLDERS AND OR OTHER PARTIES PROVIDE THE LIBRARY AS IS WITHOUT WARRANTY OF ANY KIND EITHER EXPRESSED OR IMPLIED INCLUDING BUT NOT LIMITED TO THE IMPLIED WARRANTIES OF MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE THE ENTIRE RISK AS TO THE QUALITY AND PERFORMANCE OF THE LIBRARY IS WITH YOU SHOULD THE LIBRARY PROVE DEFECTIVE YOU ASSUME THE COST OF ALL NECESSARY SERVICING REPAIR OR CORRECTION 16 INNO EVENT UNLESS REQUIRED BY APPLICABLE LAW OR AGREED TO IN WRITING WILL ANY COPYRIGHT HOLDER OR ANY OTHER PARTY WHO MAY MODIFY AND OR REDISTRIBUTE THE LIBRARY AS PERMITTED ABOVE BE LIABLE TO YOU FOR DAMAGES INCLUDING ANY GENERAL SPECIAL INCIDENTAL OR CONSEQUENTIAL DAMAGES ARISING OUT OF THE USE OR INABILITY TO USE THE LIBRARY INCLUDING BUT NOT LIMITED TO LOSS OF DATA OR DATA BEING RENDERED INACCURATE OR LOSSES SUSTAINED BY YOU OR THIRD PARTIES OR A FAILURE OF THE LIBRARY TO OPERATE WITH ANY OTHER SOFTWARE EVEN IF SUCH HOLDER OR OTHER PARTY HAS BEEN ADVISED OF THE POSSIBILITY OF SUCH DAMAGES Source code The source code is released under the Lesser Gnu Publ
7. onfailing sehemic iWiopathic Y axis Heart failure study Default Interpretation Colored by 112 Nonfailing Gene List all genes 54676 gt 4 PO PE All Data Magnification 1 2 5 2 5 1 2 5 2 The Experiments Inspector lets you change a number of annotations for the Experiment such as the Experiment Name and Project association It also lets you view and edit the Experiment Parameters Interpretations and Normalizations For more information on the Experiment Inspector see the User Manual 2 The Genome Inspector menu item is new to the Annotations window S Full Development HG_U133_PLUS2_Target Genes all genes File Edit View Experiments Colorbar Filtering Tools Euwe Window Help Gene Lists F4 GeneSpider Experiments Build Homology Tables Gene Trees Condition Trees Classifications Pathways Array Layouts Expression Profiles External Programs Bookrnarks Scripts Make Gene Lists from Annotations Build Ontology Edit Genes and Annotations Genome Inspector i MoN De AE PL TRES ap ve il os eae P J E ON he 3 i Normalized Intensity log scale Expression onfailing L sthemic L aiopathic Y axis Heart failure study Default Interpretation Colored by 112 Nonfailing Gene List all genes 54676 Kig gt Show An Data Magnification 1 The Genome Inspector lets you obtain information about the currently opened genome a
8. stored in the Affymetrix CEL files GeneSpring 7 2 supports the direct loading of CEL files from the GOCS system and the normalization of RMA or GC RMA on those CEL files The workflow differs only slightly from the workflow to import of other data files CEL files are imported by the drag and drop method or by using the File gt Import Data menu item 2 6 1 Supported formats The import of the CEL files is implemented as a pre processor plug in which recognizes the following formats 2 6 2 e Original CEL file format Version 3 Before GCOS 1 2 e New Binary CEL file format Version 4 GOCS 1 2 and later Sample attributes imported The pre processor plug in extracts a number of fields from the CEL files that are imported automatically as Sample Attributes Table 1 contains a description of the Sample Attributes that are imported Sample Attribute name Contents 2 6 3 Algorithm Name The name of the Algorithm used in the analysis Usually Percentile for the RMA algorithm Algorithm Parameters The comma separated set of parameters used for the analysis like BF Alpha1 Alpha2 Tau Gamma etc for MAS5 CEL File Name The name of the original CEL used in the analysis NOTE The complete path of the CEL file is recorded but since only the CHP file is imported it is not guaranteed that the CEL file can be found in this location Probe Level Analysis Indicates what type of probe level analysis was perf
9. Ea Previous Next Cancel Help 10 After the Sample attributes are entered GeneSpring creates the samples im Creating Samples E m EW Please wait while the samples are created 11 After the samples are created you are offered a chance to create a GeneSpring Experiment with the samples that were just loaded Click Yes to create an experiment or No to continue without creating an experiment e f you choose not to create an experiment you can access the samples through the Sample Manager See the User Manual entry for the Sample Manager for more information Import Data Create Experiment 4 new samples have been created Would you also like to create an experiment trom these samples 12 If you choose to make an experiment the Save New Experiment window appears where you can change the name of the experiment and assign it to a Project Save New Experiment KB Name ES M Project Change Project s Notes EY EE a Hematopoiesis Study 2 os pe m Le in oi T T EL a D Le m E E Sample Number 3 Save Cancel At this point a new experiment is created from the CHP files and regular GeneSpring analysis can begin 2 6 Directly load CEL files and perform RMA and GC RMA RMA Robust Multichip Average and GC RMA are alternative probe level analysis algorithms for the Affymetrix GeneChip technology These algorithms use the probe data
10. GC RMA algorithms using the special interactive plug ins The interactive plug ins were created using the GeneSpring API that was introduced in GeneSpring 7 For more information about the Page 20 of 40 GeneSpring Java API see the JAVADOCS and GeneSpring API Tutorial located in the GeneSpring docs directory The RMA and GC RMA normalization techniques use all available expression data for all of the available chips as they are loaded into GeneSpring with the preprocessor plug in as described previously If one or more of the loaded CEL files is found to contain incorrect data If the hybridization failed for instance or is incorrectly labeled this aberrant CEL file may bias the normalization of all the other samples It is recommended that you exclude aberrant CEL files from RMA and GC RMA normalization GeneSpring 7 2 allows you to select a subset of samples from the GeneSpring data repository without having to retrieve the original CEL files from the GCOS or other archival system If the CEL files are attached to the GeneSpring Samples they can be directly used in the re analysis with RMA or GC RMA To re analyze samples with CEL files that are already loaded into GeneSpring follow these steps 1 Select the interactive plug in Reanalyze samples using RMA or using GC RMA from the External Programs folder Reanalyze samples Full Development Demo Human Genes all genes File Edit View Experiments Colorbar Filtering To
11. Gene Normalize to median Per Chip and Per Gene Median polishing El OK DAAA 1 O af AN Dana QU MT A a CG J U T U Order of Normalizations to Perform Per Gene Mormalize to median Use Defaults El Use Recommended Order Get Text Description Use a Saved Scenario Save As Scenario Warnings Mo per chip normalization has been applied to any sample Help Cancel The default cutoff settings for the Per Gene Normalize the median normalization step sets the minimal value for the raw expression value to 10 This is rather high for RMA normalized data and it normalization step to 0 01 T Per Gene Normalize to median is therefore recommended to set the cutoff values in this Bee Normalize each gene to the median of the measurements for that gene Cutoff Ifthe median is below the cutoff the cutoff value will be used instead See Help for details Cutoff Value 0 01 Apply only to Specific Samples _X Raw Signal measurement values Cancel NOTE Most implementations of RMA and GC RMA including the RMA implementation in the GeneSpring R Integration package return expression values in LOG2 space The GeneSpring implementation transformation step is not needed 2 7 returns data in normal linear space A Log to Linear Re analyze samples with CEL files using RMA and GC RMA Existing GeneSpring samples can be re analyzed with the RMA or
12. PA Systolic 8 eo 2 Experiment Properties Heart failure study Experiment inspector Experiment Farameters Error Model Structure Save Save As New Help In GeneSpring 7 2 the number of genes used in drawing the thumbnail graphic is limited to a random set of a maximum of 1000 genes Performance of the Change Interpretation is increased because drawing is much faster Not all genes are visible in the thumbnail version of the graph The thumbnail graph is only intended to indicate how the graph in the main GeneSpring window will appear The graph in the main GeneSpring view is not limited by this set of 1000 genes The main graph will continue to show all the genes in the selected gene list New menu items Two new menu items were added to make navigating to the Inspectors faster 1 The Experiment Inspector menu item is new to the Experiments menu Full Development HG_U133_PLUS2_ Target Genes all genes File Edit View SSH Colorbar Filtering Tools Annotations Window Help Experiment Experiment Normalizations F i _ Gene Trees Experiment Parameters Condition T Cross Gene Error Model 1 Classificati Experiment Interpretation A Pathways _ Array Layou _ Expression Create New Experiment Duplicate Experiment Expression External Pr Sample Manager is i rth _ Bookmarks 50170 sa F Scripts 112 200 294 325 115 242 297 38650 132 225 319 64 85 98 L
13. assessment of HG_U133A spike in data Results from TableAll function in the BioConductor affycomp package Ideal indicates the number that the assessment would be if the algorithm and hybridizations were perfect HGU_133A New Assessment GeneSpring RMA BioConductor RMA null log fc IQR 0 194 0 194 null log fc 99 0 412 0 412 null log fc 99 9 0 574 0 575 low AUC 0 313 0 314 med AUC 0 851 0 851 high AUC 0 458 0 459 weighted avg AUC 0 443 0 444 25 SD 0 096 0 096 Median SD 0 114 0 114 15 SD 0 135 0 135 99 SD 0 218 0 218 low slope 0 293 0 293 med slope 0 733 0 734 high slope 0 473 0 473 low R2 0 032 0 032 med R2 0 457 0 457 high R2 0 332 0 332 0 25 0 0 238 0 236 0 5 0 25 0 291 0 291 1 0 5 0 295 0 295 2 1 0 479 0 479 4 2 0 641 0 641 8 4 0 712 0 713 16 8 0 780 0 780 32 16 0 788 0 788 64 32 0 753 0 753 128 64 0 629 0 630 256 128 0 559 0 559 512 256 0 406 0 407 1024 512 0 285 0 285 Table 7 The results from the affycomp GC RMA new assessment of HG U1535A spike in data Results from TableAll function in the BioConductor affycomp package HGU_133A New Assessment GeneSpring GC RMA BioConductor GC RMA null log fc IQR 0 087 0 081 null log fc 99 0 417 0 416 null log fc 99 9 0 647 0 641 low AUC 0 469 0 472 med AUC 0 799 0 801 high AUC 0 839 0 842 weighted avg AUC 0 552 0 555 25 SD 0 050 0 048 Median SD 0 074 0 073 3 3 2 75 SD 0 103 0 102 99 SD 0 198 0 198 low slope 0 369 0 371 med slope 0 964 0 962 high slope
14. files continues Download Progress Downloading array description for Mo UrF4Ayv2 The current list of Arrayinfo files that are provided on the Agilent Technologies website are listed in Appendix A 10 If the file cannot be found on the Agilent Technologies website or no internet connection exists and you are trying to perform a regular RMA normalization you are asked to locate a CDF file library file for the specific array type A file dialog box appears and you will be able to select the CDF file If no CDF file is available no RMA normalization is possible Please locate the CDF file for the Test3 array 1521c99hpp_av06 CEL 1521d99hpp_av06 CEL 1521e99hpp_av06 CEL 1521f89hpp_av06 CEL 1521g99hpp_av0l6 CEL 1521h99hpp_av06 CEL 1521i99hpp_avO6 CEL 1521j99hpp_av06 CEL 1521k99hpp_av06 CEL 1521199hpp_av06r CEL Page 16 of 40 1521q99hpp_z 1521r99hpp_a jil gt ns Files of type All Files Cancel NOTE CDF library files can be downloaded from the support section of the Affymetrix website File name If you attempt to perform GC RMA normalization and an Arrayinfo file is not available or cannot be downloaded an Error dialog box appears instead GC RMA normalization can only work with the Arrayinfo files that are created and maintained by Agilent Technologies If the dialog box below appears contact Technical Support at Agilent Technologies at sig support agilent com or call 1 866 744 76
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16. 0 956 0 956 low R2 0 168 0 170 med R2 0 651 0 652 high R2 0 680 0 684 0 25 0 0 164 0 166 0 5 0 25 0 201 0 201 1 0 5 0 190 0 195 2 1 0 776 0 775 4 2 1 182 1 190 8 4 1 128 1 118 16 8 0 950 0 954 32 16 0 838 0 839 64 32 1 008 1 005 128 64 1 122 1 120 256 128 1 176 1 175 512 256 0 985 0 985 1024 512 0 696 0 698 Table 8 The results from the affycomp GC RMA new assessment of HG_U133A spike in data Results from TableAll function in the BioConductor affycomp package HG_U95A spike in experiment The HG_U95A spike in experiment was used for the assessment of the RMA and GC RMA algorithm as described by Cope et al Leslie M Cope Rafael A Irizarry Harris A Jaffee Zhijin Wu and Terence P Speed A benchmark for Affymetrix GeneChip expression measures Bioinformatics Vol 20 No 3 2004 323 331 The MAS5 assessment results are also provided for the original assessments to show the improvements in accuracy of the RMA algorithm in comparison with the standard MAS5 algorithms as used in the Affymetrix GCOS system HG_U95A Original Assessment GeneSpring RMA BioConductor RMA MAS5 Ideal Signal detect slope 0 625 0 625 0 706 1 000 Signal detect R2 0 804 0 804 0 857 1 000 AUC FP lt 10 0 578 0 578 0 217 1 000 AUC FP lt 15 0 627 0 627 0 238 1 000 AUC FP lt 25 0 690 0 690 0 270 1 000 AUC FP lt 100 0 821 0 821 0 356 1 000 AFP call if fc gt 2 15 858 15 842 3108 992 0 000 ATP call if fc gt 2 11 981 11 979 12 819 16 000 IQR 0 308 0 308
17. 12 19 PM 9 HHC Mini yeast MPRO_Oday_ACEL 10 16 01 12 09 PM 9 EH Mouse brain a MPRO_Ohr_ACEL 10 22 01 1 41 PM RE FH ONE COLOR ex MPRO_Ohr B CEL 10 22 01 2 46 PM ON afly webcast MPRO_Ohr_C CEL 10 22 01 3 37 PM Ta Web MPRO_Ohr_D CEL 10 22 01 2 04 PM 1 Affy WebC MPRO_1day_ACEL 10 16 01 12 51 PM _ lt lt lt Remove Al HH CHP files MPRO_1day_B CEL 10 16 01 1 45 PM Amersham C MPRO_1day_C CEL 10 16 01 2 08 PM i b HH Mouse brain y MPRO 1dav D CEL 1016 011 57PM_ 7 Be 4 gt 4 gt F Samples have multiple files subchips Previous Next Cancel Help 7 The Preprocessing Data Files window appears to indicate that GeneSpring is loading and analyzing the files T Preprocessing Data Files ig mE Preprocessing Files RMA File Preprocessor 8 For the RMA or GC RMA analysis a special file is required that links the probe information to the gene information For some of the widely used array types these files are included in the product and no action is required The arrays that are provided with GeneSpring 7 2 are e HG U133 Plus 2 e HG _U95Av2 e MG U74Av2 e Mouse430Av2 e Rat230v2 9 Ifthe array you are using is not in this list GeneSpring will try to automatically load the appropriate file called array description or Arrayinfo files from the Agilent Technologies website A dialog box indicates that the file is loading When the file is loaded the processing of the CEL
18. 2 655 0 000 Obs intended fc slope 0 612 0 612 0 693 1 000 Obs low int fc slope 0 359 0 360 0 647 1 000 FC 2 AUC FP lt 10 0 304 0 303 0 062 1 000 FC 2 AUC FP lt 15 0 343 0 343 0 062 1 000 FC 2 AUC FP lt 25 0 401 0 400 0 062 1 000 FC 2 AUC FP lt 100 0 544 0 543 0 065 1 000 FC 2 AFP call if fc gt 2 0 929 1 000 3072 179 0 000 FC 2 ATP call if fc gt 2 1 714 1 714 3 714 16 000 AUC Area Under the ROC curve AFP Average False Positives ATP Average True Positives FC Fold Change Table 5 The results from the affycomp RMA and MASS original assessment of HG _U954 spike in data Results from TableAll function in the BioConductor affycomp package Ideal indicates the number that the assessment would be if the algorithm and hybridizations were perfect HG_U95A Original Assessment Signal detect slope Signal detect R2 AUC FP lt 10 AUC FP lt 15 AUC FP lt 25 AUC FP lt 100 AFP call if fc gt 2 ATP call if fc gt 2 IQR Obs intended fc slope Obs low int fc slope FC 2 AUC FP lt 10 FC 2 AUC FP lt 15 FC 2 AUC FP lt 25 FC 2 AUC FP lt 100 FC 2 AFP call if fc gt 2 FC 2 ATP call if fc gt 2 0 842 0 908 0 583 0 643 0 704 0 839 6 535 13 154 0 411 0 824 0 651 0 301 0 351 0 415 0 577 3 000 4 714 0 843 0 908 0 583 0 643 0 705 0 840 6 856 13 109 0 412 0 825 0 654 0 297 0 349 0 414 0 574 3 179 4 536 GeneSpring GC RMA BioConductor GC RMA MASS5 0 706 0 857 0 217 0 238
19. 38 to request the creation of an Arrayinfo file x Could not Find the appropriate array definition File Please contact Silicon Genetics technical support to request the appropriate Arrayinfo File 11 After the CEL files are processed you are given the opportunity to enter some additional Sample Attributes in the Import Data Sample Attributes window The attributes that are automatically loaded as described in section 3 2 2 are not shown in this window but will be loaded Click Next to continue e If the Import Data Sample Attributes window does not appear or contains different fields a different set of Standard Attributes may be the cause See the manual on Standard Attributes for more information e The Sample Attributes window shows the sample names as XX txt These are the new sample files that are created from the CEL files by the processor The original CEL files names are one of the automatically loaded Sample Attributes DEK Import Data Sample Attributes Please select values for sample attributes Jame name E e S New Attribute Attribute Name Array Design Author Experiment Type Labelin Delete Atribute Replace Text no no no no MPRO_Oday_A txt MPRO_Oday_B ttt Su mPro_oday_c te MPRO_Oday_D te Previous Next Cancel Help 12 The Creating Samples window appears while GeneSpring is creating the samples be Creating Samples M Ed Please wait while the s
20. BEAMPRO RMA in GS MPRC 145987 errors 1 Author Organization Folder L 4 Attachments a s 555 P TRANSCRIPTOME ANALYSIS STUDY MPRO_3d lvpxgl1 45988 Preprocessor al Thon MPRO_Sday_ MPRO_Sd vpxgh145969 Preprocessor Thon MPRO_S3day_B MPRO_3d vpxgl145990 Preprocessor Thon MPRO_3day_C Remove Remove All Configure Columns Selected tems 15 ID Notes vpxgl 145979 Preprocessor l vpxgl 145980 Preprocessor _ vpxgl 145981 Preprocessor vpxgl 145982 Preprocessor il vpxgl 145983 Preprocessor vpxgl145984 Preprocessor wel PXGE145985 Preprocessor PXGE145986 Preprocessor vpxgl 145987 Preprocessor D vpxgl 145991 Preprocessor verve 145909 iPrenracesenr Folder Attachments IMPRO_Oday_ IMPRO_Oday_B MPRO_Oday_C IMPRO_1day_ IMPRO_1day_B IMPRO_1day_C IMPRO_2day_ IMPRO_2day_B IMPRO_2day_C IMPRO_4day_ MPRA ddaw R Organization Thon Thon Thon Thon Thon Thon Thon Thon Thon Thon Than NS Se eee MPRA Ad Fig 1 All samples except those starting with MPRO_3d are added to the lower right hand panel To select samples that are not associated with an experiment click the Show All tab to show all the sam
21. Bolstad Francois Collin Leslie Cope Bridget Hobbs and Terence Speed Summaries of Affymetrix GeneChip probe level data Nucleic Acids Research 31 4 2003 3 2 3 2 1 3 2 2 3 2 3 3 2 4 3 3 GC RMA algorithm GCRMA Robust Multi chip Average with GC content background correction is a method for normalizing and summarizing probe level intensity measurements from Affymetrix GeneChips Starting with the probe level data from a set of GeneChips the perfect match PM values are background corrected normalized and finally summarized resulting in a set of expression measures The three steps of the process are outlined below Background Correction The background correction used in GCRMA is designed to account for background noise as well as non specific binding Probe affinity is modeled as a sum of position dependent base effects and can thus be calculated for each PM and MM value based on its corresponding sequence information The correction is motivated by the assumptions that observed PM and MM values consist of optical noise non specific binding noise and signal Optical noise is assumed to be normal and logged non specific binding noise from PM MM pairs assumed to be bivariate normal Using the data on a single array the corresponding model parameters can be estimated Each PM value is then adjusted by subtracting a shrunken MM value that has been corrected for its affinity Normalization Normalization is nece
22. L g a a ete e e Oe oo E a GeneSpring 7 2 Addendum te Agilent Technologies Agilent Technologies Inc 2005 sig_support agilent com Main 866 744 7638 1 Table of Contents Du DR OFC ONNI SSSR nn a D eee oe nee den 2 2 Newicawres tGene S DANS T2 renar ie a a E alien 3 2 1 ING VS ACU CS sesi dices cores EE A E E A 3 2 2 Saving an experiment directly onto Signets 4 23 Change Interpretation with OW ecos Rd 4 2A ING W THEM WSIS re de dee Seeds eal eaten nceterseae Soedontademe end orvedanh ested evade econ demeaeeea det unacetedanle 5 ZS Directly load CHP HIS sans nn nn Re ER ne nee 6 2 5 1 SUPPOMCO IO MATS eia ne diese peine irc dose in ent on 6 2 02 Sample ATIHDULCS NDONE MR di nette a a 6 25 3 PNAC HINGIS EEE J 2 5 4 IV OT KON ee E E A 8 2 6 Directly load CEL files and perform RMA and GC RMA uuRRRRR 11 2 6 1 SUPPE TONAS yeaa a a T Up au 11 2 6 2 Samplet pes IM POMC asda a E eme A 12 2 6 3 PRCA AMS NES a A Tacha deel eae tenet aca nn dede 12 2 6 4 WOOW creian a ania ae E A 13 2 6 5 Normalizations in GeneSpring after RMA or GC RMA analysis 18 2 7 _Re analyze samples with CEL files using RMA and GC RMA 19 a RMA aand CRMX Ale ori ims En cn Ad diesel 26 3 1 RNA IP OR EE en de dead ed tn den 26 3 1 1 Back eround C orrec NoN enen D nn tenir 26 3 1 2 NOMAZAUON SR Re enced Seat T A 26 3 1 3 D UNO 008012 V5 6 6 amp En eg RSD tt D Ce cee 26 3 1 4 PRB SINC CS na
23. MPRO_Oday_A CHP Sample Image MPRO_Oday_A t Data File Add File Extract File Delete File View File View Data File Format Export as Zip OK Cance Help 2 5 4 Workflow The import of CHP files into GeneSpring follows the standard workflow with only one possible new step as outlined below 1 Select the CHP files you want to load and drag them onto an open window of GeneSpring or use the File gt Import Data menu item 2 GeneSpring analyze the files Please w EJER Analyzing tiles please 3 The Define File Format window appears with all the possible import formats options for this file 7 Selectthe genome set of genes on the array for this data If your genome does not appear on the list you can create anew one by selecting Create a New Genome Select Genome EH Genomes or Arrays Atymetrix HG_U133_ PLUS Target HG_U95v2 HG U133 EG U74 Al ver gt Multik RG_U34 Demo Chips LS Demo Human C Create a New Genome EN Next Cancel Help Affymetrix CHP files are identified in the top drop down menu e lf a file cannot be uniquely identified as one format more than one format is possibly listed in the drop down menu If the default selection is not appropriate change the selection from the drop down menu The Genome that is to be used with the data is selected in the Select Genome section If the data should be l
24. amples are created 13 After the samples have been created you will be offered a chance to create a GeneSpring Experiment with the samples that have just been loaded Click Yes to create an experiment or No to continue without creating an experiment e f you choose not to create an experiment you can access the samples through the Sample Manager See the User Manual entry for the Sample Manager for more information ce Import Data Create Experiment 4 new samples have been created Would you also like to create an experiment trom these samples 14 If you choose to make an experiment the Save New Experiment window appears where you can change the name of the experiment and assign the experiment to a project Save New Experiment EEK Name ae Experiment Falder Project Change Project s Notes ES periments Hematopoiesis Study ae oi mau a aw m Le D on mn D 2 os 5 T H a E Sample Number Save Cancel NOTE The default name for the experiment is ALWAYS RMA File Preprocessor Experiment even if GC RMA analysis was performed Change the name of the experiment to something more appropriate At this point a new experiment is created from the CEL files and regular GeneSpring analysis can commence 2 6 5 Normalizations in GeneSpring after RMA or GC RMA analysis The RMA and GC RMA analyses converts the probe level expression data in
25. bs intended fc slope 0 678 0 677 1 000 Obs low int fc slope 0 305 0 306 1 000 FC 2 AUC FP lt 10 0 412 0 412 1 000 FC 2 AUC FP lt 15 0 450 0 450 1 000 FC 2 AUC FP lt 25 0 503 0 503 1 000 FC 2 AUC FP lt 100 0 643 0 646 1 000 FC 2 AFP call if fc gt 2 0 238 0 238 0 000 FC 2 ATP call if fc gt 2 10 857 10 810 42 000 AUC Area Under the ROC curve AFP Average False Positives ATP Average True Positives FC Fold Change Table 1 The results from the affycomp RMA assessment of HG_UI33A spike in data Results from TableAll function in the BioConductor affycomp package deal indicates the number that the assessment would be if the algorithm and hybridizations were perfect HG_U133A Original Assessment GeneSpring GC RMA BioConductor GC RMA Ideal Signal detect slope 0 930 0 930 1 000 Signal detect R2 0 927 0 927 1 000 AUC FP lt 10 0 531 0 531 1 000 AUC FP lt 15 0 566 0 567 1 000 AUC FP lt 25 0 622 0 622 1 000 AUC FP lt 100 0 788 0 789 1 000 AFP call if fc gt 2 2 905 2 824 0 000 ATP call if fc gt 2 36 026 36 018 42 000 IQR 0 397 0 399 0 000 Obs intended fc slope 0 929 0 929 1 000 Obs low int fc slope 0 555 0 557 1 000 FC 2 AUC FP lt 10 0 389 0 391 1 000 FC 2 AUC FP lt 15 0 428 0 430 1 000 FC 2 AUC FP lt 25 0 487 0 489 1 000 FC 2 AUC FP lt 100 0 641 0 645 1 000 FC 2 AFP call if fc gt 2 1 286 1 238 0 000 FC 2 ATP call if fc gt 2 19 000 18 905 42 000 Table 2 The results from the affycomp GC RMA
26. cannot be uniquely identified as one format more than one format is possible listed in the drop down menu If the default selection is not appropriate you can change the selection with the drop down menu 5 The Genome to be used with the data is selected in the Select Genome section but if the data should be loaded into a different genome select the appropriate genome and click Next e If no suitable pre loaded genome is available or appropriate you can also create a genome at this time Click Create a New Genome option to do so 2 You are given a choice of two supported analysis techniques to apply Choose the appropriate analysis technique from the dropdown menu and click Next GeneSpring 7 2 includes two supported probe level analysis techniques RMA and GC RMA Import Data Preprocess Data Files 0 x Do you wantto preprocess your data files before importing your samples lac REMA File Preprocessor RAIA File Preprocessor 6 The Import Data Selected Files window appears to let you add more data files to be imported The originally dragged and dropped files will already be selected If no more data files need to be added click Next Page 15 of 40 Import Data Selected Files ME Drives Selected Files WFlamingo USERS x Name Date Modified SEI BIC MPRO_Oday_D CEL 10 16 01 12 40 PM 97 pies MPRO_Oday_C CEL 10 16 01 12 30 PM 9 HCI MAGE ML from MPRO_Oday_B CEL 10 16 01
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29. e exact same function this normalization step is not required and can be removed The third normalization step Per Gene Normalize the median ensures that the expression value for one gene across the different conditions is centered on 1 by dividing the expression value by the median value of the expression values for that gene across the conditions This ensure that genes that do not change across conditions get an normalized expression values of 1 allowing for easy visual detection of differentially expressed genes Certain algorithms in GeneSpring also assume that all data is normalized on 1 and it is therefore recommended to retain the Per Gene Normalize the median normalization step after RMA or GC RMA analysis Experiment Normalizations GS GC RMA Spike in Choose a Normalization Step Start with pre normalized values Data Transformation SAGE transform Data Transformation Real Time PCR transform Data Transformation Subtract background based on negative Data Transformation Set measurements less than 0 01 to 0 1 Data Transformation Transform fram log to linear values Data Transformation Dye swap Per Spot Divide by control channel Data Transformation Reserve control channel Per Spot and Per Chip Intensity dependent Lowess normali Per Chip Normalize to a median or percentile Per Chip Normalize to positive control genes Per Chip Normalize to a constant value Per Gene Normalize to specific samples Per
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31. ect values for experimental parameters Warning Modifying parameters may invalidate existing condition trees built from this experiment se Parameter Name Sample Name CEL File Name Parameter Units Numeric Logarithmic 1 MPRO_Gday Cat MPRO_Gday_C t MPRO_Sday_C CEL 2 MPRO 6day Bist MPRO_Gday_B te MPRO_Gday_B CEL 3 MPRO_Gday Atxt MPRO_Gday_Att MPRO_Gday_A CEL 4 MPRO_4day Cixt MPRO_4day_C te MPRO_4day_C CEL 5 MPRO_4day Btxt MPRO_4day_Bte MPRO_4day_B CEL 6 MPRO_Aday Atxt MPRO_4day_Att MPRO_4day_A CEL Z MPRO_2day Ctxt MPRO_2day_C te MPRO_2day_C CEL E MPRO 2day Bixt MPRO_2day_B h MPRO_2day_B CEL 9 MPRO_2day Atxt MPRO_2day Att MPRO_2day_ACEL 10 MPRO Aday Ctt MPRO_1day_C t MPRO_1day_C CEL HT MPRO_1day_B txt MPRO_1day_B MPRO_1day_B CEL M2 MPRO 1day A txt MPRO_1day_Abt MPRO_1day_A CEL 13 MPRO_Oday C txt MPRO_Ocay Cte MPRO_Oday_C CEL HA MPRO_Oday Baxt MPRO_Oday_B h MPRO_Oday_B CEL 15 MPRO_Oday_A txt MPRO_Oday_A bt MPRO_Oday_A CEL New Parameter Import Parameter Replace Text Extract Subvalues sort set Value Order Sos aS oS Re ee eS ma Previous Next Finish Cancel Help 11 The normalization window appears with the defaults normalizations for your experiment Change the normalization settings as required See section 2 5 5 in the manual for some suggestions and click Finish to continue Create New Experiment Experiment Normalizations a Order of No
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33. hapters 13 16 of the GeneSpring manual for tools to analyze your data 3 1 3 1 2 3 1 3 3 1 4 3 RMA and GC RMA Algorithms This section describes the algorithms in the pre processor and interactive plug ins RMA algorithm RMA Robust Multi chip Average is a method for normalizing and summarizing probe level intensity measurements from Affymetrix GeneChips Starting with the probe level data from a set of GeneChips the perfect match PM values are background corrected normalized and finally summarized resulting in a set of expression measures The three steps of the process are outlined below Background Correction The background correction used in RMA is a non linear correction done on a per chip basis It is motivated by the assumption that the observed PM values consist of a background signal caused by optical noise and non specific binding plus a signal which is what we are trying to detect The signal is assumed to be normally distributed and the background noise is assumed to be exponential The parameters for these distributions are estimated using all the PM values on the chip and the background is then subtracted from the PM s Normalization Normalization is necessary so that multiple chips can be compared to each other and analyzed together It is motivated by the assumption that all n chips should have approximately the same distribution of PM values The normalization used in RMA is quantile normalizatio
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36. hms have been implemented in JAVA based on all available documentation To compare the performance of the RMA and GC RMA algorithms we performed the Affycomp assessment on data analyzed with GeneSpring s RMA and GC RMA algorithms and compared it with the implementation of RMA and GC RMA in the R BioConductor package rma and germa The results of the assessment have been submitted to the Affycomp website for comparison other probe level analysis algorithms For the spike in experiment using the HG_U95A chip the GeneSpring GC RMA algorithms scored 2 of the best new assessment scores from a total of 14 scores and for the experiment using the HG_U133A chip the GeneSpring GC RMA algorithm scored 6 of the best assessment scores 3 3 1 HG U133A spike in experiment The HG_U133A spike in experiment was used for the assessment of the RMA and GC RMA algorithm as described by Cope et al Leslie M Cope Rafael A Irizarry Harris A Jaffee Zhijin Wu and Terence P Speed A benchmark for Affymetrix GeneChip expression measures Bioinformatics Vol 20 No 3 2004 323 331 HG_U133A Original Assessment GeneSpring RMA BioConductor RMA Ideal Signal detect slope 0 678 0 678 1 000 Signal detect R2 0 898 0 898 1 000 AUC FP lt 10 0 537 0 537 1 000 AUC FP lt 15 0 572 0 572 1 000 AUC FP lt 25 0 626 0 626 1 000 AUC FP lt 100 0 787 0 787 1 000 AFP call if fc gt 2 1 711 1 711 0 000 ATP call if fc gt 2 32 901 32 908 42 000 IQR 0 248 0 248 0 000 O
37. ic License See Above and can be found in the Programs directory in the GeneSpring data directory C Program Files SiliconGenetics GeneSpring data Programs for the default installation on Windows platforms Appendix A Currently supported Affymetrix chip types The default installation of the Affymetrix Preprocessor plug ins contains the Arrayinfo files for the following Affymetrix Chips e HG U133 Plus 2 e HG U95Av2 e MG U74Av2 e Mouse430Av2 e Rat230v2 If your CEL files did not originate from any of these chip types the plug in attempts to download the necessary files from our web server Currently the following chips are available from the web server e ATH1 121501 e Celegans e DrosGenomet e Drosophila 2 e Ecoli_ASv2 e HG Focus e HG U133A e HG U133A_2 e HG U133A_tag e HG U133B e HG U133 Plus 2 e HG U95A e HG U95Av2 e MG U74Av2 e MG U74Bv2 e MG U74Cv2 e MOE430A e MOE430A_2 e MOE430B e Mouse430 2 e Pae Gla e Plasmodium_Anopheles e RAE230A e RAE230B e RG_U34A e RG_U34B e RG_U34C e RN_U34 e RT_U34 e Rat230 2 e U133_X3P e Vitis Vinifera e Xenopus laevis e YG 98 e Zebrafish The Arrayinfo files have been created by Agilent Technologies to reduce the size of the required files and to ensure users will have the appropriate probe affinity information for each of the chips for the GC RMA If the GeneChip you are using is not listed above and you want to obtain the appropriate ar
38. ilicongenetics com or call 1 866 744 7638 to request the creation of an arrayinfo file After the normalization is complete a new dialog box appears to indicate the number of samples that have been created and where you can find the samples If you want to create a new experiment from these samples you can open the Sample Manager and select the newly created samples Message Choose the menu item Sample Manger from the Experiment menu to open the sample manager Full Development MG_U74 All ver2 Genes all genes File Edit View SS Eu ue Colorbar Filtering Tools Annotations Window Help Gene Lists Experiment Normalizations Bo Experiment Experiment Parameters EHH Late A Cross Gene Error Model P Experiment Interpretation Create New Experiment ee Co eee Duplicate Experiment HA Condition TEERAA Classificatio CEL File Name HA e RnE PRO Oday ACEL MPRO_2day_C CEL MPRO_5day_C CEL Array Layouts k g a S Re aaa g Expression Profiles CHA External Programs Y axis MPRO RMA in GS Default Interpretation Calculate Power on Interpretation Colored by MPRO_Oday_A CEL 0 DifferentialExpression LPE Gene List all genes 36767 102744 at selected Q DifferentialExpression SAM E a Trust ea 11 Show RMA in GeneSpring af JE Magnification 1 To find your newly created samples sort the samples in the Show All tab by the Creation Date column and select the samples that were created most recentl
39. in the Preference setting e Direct load of CHP file into GeneSpring e Direct load of CEL files into GeneSpring with RMA and GC RMA normalization e Re analysis of already loaded samples with CEL files attached with RMA and GC RMA The next sections describe the new functionality in more detail 2 2 2 3 Saving an experiment directly onto Signet GeneSpring experiments can be saved both locally on the hard drive of the personal computer running GeneSpring or on the Signet server for centralized storage When an experiment is created in GeneSpring that is intended to be saved only on the Signet server from existing samples using the SampleManager GeneSpring 7 2 does not create the experiment locally first but saves the experiment directly onto Signet GeneSpring does not make an intermediate local copy of the experiment If samples are not yet loaded into GeneSpring but are loaded from tab delimited files or from a database the normal local normalization are performed as before Because the experiment is no longer created on the local machine first the experiment graph is no longer shown in the Save New Experiment window and is replaced with a list of the samples that make up the experiment Save New Experiment M x EH Experiments Sample Name I Public Signet 5 MPa N 148 TXT PA M_118 TxT 3 PA N_112 1 TxT 4 PA D_85 TxT Previous pave Cancel Because the experiment is not created locally when the ex
40. ion of software functions and or data prepared so as to be conveniently linked with application programs which use some of those functions and data to form executables The Library below refers to any such software library or work which has been distributed under these terms A work based on the Library means either the Library or any derivative work under copyright law that 1s to say a work containing the Library or a portion of it either verbatim or with modifications and or translated straightforwardly into another language Hereinafter translation is included without limitation in the term modification Source code for a work means the preferred form of the work for making modifications to it For a library complete source code means all the source code for all modules it contains plus any associated interface definition files plus the scripts used to control compilation and installation of the library Activities other than copying distribution and modification are not covered by this License they are outside its scope The act of running a program using the Library is not restricted and output from such a program 1s covered only 1f its contents constitute a work based on the Library independent of the use of the Library in a tool for writing it Whether that is true depends on what the Library does and what the program that uses the Library does 1 You may copy and distribute verbatim copies of the Library s complete
41. matically terminate your rights under this License However parties who have received copies or rights from you under this License will not have their licenses terminated so long as such parties remain in full compliance 9 You are not required to accept this License since you have not signed it However nothing else grants you permission to modify or distribute the Library or its derivative works These actions are prohibited by law if you do not accept this License Therefore by modifying or distributing the Library or any work based on the Library you indicate your acceptance of this License to do so and all its terms and conditions for copying distributing or modifying the Library or works based on it 10 Each time you redistribute the Library or any work based on the Library the recipient automatically receives a license from the original licensor to copy distribute link with or modify the Library subject to these terms and conditions You may not impose any further restrictions on the recipients exercise of the rights granted herein You are not responsible for enforcing compliance by third parties with this License 11 If as a consequence of a court judgment or allegation of patent infringement or for any other reason not limited to patent issues conditions are imposed on you whether by court order agreement or otherwise that contradict the conditions of this License they do not excuse you from the conditions of t
42. n This is a generalization of the idea behind quantile quantile plots to more than two dimensions The quantiles for each PM value are plotted in n dimensions and projected onto the diagonal The final result is that the PM values on each chip will have the same distribution Summarization Once the probe level PM values have been background corrected and normalized they need to be summarized into expression measures so that the result is a single expression measure per probe set per chip The summarization used is motivated by the assumption that observed log transformed PM values follow a linear additive model containing a probe affinity effect a gene specific effect the expression level and an error term For RMA the probe affinity effects are assumed to sum to zero and the gene effect expression level is estimated using median polishing Median polishing is a robust model fitting technique that protects against outlier probes References B M Bolstad R A Irizarry M Astrand and T P Speed A comparison of normalization methods for high density oligonucleotide array data based on variance and bias Bioinformatics 19 2 185 193 Jan 2003 Rafael A Irizarry Bridget Hobbs Francois Collin Yasmin D Beazer Barclay Kristen J Antonellis Uwe Scherf and Terence P Speed Exploration normalization and summaries of high density oligonucleotide array probe level data Biostatistics 2003b To appear Rafael A Irizarry Benjamin
43. nd edit the Web links Directly load CHP files In GeneSpring 7 2 you can import the CHP files from Affymetrix GeneChip gene expression chips directly into GeneSpring in the same manner as any other data files Previously to load data from a MAS5 analysis into GeneSpring required a text version of the CHP files Supported formats The import of CHP files is implemented as a pre processor plug in and recognizes the following formats e Original CHP file format Before GCOS 1 2 e New XDA file format GOCS 1 2 and later Sample attributes imported The pre processor plug in extracts a number of fields from the CHP files that are stored as Sample Attributes for the imported samples Table 1 contains a list of the Sample Attributes that are imported with a description of the contents Sample Attribute name Contents CHP File Name The name of the original CHP file that was imported CEL File Name The name of the original CEL used in the analysis NOTE The complete path of the CEL file is recorded but because only the CHP file is imported the CEL file is not guaranteed to be found in this location 2 5 3 Array Design Algorithm Name Algorithm Version Algorithm Parameters Algorithm Summary Table 1 Imported Sample Attributes Attachments Each of the samples that is created by the CHP preprocessor plug in has two attachments e Original CHP file e Data file The original CHP file is attached to the sample a
44. nd can be retrieved at any time by extracting the file from the sample in the Sample Inspector See the GeneSpring User The name of the Affymetrix Chip The name of the Algorithm used in the analysis Usually ExpressionStat for the MAS5 algorithm The version of the algorithm used in the analysis Usually 5 0 for the MAS5 algorithm The comma separated set of parameters used for the analysis like BF Alphat Alpha2 Tau Gamma etc for MAS5 A summary of the results of the analysis such as background Noise and RawQ Manual for more information about the Sample Inspector The data file is a new text file that is created by the preprocessor plug in It contains all the columns of the original CHP files and can be used to view or filter In addition to the attachments and the Sample Attributes each of the Samples loaded with the CHP plug in also contains a note in the Notes section of the sample to indicate the preprocessor that was used to import the sample Page 8 of 40 ay Sample Inspector Sample Name Author s Research Group Project s D Change Project s Organization Thon Identifier vpxgl 173954 Created Fri Sep 3 16 51 00 PDT 2004 Application Full Development 7 Notes Preprocessor used CHP File Importer Attributes and Parameters Sample Correlations Associated Files Graph To add an attachment drag a file into the table below or click on the Add File button File Name
45. ne unie te ne 26 3 2 CCRMA AIS OI a tease adete son enr acte teste 27 3 251 BACKS OUR COI CHONE Sis cad Sate ne nd seen 27 22 NO MA ZAUHONME en edit 2 3 2 3 MUR EU LE MAS LA O etn ech an saree a acter ele ucts cemlees demande E wun silante 27 3 2 4 RETTEN CS Sd nn deenc caen ten erence eaten 27 3 3 Performance Comparison Senario EA N N 27 3 3 1 HG UTSA spike In experimen esa a ea n nn laide anti 28 332 HG UVA SPIKE INEP MEN enan e N At ae ees 30 3 3 3 OTIC TO MS enea E E ees ees 32 A Pl 8 1 SOULCE Code And cen iho aoui a a 33 4 1 CORET N Ce a E E EE E 33 4 2 OEE O E E E E EATA T 38 AD E a A A A S A E E TRIER I 39 Currently supported A fy metrix Chip types siaran e ET a E dis it 39 2 1 2 New features in GeneSpring 7 2 This document describes the functions that are new to version 7 2 of GeneSpring It combines the information from the addendum for version 7 1 with the new information for version 7 2 GeneSpring 7 1 introduced a number of new features that are specifically designed to enhance the experience of Affymetrix users The new features are all implemented with the new external JAVA API which allows for the rapid development of new functionality for GeneSpring without having to change any of the core functionality of GeneSpring The new functionality of GeneSpring 7 1 is implemented as a set of pre processor plug ins and one interactive plug in The plug ins provide GeneSpring users with new functionality and the JAVA
46. oaded into a different genome select the appropriate genome and click Next e f no suitable pre loaded genome is available or appropriate you can also create a genome at this time Click Create a New Genome option to do so If more than one preprocessor can analyze a CHP file you are shown the Import Data Preprocess Data Files window see figure below The drop down menu can be used to choose the analysis that you want to use on the CHP file In most cases this window does not show up because only one CHP preprocessor is available in GeneSpring 7 2 Kz Import Data Prepr POCO COC CECE ECC EEE CCE CEEEECEECCCCECCCECEEECCEEEEECEE Do vou wantto preprocess your data files before importing your samples Choose Plug In Po The Import Data Selected Files window appears and lets you add more data files to be imported The originally dragged and dropped files are already selected If no more data files need to be added click Next 8 Page 10 of 40 Import Data Selected Files Drives Selected Files WFlamingo USERS Name isis Date Modified Size MG_U74Av2 CDF 3 15 01 MPRO_Oday_D CHP 7 13 021 27 PM 116 Directories MG_U74Bv2 CDF 3 21 01 MPRO_Oday_C CHP 7 13 021 27 PM 116 Mouse brain M MG_U74Cv2 CDF 3 21 01 MPRO_Oday_B CHP 7 13 021 26PM 116 FHS ONE COLOR ex Oday_A CI 7113 02 __ Add MPRO_Oday ACHP 17 13 02 1 25 PM 116 FHA Afly webcast MPRO_Oday A vOhrACHP 7 14 02 C any wenc _
47. ols Annotations Window Help Array Layouts El 1 ISSN ME ores i Expression Profiles 4 TIRIS E HEHE i EE i EE i BG Einm HH FiSH A l MMH ttt ee EH External Programs ee A Load Classification From File 6 E Load Experiment From File A Load Gene List From File 11 BELEH EI H l Tee el UE IE INH Hil U H RII Ee eee z E eH i R 1c EIR ieS 110 2 12 WH IER ss llm a ee ae E Load Gene List With Numbers From File A Load Gene Tree From File 16 Sam 23 IS RMI IR MIN If Er B EE Hier UE ONE llil ER EN i CH im Expression A Reanalyze samples using GC RMA A Reanalyze samples using RMA amp save Classification To File A Save Experiment To File Save Gene List To File A Save Gene List With Numbers To File Save Gene Tree To File Bookmarks Srrints a Trust fi Show All Data Colored by Demonstration Experiment Default Interpretation Gene List all genes 159 A a 1 2 The Select Sample window is used to choose which samples to use in the RMA or GC RMA analysis Select Samples MISE CHR NT SEE N 68 vi C Show All Fitter On Experiment Filter on Attribute Fitter Results 21 items 0 selected 4 Experiments 4 All Objects Name MPRO_Od ID vpxgl 145979 Notes Preprocessor Organization Folder Attachments Thon IMPRO_Oday_ C3 Hematopoiesis Study MPRO_Od vpxgl 145980 Preprocessor Thon MPRO_Oday_B
48. on IMPRO_Oday_C vpxgl 145982 p3 Preprocessor Thon IMPRO_1day_ Preprocessor vpxgl 145963 vpxgl 145984 Preprocessor T hon Thon vpxgl 145985 oe Preprocessor Thon IMPRO_2day_ MPRO_1day_B IMPRO_1day_C vpxgl145986 Preprocessor ie Thon MPRO_2day_B pxgl145987 pxXgh145991 Preprocessor Preprocessor Thon 7 T 7 f T 4 T T 4 Thon MPRO_4day_ IMPRO_2day_C MPRA Ad nv 1 45007 IPrenrnressnr Than MPRA ddaw R the normalization click the Cancel button 3 After you select samples by adding to the lower right hand box click OK to start the 4 A new dialog box appears that shows the progress of the RMA normalization To cancel Page 22 of 40 ar n Progress Q Performing RMA 92 complete 5 If the appropriate array definition arrayinfo files are not available GeneSpring tries to get the appropriate files from the Agilent Technologies web server A dialog box appears to indicate the progress of the download Downloading array description for Mo UF4Ayve2 Cancel 6 If no internet connection is available or the array definition for the chip is not present on the web server a Locate CDF file dialog box is displayed to let you select a CDF file stored on your computer to be used as the ar
49. or GC RMA null log fc IQR 0 069 0 062 null log fc 99 0 506 0 498 null log fc 99 9 0 793 0 788 low AUC 0 503 0 494 med AUC 0 906 0 908 high AUC 0 378 0 379 weighted avg AUC 0 598 0 592 25 SD 0 069 0 065 Median SD 0 096 0 091 75 SD 0 133 0 129 99 SD 0 266 0 262 low slope 0 512 0 509 med slope 1 017 1 017 high slope 0 547 0 548 low R2 0 193 0 191 med R2 0 646 0 642 high R2 0 449 0 452 0 25 0 0 591 0 582 0 5 0 25 0 483 0 465 1 0 5 0 541 0 553 2 1 0 849 0 854 4 2 0 978 0 968 8 4 1 032 1 050 16 8 1 043 1 028 32 16 0 995 1 000 64 32 0 896 0 888 128 64 0 728 0 756 256 128 0 634 0 627 512 256 0 468 0 468 1024 512 0 356 0 343 Table 8 The results from the affycomp GC RMA new assessment of HG U95A spike in data Results from TableAll function in the BioConductor affycomp package 3 3 3 Conclusions The results of the affycomp assessment show that the GeneSpring RMA and GC RMA implementation performs as well as the BioConductor RMA and GC RMA implementation 4 1 4 Plug in source code and licensing This section describes the source code and licensing issues for the provided plug ins LGPL license 0 This License Agreement applies to any software library or other program which contains a notice placed by the copyright holder or other authorized party saying it may be distributed under the terms of this Lesser General Public License also called this License Each licensee is addressed as you A library means a collect
50. ormed RMA or GC RMA Table 1 Imported Sample Attributes Attachments Each of the samples that is created by the RMA and GC RMA preprocessor plug in has two attachments e Original CEL file e Data file The original CEL file is attached to the sample and can be retrieved at any time by extracting the file from the sample in the Sample Inspector See the figure below and the GeneSpring User Manual for more information about the Sample Inspector The attached CEL file can be used by the interactive plug in to re analyze samples that have already been loaded into GeneSpring The data file is a new text file that is created by the preprocessor plug in The data file consists of two columns the Affymetrix Probe identifier and the Signal value as determined by the RMA or GC RMA analysis The data is provided as linear values and not as LOG2 Some other implementations of RMA and GC RMA use LOG2 values In addition to the attachments and the Sample Attributes each of the Samples loaded with the RMA or GC RMA plug in also contains a note in the Notes section of the sample to indicate which preprocessor was used Page 13 of 40 LEA Sample Inspector Sample Name MPRO_Oday_Att Web Links Author s EEE Googie Research Group Project s Change Project s Organization Thon Identifier vpxgl 145979 Created Mon Aug 30 18 06 49 PDT 2004 Application Full Development 7 Notes Preprocessor used RMA File Preprocessor
51. periment is saved to Signet directly none of the normal checks are done locally In the case that the Normalization or other action fails or would produce a warning like Not enough genes to perform a Lowess the normal warnings or error messages are not shown although a generic error message stating Error loading experiment X will be shown If this error message appears check the normalization window and correct the problem Change Interpretation window The Change Interpretation window allows you to edit the Experiment Interpretations The Interpretation determines how the data is displayed and analyzed in many of the views and analysis When a genome contains many genes changing an interpretation could be a slow process because each time the Interpretation is changed the small thumbnail graph is updated to draw the expression values for all of the genes 2 4 Page 5 of 40 Change Interpretation for Heart failure study Default Interpretation Vertical Axis Range Lower Bound 0 01 Upper Bound 100 Analysis Mode Log of ratio al Use Measurements Flagged All Measurements 0 conditions excluded Exclude Conditions O01 Use Cross Gene Error Model in This Interpretation 112 325 314 2275 82 ef Se How to Display Parameters Parameter Name Continuous Non continuous Color Code Do Not Display Sample Name 2 i Patient ID o Age Gender CHF Etiology 0 Tissue Source Diseased Normal
52. ples associated with the Genome To select samples based on their attribute value click the Filter on Attribute tab and select the attributes that best represent the samples MES Show Al Filter On Experiment Filter on Attribute Filter Results 24 items 0 selected Select Samples Select Sample Attribute of Interest Experiment TimeCourse TimeStr DataType Array Design RNA type Analysis Operator Sample Name Select Attribute Values 0 24 48 12 72 96 4 Select All Clear All analysis of the samples Name RMA norm ID peznc 149783 Notes Organization Thon Folder Attachments IRMA normalize RMA norm peznc 149764 Thon IRMA normalize RMA norm RMA norm peznc 149785 _ Thon Thon RMA normalize RMA normalize 47 peznc 149786 45 jpezne 32783 peznc 32764 Thon Thon 47 txt 48 txt 49 jpezne 32785 Thon aabt jpezne 32786 Thon f J 50 tt Selected tems 15 ada an Remove Thom Remove All Configure Colurnns JER tvt ID vpxgl 145979 Notes Preprocessor Organization Thon Folder Attachments MPRO_Oday_ vpxgl 145980 vpxgl 145981 Thon MPRO_Oday_B Preprocessor ES cer Preprocessor Th
53. ray definition file Download Progress KES Please locate the CDF file for the Test3 array Look in LD HG U95A Up One Level Bhpp E 152in99hpp_e 1521999hpp_av06 CEL 1521h35hpo_av06 CEL 1521c99hpp_av06 CEL 1521d99hpp_av06 CEL 1521e99hpp_av06 CEL 1521f99hpp_av06 CEL 1521i99hpp_av06 CEL 1521j99hpp_av06 CEL 1521k99hpp_av06 CEL 1521199hpp_avO r CEL 1521099hpp 1521p99hpp 1521g99hpp _ lt 1521r99hpp_a Ju gt ae Cancel NOTE CDF files can only be used with the RMA normalization method When you want to perform GC RMA normalization an appropriate Arrayinfo file from Agilent Technologies is required File name Files of type If you attempt to perform GC RMA normalization and an Arrayinfo file is not available or cannot be downloaded the CDF dialog box will not appear but an Error dialog box as shown below is displayed GC RMA normalization can only work with the Arrayinfo files that are created and maintained by Agilent Technologies If the dialog box below appears contact Technical Support at Agilent Technologies at support silicongenetics com or call 1 866 744 7638 to request the creation of an Arrayinfo file x Could not Find the appropriate array definition File Please contact Silicon Genetics technical support to request the appropriate 4rrayinfo File 8 9 Page 23 of 40 If the arrayinfo file is not available contact Agilent Technologies tech support at support s
54. ray definition files please contact Agilent Technologies tech support at support silicongenetics com or call 1 866 744 7638 to request the creation of an Arrayinfo file
55. rmalizations to Perform 14 Data Transformation Set measurements less than 0 01 to 0 01 Choose a Normalization Step FN Per Chip Normalize to 50th percentile Start with pre normalized values 3 Per Gene Normalize to median Data Transformation SAGE transform Data Transformation Real Time PCR transform Data Transformation Subtract background based d Use Defaults Data Transformation Set measurements less thar Data Transformation Transform from log to linear y Data Transformation Dye swap Per Spot Divide by control channel Data Transformation Reserve control channel Per Spot and Per Chip Intensity dependent Lowes Per Chip Normalize to a median or percentile Per Chip Normalize to positive control genes PRE Get Text Description Per Chip Normalize to a constant value Per Gene Normalize to median Warnings Per Chip and Per Gene Median polishing No warnings E Previous Finish Cancel Help 12 The Save New Experiment window appears to let you enter a new name and project association for the Experiment Save New Experiment E Eg Name MPRO RMA in GS no 3 and 5 days Folder Project Change Project s Hematopoiesis Study Cee de m Le an om W D Boe a a td m o 0 Sample Mame MPRO Oday Att MPRO_iday Bit MPRO_ day_C tet MPRO_Gday_A tet Previous Save Cancel Your experiment with the re analyzed samples is now saved and normalized See c
56. rms of the ordinary GNU General Public License instead of this License to a given copy of the Library To do this you must alter all the notices that refer to this License so that they refer to the ordinary GNU General Public License version 2 instead of to this License If a newer version than version 2 of the ordinary GNU General Public License has appeared then you can specify that version instead if you wish Do not make any other change in these notices Once this change is made in a given copy it is irreversible for that copy so the ordinary GNU General Public License applies to all subsequent copies and derivative works made from that copy This option is useful when you wish to copy part of the code of the Library into a program that is not a library 4 You may copy and distribute the Library or a portion or derivative of it under Section 2 in object code or executable form under the terms of Sections and 2 above provided that you accompany it with the complete corresponding machine readable source code which must be distributed under the terms of Sections 1 and 2 above on a medium customarily used for software interchange If distribution of object code 1s made by offering access to copy from a designated place then offering equivalent access to copy the source code from the same place satisfies the requirement to distribute the source code even though third parties are not compelled to copy the source along with the obje
57. source code for the plug in is freely available and allows for you to use the code in your own GeneSpring plug ins The JAVA source code is released under the LGPL license For more information about the LGPL see Appendix A or http www gnu org copyleft lesser html New features GeneSpring 7 2 provides better performance for large experiments Performance enhancement features include e Improved the speed of experiment creation when you save to Signet by bypassing the normalization step in GeneSpring o Bypassing the normalization step in GeneSpring when saving an experiment to Signet allows for faster processing of experiments The graph in the Save Experiment window e Performance improvements in the binary cache loader to allow for faster loading of experiments e Optimized memory management when saving files e Improved performance of the Change Interpretation window to make easier quickly changes in Experiment Interpretations o The thumbnail view of the experiment in the Interpretation window no longer shows all the genes in the experiment but a random set of 1000 genes to allow for faster redrawing of the graph e Optimized the drawing speed in the Blocks Physical Position and Ordered list view Other new features of GeneSpring 7 2 include e New Experiment Inspector menu item in the Experiments menu e New Genome Inspector menu in the Annotations menu e Location of temporary file directory can be changed
58. ssary so that multiple chips can be compared to each other and analyzed together It is motivated by the assumption that all n chips should have approximately the same distribution of PM values The normalization used in RMA is quantile normalization This is a generalization of the idea behind quantile quantile plots to more than two dimensions The quantiles for each PM value are plotted in n dimensions and projected onto the diagonal The final result is that the PM values on each chip will have the same distribution Summarization Once the probe level PM values have been background corrected and normalized they need to be summarized into expression measures so that the result is a single expression measure per probe set per chip The summarization used is motivated by the assumption that observed log transformed PM values follow a linear additive model containing a probe affinity effect a gene specific effect the expression level and an error term For RMA the probe affinity effects are assumed to sum to zero and the gene effect expression level is estimated using median polishing Median polishing is a robust model fitting technique that protects against outlier probes References Wu Zhijin Irizarry RA Gentleman R Martinez Murillo F Spencer F 2003 A Model Based Background Adjustment for Oligonucleotide Expression Arrays To appear in JASA Performance comparisons The GeneSpring versions of the RMA and GC RMA algorit
59. to Probe set or Gene level expression data that is normalized to a certain extent The normalizations that are performed in the RMA normalization steps ensure that the distribution of the expression values is comparable across the different chips or samples Additional normalization is applied to experiments that are created with samples that have been normalized using RMA or GC RMA using the standard GeneSpring normalizations for one color data The GeneSpring normalization steps ensure that there are no negative values and that the data is centered on the value 1 These normalization steps are perfectly acceptable normalization steps even though the data has already been normalized with RMA or GC RMA The GeneSpring normalizations will not negate or alter the RMA or GC RMA normalizations in any way since the normalizations only involve a simply division by the median of the chip and gene expression values The normalization steps that are performed by default are shown in the figure below S Experiment Normalizations GS GC RMA Spike in Order of Normalizations to Perform Data Transformation Set measurements less than 0 01 to 0 01 Choose a Normalization Step Per Chip Normalize to 50th percentile Start with pre normalized values Per Gene Normalize to median Data Transformation SAGE transform Data Transformation Real Time PCR transform Data Transformation Subtract background based onn Data Transformation Set measurements less than 0 0
60. xample a function in a library to compute square roots has a purpose that is entirely well defined independent of the application Therefore Subsection 2d requires that any application supplied function or table used by this function must be optional if the application does not supply it the square root function must still compute square roots These requirements apply to the modified work as a whole If identifiable sections of that work are not derived from the Library and can be reasonably considered independent and separate works in themselves then this License and its terms do not apply to those sections when you distribute them as separate works But when you distribute the same sections as part of a whole which 1s a work based on the Library the distribution of the whole must be on the terms of this License whose permissions for other licensees extend to the entire whole and thus to each and every part regardless of who wrote it Thus it is not the intent of this section to claim rights or contest your rights to work written entirely by you rather the intent is to exercise the right to control the distribution of derivative or collective works based on the Library In addition mere aggregation of another work not based on the Library with the Library or with a work based on the Library on a volume of a storage or distribution medium does not bring the other work under the scope of this License 3 You may opt to apply the te
61. y aNormalized Intensity log scale Expression Sample Manager Filter on Experiment Filter on Keyword Filter Results 367 Samples Filter on Parameter Filter on Project Filter on Attributes Show All Creation Date Aug 30 2004 6 08 26 PM MPRO_6day_ Aug 30 2004 6 08 22 PM MPRO_6day_ Aug 30 2004 6 08 17 PM MPRO_6day_ Aug 30 2004 6 08 12 PM MPRO_Sday_w gt Add All x Remove Al Configure Columns Selected Samples 15 Samples All Samples are displayed Projects z MPRO_6day_C tt Aug 30 2004 6 32 16 PM MPRO_6day_ m MPRO_6day_B t Aug 30 2004 6 32 44 PM MPRO_6day_ fat MPRO_Gday_A tt Aug 30 2004 6 32 39 PM MPRO_6day_ ia MPRO_4day_C bt Aug 30 2004 6 32 37 PM MPRO_4day_ iat MPRO_4day_B t Aug 30 2004 6 32 46 PM MPRO_4day_ m MPRO_4day_Atet Aug 30 2004 6 32 27 PM MPRO_4day_ T MPRO_2day_C tt Aug 30 2004 6 32 20 PM MPRO_2day m MPRO_2day_B tt Aug 30 2004 6 32 18 PM MPRO_2day_ MAMA Ada A hd Aara ANO ANNA RAIA MAA RADIO aas ia Create Experiment Edit Attributes Assign Projects Publish to Signet Close Help Use samples stored Locally 7 L 10 You can create a new experiment by clicking the Create Experiment button The Edit Parameters window appears and you can enter parameters for the experiment After entering or importing the relevant parameters click Next to continue Create New Experiment Edit Parameters Seles Please sel

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