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1. CGHSampleAcys tif seg Plots Normalized Data Overview Whole Genome Chromosome CGH Report Summary En m MC Genem chromos Basepar Flag iN SgnalMean cht S a A h o no f Rema k pisemnie BEB TETIN aA n A f BR Remamms po fassaooea O Z82998 o O oo Fa A Ao n fo B Rema Ko o pazrear7 8ST A n fA f RPmaso2o fa 22574119185 6 1831 A Ao A BRUSH att A nA A f B Remas Ko 96737813 D 784162 143 A Ao fno fo o p RP1 205c23 A3 p2218390 BZD aA n A f BRP atest a g1asrora D 72463737 MAE Ag patra 27669953 82S 285 A 1 1 1 10 RP11 16251 4 139575905 0 6 7721334 6 6680 A Ao A fpo o m Rons Ko o pizo IONE A Mo fA f f2 RPmaesa20 19 fosreses O 68393 Z o BE A A aA fo f3 RPm 53F1g 3 Sg D B695714 8 A Mo A fo RP1 163L23 f3 61473828 SFE BBL A Ao n fpo fs RPMa7aa A3 p3zesoeo O 8736278 BAL A Ao A fo f6 RP11 164G18 f1 64184899 D 7587011 435 A Ao A fpo Aa Raza As paoamy7 D 8493798 9 503 A Mo A fo fe RP11 162F14 f5 3956624 R 56447306 m A A n fp fs Rema SEIS SES A Mo f fo po RP11A163612 6 04795721 D BAS43AT y y y y Paet aA h h fh m Rm sozn Sess A Mo fA fo o p2 RP1A163013 6 62904636 O W6761723 SE A Ao n fo o B RPN te poe69243 D 6676882 8 662 A Mo fA fo o p4 RP11A164H13 f4 85199492 D 8063788 6 067 A Ao nA fo 25 RPm 2017 STI 09246788 9 244 A Mo A fo pe RP1A183m19 B 2654725 FD iB A Ao n fo Reage As 61375336 0 8584351 2 O o Brst A Mo A f pe RP1AB4M3 f2 37
2. R1214 Remove Flag R121442 Restore default column order returns the columns to the default order 1f the columns have been reshuffled Freeze row selection memorizes currents row selection for future use When this command is activated the table rows will be highlighted in dark blue color and flags of 1 for these rows will appear in the Selected column at the right hand end of the table Clicking on the Selected column heading will move the selected rows to the bottom of the table Meanwhile Freeze row selection toggles to Unfreeze selection Selecting Unfreeze selection cancels the grouping of the selected rows and they will become highlighted with a light blue selection color This feature is useful if spots were selected using one of the plots for example selected regulated genes using Histogram and want to analyze their images segmentations and quantifications results The selected rows can be saved into txt file using Save frozen rows to file Also user can manually flag the selected rows spots by selecting Flag selected and its subsequent flagging options Using this option user can remove flags as well If automatic flags are removed through this option they can be re generated by changing flagging options or re quantifying images 38 1 2 3 Normalization Tab This functionality as of ImaGene version 7 0 has been moved to the Settings tool panel In this
3. d The computer s Ethernet Address is used as the computer ID e The Ethernet Address is a 12 digit hexadecimal number In this example the address is 00065b1bb4ee f The computer s Hostname is used to identify the computer on the network In this example the hostname is BIOINTERN g You can close LMTools by select Exit from the File menu or clicking the small X button in the top right corner 5 Request license file from BioDiscovery a Email support biodiscovery com for your customized license file b Please include 1 The Computer s Ethernet Address from step 4 2 The Computer s Hostname from step 4 3 Your name or the name of who purchased the software 4 Your company 5 Your BioDiscovery Product Serial Number if you ve purchased a copy You can find your serial number on the case of the CD shipped to you or in the accompanying User Registration form If you haven t purchased a copy just list the products you wish to evaluate c BuioDiscovery will reply with a small text file called license lic This file contains all of your licensing information 6 Copy this license file into the FlexNet directory you specified in step 2 169 You should keep a backup copy of this license file on another computer or floppy disk in case of an emergency 7 Configure the license services a Start the LMTools program by double clicking on Imtools exe in the FlexNet directory Click on the Con
4. i 12 3 RP11 8 nane 4 3 12 4 RP11 1 7 1 12 5 RP11 8 14 4 h2 6 RP11 1 8 vee i 12 7 RP11 8 19 i j2 8 RP11 1 i 12 9 RP11 8 z i 13 RP11 1 13 A 4 3 13 RP11 8 X A i B 13 RP11 1 16 ramman n an a Fa 2 6e8 4 b Selected rows 1 Save Export XML Close Right click to freeze selection Another useful feature of the program is the ability to launch a web browser and immediately go to Ensemble UCSC or Google for a search on the selected probe ID The CGH Report results table can be saved in a text file by clicking on the Save Report icon at the bottom of the panel The results contain fields for Gene ID Chromosome Base Pair Value normalized transformed and replicate combined ratio values Cluster Value and Algorithm Calls 32 CoHsampleBbCys tif seq Plots Mormalized Data Overview Whole Senome Chromosome Cluster Valle RP41 746019 oH es ss i 1 4211 8334770202637 0 42118334770202637 0 0 RP11 629G14 5859133 00 6035451 889038086 0 6035451689038086 00 RP141 353018 2138316 0 530672550201 416 0530672550201416 DO RP11 799F1 6256010 23256301 87988281 2 0 23256301679662612 0 0 RPA 1 875G4 7648591 0 3046150207519531 0504615020751953 0 0 RP141 14K12 6184471 0 4 262001 991 271 9727 042620019912719727 0O RP41 145C4 f 6771772 1 3313205242156982 1 0 RP 1 1 05862 20249563 0 3050556182861328 0 3050556152661328 0 0 RP11 57F20 23544768 0 22323107719421367 0
5. xi Gene ID a 88 Gene101 d 0 4616990 0 1572884 0 3044106 E Condition 1Mean locBc 127 Gene101 F 0 0475235 0 0479679 0 0954914 2 2789508 5 131 Gene101 F 8 2535989 0 2426557 0 2434810 Gene101 F 0 0149428 0 3241027 0 3091599 35 589990 Gene101 F 0 2567415 0 0404948 0 2162467 Gene101 g 0 1826571 0 1383636 0 0442935 1 4444261 Gene101 h 0 2660861 0 6196911 0 3536060 Gene101 r 0 2963549 0 0146570 0 2816978 1 1495416 4 f 0 3777735 0 6813168 0 3035433 0 1223344 j 0 0290404 0 9124788 0 8834383 0 0980410 0 0632792 0 0347618 0 47 08484 0 2997654 0 7 706138 1 1783400 5 Gene101 n 0 1087322 1 0954607 0 9867284 n 0 3130406 0 3288873 0 0158466 we p co T 22697 66 2 2 Pre manufactured Array Analysis ImaGene provides the ability to load templates for many commercially available arrays A template within ImaGene is the combination of a grid and the gene list or Gene IDs These are required to easily quantify and name the various probes on the array BioDiscovery provides a selection of these templates to its customers as a service to aid their array analysis For a complete listing of templates currently available contact BioDiscovery
6. Choose the plot type Scatter Plot K Any measurement available from the results table can be plotted in one of the tools Histogram Scatter Plot M A Plot Box Plot and GenePie Once a desirable type of visualization is chosen choices of the measurements become available Measurement coming from a specific channel image will be denoted by suffix ch1 ch2 etc Also normalization options are available if signal mean median or mode is chosen for the plot x axis Y axis Signal Mean cht UL Normalized Signal Mean ch2 Normalized Change in measurement selection or normalization option will be followed by update of the current plot Any plot can be printed or saved as an image file Histogram Histogram allows users to choose only one measurement Normalized data can be displayed if the Normalized box is checked 57 Choose the plot type a Histogram Bin number 100 log Bd Tails eT coe Total sor Left selected Genes 0 Middle selected Genes Right selected genes fo Total selected genes fo xX axis Signal Mean ch1 v x Normalized The histogram demonstrates distribution of the measurement values over their domain One can make an interval selection on the histogram and visualize how many genes fall into that selection By changing selection of Tails check box genes will be selected either inside the interval or outside Toggling between untransformed measurements and log transformed
7. European Organization for Nuclear Research Permission to use copy modify distribute and sell this software and its documentation for any purpose is hereby granted without fee provided that the above copyright notice appear in all copies and that both that copyright notice and this permission notice appear in supporting documentation CERN makes no representations about the suitability of this software for any purpose It is provided as is without expressed or implied warranty Package com imsl math Written by Visual Numerics Inc Check the Visual Numerics home page for more info Copyright 1997 1998 by Visual Numerics Inc All rights reserved Permission to use copy modify and distribute this software is freely granted by Visual Numerics Inc provided that the copyright notice above and the following warranty disclaimer are preserved in human readable form Because this software is licenses free of charge it is provided AS IS with NO WARRANTY TO THE EXTENT PERMITTED BY LAW VNI DISCLAIMS ALL WARRANTIES EXPRESS OR IMPLIED INCLUDING BUT NOT LIMITED TO ITS PERFORMANCE MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE VNI WILL NOT BE LIABLE FOR ANY DAMAGES WHATSOEVER ARISING OUT OF THE USE OF OR INABILITY TO USE THIS SOFTWARE INCLUDING BUT NOT LIMITED TO DIRECT INDIRECT SPECIAL CONSEQUENTIAL PUNITIVE AND EXEMPLARY DAMAGES EVEN IF ADVISED OF THE POSSIBILITY OF SUCH DAMAGES Packages jal Written by Mat
8. Quality Flags Once the segmentation is completed and the spot expressions are extracted the next question would be how reliable is the expression data In ImaGene the user can use various types of automated spot flagging schemas to help remove suspicious spots from consideration Alternatively manual flags of different types can be applied for quality control 69 eee ImaGene Parameter Settings l ioj x Normalization Analysis Controls and Alerts see _ Image Preferences Spat Finding Segmentation P Quality Flags Measurements T a Spot Quality Labeling Threshold signal strength Empty Spots m 00 10 20 30 4 0 2 0 Change Parameters Multi Channel Flagging Poor Spots a Negative Spots al Load save As Close Threshold signal strength Empty Spots yj 0 0 1 0 3 0 4 0 Empty spots woo o this option is utilized to automatically flag low expressed spots or missing spots This tool needs to have a sensitivity threshold determined For each spot the following ratio is compared to the threshold R signal mean background mean background standard deviation If for some spot this ratio appears to be lower than the threshold value the spot will be flagged with a green X mark on image It will also have flag value 2 assigned in the corresponding column in results table Threshold values can be chosen anywhere between 0 and 4 The user can either
9. 7 8 9 of the Binary Code License and Sections 1 and 2 of the Supplemental terms in Licensee s license agreement for the Program Agrees to indemnify hold harmless and defend Sun and its licensors from and against any claims or lawsuits including attorneys fees that arise or result from the use or distribution of the Program Does not modify or authorize its licensees to modify the Java Platform Interface JPI identified as classes contained within the java package or any subpackages of the java package by creating additional classes within the JPI or otherwise causing the addition to or modification of the classes in the JPI and Only distributes the Licensed Software pursuant to a license agreement that protects Sun s interests consistent with the terms contained in the Agreement In the event that Licensee creates any Java related API and distributes such API to others for applet or application development Licensee must promptly publish broadly an accurate specification for such API for free use by all developers of Java based software and Licensee must incorporate this term into its license agreements Trademarks and Logos This License does not authorize Licensee to use any Sun name trademark or logo Licensee acknowledges that Sun owns the Java trademark and all 206 Java related trademarks logos and icons including the Coffee Cup and Duke Java Marks and agrees to e Comply with the Java Trademar
10. As the batch entries are added information is displayed within the Batch Table to the left of the button Remove Selected This will remove the selected entry from the batch Multiple entries may be highlighted and removed through the use of lt shift gt and lt ctrl gt keys Edit Selected Entry This opens up a window that allows the batch parameters to be changed For example if the location of the images changes this can be used to update this field If a batch file template is made this tool would be used to modify any default values if needed Show Full Path By default ImaGene will only display the name of the files within the Batch Table Select this option to display the entire path within the table For example select this checkbox to display the Grid file as C ImaGene BatchImaGeneGridFile grd Load Batch Save Batch Run Load Batch This loads a batch file from the file system that had been previously saved ImaGene batch files are simple XML files with a bch file extension Please see Section 5 1 regarding ImaGene file specification for additional information on ImaGene batch files Save Batch The batch including information about all the entries will be saved to a file on the hard disk This file can later be reloaded using the Load Batch Button Note When saving the batch only basic information about the location of configuration files and image files is included The actual files r
11. Flagging codes Manual flags color red 1 Flag spots x 5 Empty spots x 6 Poor spots 7 Negative spots Automatic flags color green 2 Empty spots x 3 Poor spots 4 Negative spots Note Manual flags override auto flags 98 Note For flagging of a group of spots you can use lasso flagging tool 1 8 9 Undo and Redo Tools ImaGene supports an unlimited number of undo and redo operations Anytime clearing the previous action 1s desired this tool will provide means to do so am a Note The action to be undone or redone is displayed next to the text within the menu bar For example the grid was moved by mistake To undo this action from within the Tools Menu the text will read Undo Move Grid This is a convenient means to visualize what action will be reversed 1 8 10 Grid Tools Adjust Metagrid This tool allows manipulation of entire metagrids The metagrid can be moved stretched or resized Adjust Subgrid This tool allows manipulation of each subgrid The subgrids can be moved stretched or resized am FE i Adjust Spot This tool allows for individual spots to be moved and resized 99 Lasso Adjust This tool is utilized to select irregularly shaped regions of circles for adjustment Instead of shifting entire subgrids unusually shaped regions of circles can be selected for fine tuning For example this tool is ideal for use with membrane
12. P 01921 2818902 2723005 _ me a eee RP11 5 72744 0 7194 0 550464 0 1689735 UNCHANGED RP11 20 453971 11 45102 1 88274160 43171883 UNCHANGED RP11 7 156680 41081 839704 42889336 UNCHANGED RP11 6_ 141408 0 18330 0 26444 0 44775835 UNCHANGED RP11 10 _ 508574 0 0875 0 570786 0 48319483 UNCHANGED RP11 2 122387 2 88228 B 564077910 6617961 UNCHANGED RP11 K 295933 8 198697 8 675418 047672066 UNCHANGED _ RP11 5 1158743 0 24620 0 58295 1 33675843 UNCHANGED RP11 X 1144452 fi 56000 1 644601600 08459973 UNCHANGED RP11 5 872096 22082 D 8942299 0 3265946 UNCHANGED RP11 10 _ f16909 0 80958 fI 4856436 0 6760549 UNCHANGED _ RP11 5 569081 0 01065 0 63696 0 6476203 UNCHANGED RP11 12 37544 2 4064 3 33061 0 92414635 UNCHANGED RP11 5 156273 8 25066 4 02191640 7712507 UNCHANGED RP11 B f104150 0 26463 0 35535 0 61998814 UNCHANGED RP11 8 237598 2 45670 5 126356 0 66764945 UNCHANGED RP11 2 999159 0 4727 0 92840 0 4556974 UNCHANGED RP N07487 0 1000 0 24771 D 3478012 UNCHANGED RP11 X 126442 D 06606 0 41005 0 4761293 UNCHANGED RP11 4 B71676 0 5915 1 53204 1 0404966 RP11 B 143160 D 25022 0 322477 0 07226031 UNCHANGED RP11 6 228216 0 77019 f 4616354 0 6914431 UNCHANGED F117 oiae joes Ise o aseesf NcHaNGED RP11 A 204192 1 6766 1 7
13. Select the data set and go to Tools menu of GeneSight Lite Select Data Preparation tool The window for Data Preparation tool will pop up Select one of the preset data preparation sequences from Select Transformation Sequences menu bel Data Preparation DefaultDS gs Select Transformation Sequence es E Spotted Array LOVWWESS X rar T 1 Apply Data Preparation 1212 Genet 1 2716279444760525 0 9974481792131189 _ _ Ja Gene9 0 9108367550565465 0 9310135967752042 _ _ E Genet 1 4855759206322514 0 9829116825740839 Gene25 1 507961329305136 0 9752853342486913 Gene33 0 5245919621800009 0 9633982820029332 ___Gene41 0 40539826921996136 0 9656087441821283 1 6637076295034872 1 0172577280485493 1 5745028331580415 0 963530269876003 P R Or alternatively you can create your own sequence by dragging and dropping the preparation icons into the area under toolbar where the 156 8 sequence will be displayed Click on Apply Data Preparation button Close data preparation window when the transformations have been applied For more information on how to construct data preparation sequence please refer to GeneSight manual Chapter 7 Select any pair of measurements in sone main window Click Scatter Plot button on the toolbar Seater 2 D scatter plot window will pop up E Gonesh 4 8 Lite on Stat Eitin Filo View Tools Picts Unlities Help Condition 1 pair E Mean docRGc
14. The Find Negative Spots option is not intended for use in these situations 65 e Enforce Grid Constraints With this option selected ImaGene will use Local and Grid Flexibility when performing spot finding When not selected ImaGene will perform spot finding with no constraints Please see below for additional information regarding Local and Grid Flexibility Note Under most circumstances enforce grid constraints should be selected as this will limit the movement of circles during spot finding and help prevent erroneous spot finding due to dust and other contaminations e Local Flexibility Local Flexibility defines the radius measured in pixels that ImaGene is allowed to search for spots The origin for the search is the initial spot location as determined by grid placement From here ImaGene will search for a spot with X pixels distance where X is defined in the local flexibility parameter e Grid Flexibility Grid flexibility is an indication of the extent to which ImaGene should deform the grid to match a given set of spots The measurement is qualitative based on the unique properties of the image Most users should set this to the middle values Note Use the End key to move the slider all the way to the right or the Home key to move the slider all the way to the left Segmentation Segmentation is the differentiation of signal and background values within the array image and constitutes one of the most important a
15. This value is used to adjust the sensitivity of the clustering algorithm described above The smaller the number the less sensitive 134 the algorithm is in creating a new cluster So if some known aberrations are not being called because they are too small increase this value We have found a value of 0 005 to provide a good balance 2 Deletions lt This will indicate the threshold for a homozygot loss both copies deleted Theoretically this would be log 0 x or a very large negative number A threshold of 0 9 offers a good performance I Deletions lt This will indicate the threshold for a heterozygot loss one copy of the pair is deleted Theoretically this would be log 1 2 or 1 Due to the inherent noise in the experiment a threshold of 0 4 offer a good performance I Amplification gt This will indicate the threshold for a single copy gain Theoretically this would be log 3 2 or 0 585 Due to the inherent noise in the experiment a threshold of 0 4 offer a good performance 2 Amplification gt This will indicate the threshold for two or more copy gain Theoretically this would be equal or greater than log 2 or 1 Due to the inherent noise in the experiment a threshold of 0 8 offer a good performance Image Loading and Manipulation 1 Launch ImaGene from the desktop button or from the ImaGene folder within the Windows Start Menu 2 Click on Load Images icon under the Main tab e amp Images Grid Gene Info
16. a Fal s ole ele Images Grid Gene Info Process 7 Enter the following information into the Create Grid Window that appears oe eS Field Name Main Rows 16 Columns 11 Min Diameter 16 Max Diameter 18 Rectangular Grid is selected 106 Create Grid X Field Mame Man Rows jie Columns hooo Min Diameter hed o Max Diameter is f Rectangular Grid f Staggered Left Grid f Staggered Right Grid f Staggered Up Grid Staggered Down Grid Place Grid Cancel 8 Click the Place Grid Button in the Create Grid Window 9 Use the mouse cursor and click on the four corner spots of the sub grid Upon placement of the fourth spot the grid becomes visible on the sub grid 107 10 11 12 13 14 15 Click on the name Main in the Grid Panel Wain 16x11 Create MetaGrid Delete Selected Fred With Main selected right click and select Create Metagrid In the window that appears enter the following information a Metarows 6 b Metacolumn 1 Click Place MetaGrid In the top sub grid where we just placed the grid click on the top left most spot Using the scroll bar located along the right of the main image panel scroll downward until the last sub grid is reached 108 16 Click the top left most spot within this sub grid as well ImaGene automatically places the remaining sub grids in the proper
17. ditter CENNO E Maan ocRGC dimer A Condition 1 g E Mean ochGC ev F Condition 1 canton g Mean ocBGC cv 9 Color Se 9 Gene Partitons Cerditiss tMear locDOC Condition 1 contolhMean GocRGC LJ Show Gane Os Reuression Shi 2 132 Slope 0 625 R2 0 389 pa l t Find Gene Reset Schemes Create Parftion __vavon_ atorscnon Calu add sources 2 F actve genes 96 For manipulations within Scatter Plot window refer to GeneSight manual Chapter 9 Histogram 1 Follow steps 1 5 from the previous section 2 Select the measurement of interest in GeneSight Lite main window 3 Click Histogram button on the toolbar Histogram Histogram window will Pop up 157 B Detauliie gs EHE Condition 1 pair 7 E m an ockGC daan cy ge or gee Zoom Print gave Fina Gane Goto viel Prone Omin iena Lari Condtion pairhiean locBGC difference E wean foceoc 5 LE mean dibatte p Erecting Gulor Eit Gene Partons 4 For manipulations within Histogram window refer to GeneSight manual Chapter 9 Go To Web When using any of the tools mentioned above select a gene or set of genes N and click on the Go To Web button EA You will be automatically directed to the web based public genetic data Report GeneSight Lite also has various ways of generating reports please refer to GeneSight manual Chapter 10 Not
18. measurements can be accomplished by using log check box however this option will become available only if logarithm transformation was not applied to the chosen measurement during normalization Changing bin density printing the histogram and saving the image this feature is especially useful for publications can also be accomplished The Histogram provides not only the ability to plot any of the measurements available in quantification table but also provides access to useful values such as inter channel signal ratios including normalized Using these values in combination with the logarithmic option a histogram of a log ratio natural logarithm will be used can be obtained Selection of the tails of 58 such distribution provides a quick way to analyze up and down regulated genes Note When a selection is made on the histogram the same spots will be selected in measurements table and on the image allowing analysis of the selected spots in full scale Chl and ch2 stand for Channel I and 2 that refer to the order of the loaded images For example the first image in the image panel corresponds to Channel 1 Scatter Plot Scatter plot offers visualization of one measurement plotted against any other The normalized measurements can be used by checking the Normalized box next to either measurement Choose the plot type Scatter Plot v BEY la Scatter plot N lt x o c m a T c Ww D a N
19. more specific information For example start by selecting the Overview tab To view the gains and losses for the number 5 chromosome left click on that chromosome ImaGene instantly displays the single Chromosome tab with the corresponding ideogram and plot As with all ImaGene plots selection of a single probe causes selection of the raw data and spot image See section 1 2 4 for full descriptions of the various CGH panels including a description of the colors and lines within the plots Save the Raw results by selecting the Save button under the Raw Data table Save the Normalized Data and CGH Report by choosing the Save Report button beneath the tables Pictures may be printed or saved using the tools available in the various plotting panels 143 2 4 Batch Processing This tutorial demonstrates how to use batch processing within ImaGene Batch processing is an additional module which in not included by default with the base ImaGene Please contact BioDiscovery support with any question regarding eligibility The lessons learned within this tutorial include Preparing for batch processing Prior to performing batch processing several files need to be made available to ImaGene Adding Entries to the Batch Entries are the images and the corresponding settings to be used when performing batch processing Post Processing Start times stop times and any errors that occurred during the batch are recorded to a simple text file fo
20. rectangle around the section of interest from within the either the main image panel or the Image Map View e Drag the Zoom slider bar located directly below the Main Image Panel Note To return to the original image size double right click the mouse 1 8 5 Scroll Tool mm The Scroll Tool provides a convenient way to move about the image without zooming out of the current zoom level To pan the image simply click and drag the image around as you would any other object The image will automatically scroll left and right as needed If at any time you wish to know where within the image the current view is situated simply look for the location of the yellow box within the Map 96 View The yellow box indicates the region currently being viewed within the Main Image Panel 1 8 6 Ruler Tool The Ruler Tool ail allows objects to be measured within the Main Image Panel The most common use of this tool is to determine the minimum and maximum diameter for spot sizes and distances between the spots The tool can also be utilized to determine the general resolution of the image and how many pixels are included within each spot The Ruler Tool displays the results within the Status Bar located under the Main image Panel The information displayed includes e Dx the distance moved on the x axis as measured in pixels e Dy the distance moved on the y axis as measured in pixels e Distance the absolute distance moved as mea
21. the software will start up normally 173 4 4 FlexNet Server Installation Guide for Windows The default install directory for FlexNet is C FlexNet In the following examples it is assumed that this is the directory being used If FlexNet is installed into another directory then make the appropriate modifications to the path 1 Extract FlexNet Server files a Double click on the self extracting executable FlexNet zip b You will see the following screen Winzip Self Extractor BDFleximWink 1 exe xX To unzip all files in BOF lesimvinA exe to the specified folder press the Unzip button a sa a ae be Aun Winzip Browse Close W Overwrite files without prompting About Unzip to folder f C SFlesM et Help Select Unzip to folder C FlexNet by default Click the Unzip button to extract the files Click OK once the extraction is complete Click Close to exit the extraction program mo ao 2 Do you already have a license file called license lic a If yes then skip to step 5 b If no then proceed to step 3 174 3 Determine the server s Ethernet Address and HostName a Start the LMTools program by double clicking on Imtools exe in the FlexNet directory b Click on the System Settings tab c You will see the following screen Senvice License File System Setting Unites Start Stop Fleresd Server Status Server Diag Config Services Borro
22. 12 13 14 expiration of this Agreement including without limitation the provisions set forth in Sections 2 3 4 5 7 8 9 10 11 12 and 13 Export Regulations The Licensed Software and technical data delivered under this Agreement are subject to U S export control laws and may be subject to export or import regulations in other countries You agree to comply strictly with all such laws and regulations and acknowledge that you have the responsibility to obtain such licenses to export re export or import as may be required after delivery to you U S Government Restricted Rights If the Licensed Software is being acquired by or on behalf of the U S Government or by a U S Government prime contractor or subcontractor at any tier then the Government s rights in the Licensed Software and accompanying documentation shall be only as set forth in this Agreement this is in accordance with 48 C F R 227 7201 through 227 7202 4 for Department of Defense DoD acquisitions and with 48 C F R 2 101 and 12 212 for non DoD acquisitions Governing Law This Agreement will be governed by California law and controlling U S federal law Neither the United Nations Convention on the International Sale of Goods nor the choice of law rules of any jurisdiction will apply Any dispute relating to or arising out of this Agreement shall be resolved solely by an action filed in the Santa Clara County Superior Court or the United States District Cou
23. 43_635 tif HB19K 17 43_532 tif seg HB19K 17 43_635 tiF seg Plots Normalized Data Overview Whole Genome Chromosome CGH Report Summary S a 1 2 3 4 5 6 7 8 9 10 11 12 i bdii ri s pd A E R aF hoon oH on 8 4 J L lt ti i i s Z A l pa H l i 7 i gt gt 7 3 Fs Hi F i ri z i H 1 Bi c r pa i OS pa H Br bi P y 13 14 15 16 17 18 19 20 21 22 x Y f3 S A f f z ms Ta g 4 a U B H Ae Uf nf Ld j g 1 pa i a x i J Fi 3 H ka The Whole Genome panel displays the entire genome with chromosomes lined end to end as shown below Each open circle corresponds to a probe on the array If the same probe is spotted in multiple locations replicates the circles shown in the plots below are the combined value of all the replicates The call thresholds for single and higher copy gain or loss are shown as green and red horizontal lines respectively The yellow line in the plot depicts a moving average value and the magenta colored line is the output of BioDiscovey s call algorithm 49 Composite HB1SK 17 43_ S32 tif HB1okK 1 43 635 tif HB1oK 1 43 532 tif seq HB19K 17 43_635 tif seg Plots Normalized Data Overview Whole Genome Chromosome CGH Report k ig eie ii a nnsnnnsnnnsunnnnnnnunnnnnnnnng gnn eo aE TT hJ The Chromosome panel enables the user to zoom in and view selected
24. 5000 0 lt STANDARD_ LOW gt lt CONTROL_TYPE_IFUPALERT_1 gt true lt CONTROL_TYPE_IFUPALERT_1 gt lt FIND_NEGATIVES gt false lt FIND_NEGATIVES gt lt EMPTY_AND gt true lt EMPTY_AND gt lt HOT_HIGH gt 100000 0 lt HOT_HIGH gt lt SIGNAL_THRESH gt 0 9995 lt SIGNAL_THRESH gt lt SATURATION_THRESH gt 50 0 lt SATURATION_THRESH gt lt PERIMETERTOAREA_FLAG gt false lt PERIMETERTOAREA FLAG gt 187 lt GENES_DEDICATED_CTRL gt false lt GENES_DEDICATED_CTRL gt lt OMIT_FLAGGED_SPOTS gt true lt OMIT_FLAGGED_SPOTS gt lt IGNORED_THRESH gt 25 0 lt IGNORED_THRESH gt lt HOT_CV gt 3 0 lt HOT_CV gt lt TOLERANCE gt 5 0 lt TOLERANCE gt lt CONTROL_TYPE_NAME_0 gt POSITIVE lt CONTROL_TYPE_NAME_0 gt lt LOG_SCALE gt false lt LOG_SCALE gt lt MEASURE_MEANS gt true lt MEASURE_MEAN gt lt SHAPE_REGULARITY gt true lt SHAPE_REGULARITY gt lt LOG gt Base 2 lt LOG gt lt SMOOTHS gt 0 1 lt SMOOTH gt lt CONTROL_TYPE_UPTHRESH_2 gt 0 0 lt CONTROL_TYPE_UPTHRESH_2 gt lt OFFSET_FLAG gt true lt OFFSET_FLAG gt lt CHANNEL2 gt true lt CHANNEL2 gt lt OVERALL_FLAG gt false lt OVERALL_FLAG gt lt CONTROL_TYPE_COLOR_0 gt 65536 lt CONTROL_TYPE_COLOR_0O gt lt CONTROL_TYPE_NAME_1 gt BLANK lt CONTROL_TYPE_NAME_1 gt lt CONTROL_TYPE_ID_0_1 gt 2000 lt CONTROL_TYPE_ID_0_1 gt lt MEASURE_TOTAL gt true lt MEASURE_TOTAL gt lt BACKGROUND_LOW3 gt 0 0 lt BACKGROUND_LOW gt lt PERIMETER_THRESH gt 20 0 lt PERIMETER_THRESH gt lt ENFORCE_
25. Diameter 30 0 Ensembl UCSC Metacolumn 1 google Column 8 Name Original Segmen Composite Histogram Ss FCI CIC ICICI ICI CIC IC a lolol loo lS oS lS SS oo TTRS TRI TS PS os oS os os os oo Selected rows Save Export XML Close Right click to freeze selection 29 Segmentation Preview Use the Segmentation Preview to view the effects of current parameter settings on the segmentation both before and after quantifying the data Select the spot to view and the corresponding information about the spot will be displayed in the segmentation panel Concurrently the selected spot can be seen on the image and four of the plots Scatter plot M A plot Box plot and GenePie This interface also supports dynamic analysis within ImaGene You can adjust settings such as the maximum and minimum signal values and see the effects of these changes in real time via the dialog box Gene D Genet01 k 13 a11 Field A Wetarow 1 Metacalumn 1 F ow 16 Column 5 Diameter 31 0 Name Original Segm Camp Histog Sampler tit Sam pleg tit The following textual spot information is available via the Segmentation Preview e Gene ID Lists the corresponding information typically name or accession from the Gene ID file only available if a Gene ID file was previously loaded into ImaGene
26. E EN OEE OES 5S LOS Creatine and Runnin BIC icin Aaa ce av N AOS 90 TOA SAVING DONG EE AIE T ied a Oats 90 150 LOOQUI2 G BO V EREE E EE Cee Pa ee 9 LOO TEGO DIEM surest E EE E E ee cea end een eve eee 9 1 6 7 Normalization and analysis in batch mode viccccccccccsssecccccccceeeeeeseeccseseaaaeesseeeeeeaas 9 TiS VIE WIN GARE S UIC ics cca chet a NTE A cele vevunieioeenenaestaaneacs 92 LP NESNA KOT UENS JIC E EEEE E EE NN EE N 92 TAa LOGGING RESUS tO IRCVICW siai an e EEEE NE IUE EN 93 VS IMAGENE TOOLS i eatear ei ea Uanp aah aly areseal a Maleatieelas Matyas 94 EO LAULO AU CRIMENE LOOP sre nc EEE ecommetendeion 94 Fz AWO Grid Placement T OOl rin Tae E tedster secs eda ae 94 Foa SAVED ISDIGY TING CE TOOL STEE E EEO EES 95 FOA LOON LOOM AEREE E E E E E eae 96 PODS CVOLE TD OOla E EE E E EO OE 96 FRO RME TOO a T E E Eee 97 PE A VOI AIEN ENE VEA AET EA NA TAON EA OE AAA 97 Peo FAG 2 CUNO T OOl irii E EE E E E AE E ER 97 Lo FUndodnd ICAO TOOLS arnir ine ra E EEEE E A E S 99 FOJO Grd 1 OOUS ciad ii N EEE E EEEN E EN EE 99 PART 2 IMAGENE TUTORIALS sisesscssscssexsstvesdendonssecovnnonssccosatesssteersdnsssevennsoaesssessesdocsse 103 2 AIS ASICAAINA ICY SIS voie spied anianine ua intasanaias nintas tal aiculcduudi sud taniuieaninnaakatia satveoaties 103 2 2 PRE MANUFACTURED ARRAY ANALYSIS vos susteshicteitparuetsscadudsveneatecnechiadsleeetiadin sadesaxavax 120 2S GL UT ORIA Rik desactetnpiatsaulasibiotan a T E wiveomsen 131 2
27. Expression MOdule cccccccccseeccccccceeenneeseceeeeeeaaaeees 184 DLI OPT U oan a E E E TE A E A EE E 185 FLEC ONRU ONT E EREE EEEE EE E EEE AE 186 JLo 0701 6 2 CP a a eA A a E E E ee 189 32 TECHNICAE SOPPOR Treron days E r E hanes aaa ee Unoneon 193 5 5 WARRANTY INFORMATION zrenie N A E E NAAN 193 SOFTWARE LICENSE AGREEMENT AND LIMITED WARRANTY ccc000 194 PACKAGES CERN COL T CERN JEU ccciscssceccsscccersidesvcsseccsceccnssisessadeocensideasedecdeseotses 202 PACKAGE COM IMSL MA TD Ty ivscecseeceeceseasedsascecccasstesteccecscassseievincsdesseceiessseneceasecdedeassees 202 PACKAGES TAL na aa a ra Sa E aai JAVA 3D 1 2 1_01 BINARY CODE LICENSE AGREEMENT sss sssssoessssssssoosscsssssso JAVA 3D TM SOFTWARE VERSION 1 2 1_01 SUPPLEMENTAL LICENSE Introduction to ImaGene ImaGene is BioDiscovery s solution for a fast and accurate microarray image analysis software system With ImaGene researchers can generate quantified data within seconds while generating extensive quality control information Microarray image analysis is utilized to quantify the relative expression levels existing within a microarray scan As such the role of ImaGene within the microarray process is post hybridization and scanning The subsequent results of ImaGene are quantified expression values that are saved to a text file These results can then be used in any data analysis package such as BioDiscovery s GeneSight for further analy
28. HB19K 17 43_635 tif Brightness Contrast IT Reverse Display Colors Grid A 12 x 4 x 29 x 29 GenelD HB19K_gid_chromtxt Clear All 15 Image Analysis Workflow Icons mages rid Gene Info Process The essential tools for image analysis are grouped together as a line of icons to load and align images to create or load grids to load templates or Gene IDs to auto adjust grids or spots and to quantify the image spots Load Images Opens the Load Images Dialog and allows the selection of desired image files Multiple files can be selected by holding the lt shift gt or lt ctrl gt keys and clicking on the image names Note ImaGene supports the following image file formats e Tiff uncompressed The file extension is tif MD Gel The file extension is gel Fuji Bas This is a two file format with one file ending in inf and the other ending in either img or bas Auto Align Images Aligns the displayed images automatically if they are misaligned to each other after being loaded EJ Create Grid Launches the Create Grid window where the essential information about the design of the grid must be entered Load Grid Displays the Load Grid dialog box Use this interface to select and open a previously saved grid grd file 16 1 Load Import Template Displays the Load Template dialog box Use this interface to select and open a template tpl gal xml for GE
29. Mame BIOGREEN User Namel Joe Green Company Name Green Corp Serial Number BDn 2345 See reating a 4 Enter the name of the computer running the license server a This can be the computer s network name like BIOGREEN b This can be the TCP IP address of the computer like 192 168 1 6 c This can be an Internet domain name like www biodiscovery com d This can be this local computer Use THIS HOST to indicate that this is the very computer serving the license files Enter your User Name Company Name and Serial Number 6 Itis important that you enter your actual Serial Number since this information is used to determine eligibility for upgrades using the BioDiscovery Auto Update feature 7 The serial number can be found on the case of the CD shipped to you or in the accompanying User Registration form 8 Click the Save Floating button to save this information and exit 180 9 Start the BioDiscovery software If the license server is running the software will start up normally 181 Part 5 Appendices 5 1 File Specifications While ImaGene is designed to be flexible with regards to information imported and exported you must adhere to certain standards regarding file formats 5 1 1 Gene ID File ExpressionModule Expression Module The Gene ID file is utilized for track information about the material spotted at each location within the array This information will be saved along wi
30. Method auto Signal Low 0 0 Signal High 0 0 Background Low 0 0 Background High 0 0 Background Buffer 1 0 Background Width 10 0 End Measurement parameters Begin Alerts Control Type Minimum thresholdlIf tested Percentage allowed If failed Maximum threshold If tested Percentage allowed If failed CV threshold If tested If failed Bright 0 0 false 0 0 false 10000 0 false 0 0 false 10 0 false false End Alerts Begin Quality Flags 185 Begin Flagging Settings Empty Spots true Threshold 2 0 Poor Spots false Negative Spots true End Flagging Settings Begin Flagged spots of Empty Spots 262 of Poor Spots 0 of Negative Spots 0 of Manually Flagged Spots 0 End Flagged spots End Quality Flags End Header Begin Raw Data Field Meta Row Meta Column Row Column Gene ID Annotation 1 Flag Signal Mean Background Mean Signal Median Background Median Signal Mode Background Mode Signal Area Background Area Signal Total Background Total Signal Stdev Background Stdev Shape Regularity Ignored Area Spot Arealgnored Median Area To Perimeter Open Perimeter XCoord YCoord Diameter Position offset Offset X Offset Y Expected X Expected Y CM X CM Y CM Offset CM Offset X CM Offset Y Min Diam Max Diam Control Failed Control Background contamination present Signal contamination present Ignored failed Open perimeter failed Shape regularity failed Perim to area failed Offset failed Empty spot Negative spot Selected spot Saturated spot A 1 1 1 1 null
31. Please see the FlexNet Client Installation Guide for details b If no then you need to do some troubleshooting Proceed to step 10 10 Troubleshooting the FlexNet license server a Double check that you entered the correct file paths in step 6 b View the license file by right clicking on the license lic in the FlexNet directory and selecting Open With c Select Notepad from the list d Place a checkmark next to Always use this program to open these files e Click the OK button 178 oe The text file will contain a line that looks similar to this SERVER HOSTNAME 00065b1bb4ee Make sure that HOSTNAME is your computer s Hostname from step 3 Make sure that the following Ethernet Address matches your computer s Ethernet address from step 3 If it still doesn t start then email the C FlexNet debug log file to support biodiscovery com 179 4 5 FlexNet Client Installation Guide all platforms These instructions apply to all BioDiscovery products running on any supported platform In some places the tag Product will appear This represents the name of the BioDiscovery product being installed like CloneTracker for example 1 Start the License Wizard by clicking on Start gt Program Files gt Product gt License Wizard 2 Click on the Floating tab 3 You will see the following screen BioDiscovery License Management W zart 3 sia ol x Locked Floating Server
32. Process 3 Browse to the Samples folder within the ImaGene folder The location is typically C Program Files BioDiscovery ImaGene SampleData CGHdemo 4 Select CGHSampleACy5S tif and CGHSampleACy3 tif images Multiple selections can be performed by holding the lt ctrl gt key on the keyboard and left click 135 Load images X Look in E CGHdema im EA Recent if results l fe CcGHSampleACy5 tif Desktop E CGHSampleb ys tif My Network File name Open Files of type Image Files tif gel inky Cancel 5 Click the Open Button to complete the image loading 6 Select the Load Grid tool and choose from the same directory the file cghdemo grd Select the Place in saved position option 7 Images Grid Gene Info Process 136 Load Grid Look in E CGHderma 7 cshdero ad Place manually f Place in saved position File name eghdemo ard Open Files of type ImaGene Grid Files grd se Cancel Ahrar 7 Load the CGH gene ID file cghdemo geneid txt from the menu bar ajel Images Grid Gene Info Process 137 E imoGene 7 0 Standard watch Edition Fle Ect Git Selection Auto Mespure Toos Heb p io E a ao Ja w ble te lt U Undo Remove Images m 2 a Man Preen Cogote CIAC yI COANA i tE images Bi COtsampletty3 F Di CatoaempleAscys tF Dnightness Corer ast F Rerne Oepbey Col
33. aid them in going through the necessary steps The Wizard can be switched on and off at any moment through the Help menu Even when the Wizard window is switched on it does not limit user s access to any of the ImaGene tools ImaGene wizard YOu are using Change settings tool Here js how to use it Change advanced settings for gridfspot finding segmentation quality flagging controls To launch the Wizard select Wizard On option from Help menu Wizard window will be shown in the bottom right part of your screen The ImaGene Wizard works in two modes First mode switches on whenever Wizard detects that user performed some action that should be logically followed by another action Then the Wizard will prompt what action was performed and what should be done next To follow this advice the user can either use the tool that the Wizard refers to or just click on Follow Advice button in the bottom part of Wizard window The latter will result in the recommended action being performed automatically If the recommended action is not necessary the user will have an option to go to the next advice A sequence of recommended actions will start immediately after ImaGene launches with the advice to load the image Thus virtually any user can just follow the Wizard prompts to learn how to use ImaGene 82 If the user tries to apply some tool that does not fall into a pre defined sequence of actions Wizard will switch
34. an image there is a need for further data processing ImaGene incorporates a powerful tool that provides creating simple types of experimental design data cleansing transformation normalization visualization etc This tool is called GeneSight Lite and can be launched at any time using its corresponding button on the toolbar Please be patient while it launches might take up to 20 sec for some machines GeneSight Lite will start with data set builder GUI where you can build your data set for further analysis Data set may include data from multiple images you processed using ImaGene To build data set you will have to arrange ImaGene output files tab delimited text files into a specific experimental design Once data set is built you can use Data Preparation tool for data cleansing transformation and normalization After all necessary preprocessing are applied to the data you can start using various visualization and analysis tools of GeneSight Lite Aforementioned tools include Histogram 2 dimensional Scatter Plot Box Plot Partition Manager K means clustering Time series visualization Reports generation 3 2 How to use GeneSight Lite For details on the usage of GeneSight Lite please refer to GeneSight Lite user s manual included in ImaGene distribution package In this section we will provide some guidelines for using basic visualization tools 153 Scatter plot To build a scatter plot on a data you
35. and directly touching signal area Spot area ignored area plus signal area Ignored median median of pixel intensity in ignored area Area to perimeter ratio see Quality Flags section above Offset from expected position multiple offset measures get outputted including X Y and overall offset from expected position for the spot s adjusted circle and spot s center of mass Shape regularity see Quality Flags section above Open perimeter see Quality Flags section above measure is outputted as a decimal fraction corresponding to Open perimeter percentage 0 0 0 100 1 0 These measurements may be checked and unchecked at any time results table will be updated immediately 76 Controls and Alerts ImaGene incorporates a powerful tool for testing expressions of control spots located on image This tool allows groupings of several gene IDs into a unique control type and to set up multiple test parameters for the type Be ImaGene Parameter Settings O x Spot Finding Segmentation Quality Flaas Measurements Normalization Analysis Controls and Alerts Image Preferences Control type list Positive Remove Minimum expression allowed 0 0 Maximum expression alowed SOOO Maximum CY allowed 1 0 Gene ID list EMPTY Add Gene ID Remove Gene ID Load Gene IDs From file Update Visual Load Save 45 Close Using Add button on the panel a ne
36. arrays because edges could become warped quite often Rectangle Adjust This tool which is similar to the lasso adjust tool is utilized to sub select circles to adjust from within the array However this tool employs a rectangular shape to highlight given circles A possible use of this tool would be with microarrays produced by using a bent pin mounted on the pin head In such a case an obvious rectangular anomaly might exist within the array making it ideally suited for the Rectangle Adjust tool mee L ar _ Duplicate MetaGrid Duplicating an existing metagrid can save time and produce consistent results rather than manually re creating the metagrid several times To use it click on the button and select a metagrid to duplicate by left clicking on it Then this metagrid can be copied to any place of the image by left clicking there To stop copying perform a right click _ H E 100 Show Hide Grid ImaGene allows the grid to be hidden enabling a better view of the spots behind Hiding the grid hides lines circles and flagging that may exist within the Main Image Panel Auto Adjust Spots Select this option to have ImaGene perform automatic spot localization ImaGene searches the area around grid placement for a spot within the predefined minimum and maximum diameters Adjustment accuracy also depends on grid constraints and the quality of your images For example if spots with irregular
37. below is a sample batch file Note that the information contained here matched the information required within the Batch Editor Note There are options in the XML file below that are not accessible through Batch Editor GUI These options can be used by advanced users through manual creation of the file lt xml version 1 0 encoding UTF 8 gt lt Batch gt lt Entry gt lt Image gt C ImaGene Sample Data 1205 1205_ TIF lt Image gt lt Image2 gt C ImaGene Sample Data 1205 1205_r TIF lt Image2 gt lt Template gt C ImaGene Sample Data 1205 1205_new gal lt Template gt lt Configuration gt C ImaGene Sample Data 1205 1205_config_with_norm xml lt Configuration gt lt Destination gt C ImaGene Sample Data 1205 Out_batch_norm lt Destination gt lt Channel gt 0 lt Channel gt lt ChannelName gt lt ChannelName gt lt Channel2 gt 0 lt Channel2 gt lt ChannelName2 gt lt ChannelName2 gt lt SubstituteGridImage gt null lt SubstituteGridImage gt lt SubstituteGridChannel gt 0 lt SubstituteGridChannel gt lt AlignImages gt true lt AlignImages gt lt AdjustGrid gt true lt AdjustGrid gt lt AdjustSpots gt true lt AdjustSpots gt lt Normalize gt true lt Normalize gt lt Analyze gt true lt Analyze gt lt Entry gt lt Entry gt lt Image gt C ImaGene Sample Data 1205 1205_r TIF lt Image gt lt Template gt C ImaGene Sample Data 1205 1205_new gal lt Template gt lt Configuration gt C ImaGene Sample Data 1205
38. chromosomes as shown The zoom tool can be used to zoom into an area on the plot or on the ideogram 50 Composite HB19K 17 43_532 tiF HB19K 17 43_635 tif HB19K 17 43_532 tif seg HE19K 17 43_635 tif seg Plots NormalizedData Overview whole Genome Chromosome CGH Report Summary Chromosome A very useful feature in all the graphic plots is the ability to select a point open circle in the plot and then be able to drill down in other screens as shown below As can be seen the original spot image segmentation overlay and quantification values are all immediately accessible If there are multiple spots combined together all the spots are selected in the quantification table at the bottom of the Raw Data tab 51 Main Raw Data Composite HB19K 17 43_532 tif HB19K 17 43_635 tif HB19K 17 43_532 tif seg re sti i i Chromosome Gene ID RP11 1100M9 HB19K 17 43_635 tif seg Plots NormalizedData Overview Whole Genome CGH Report Summary Field UCSC Ch 1 ea Metarow 1 Metacolumn3 google ee X a l df Row 12 Column 14 Diameter 17 0 HB19K 17 43_532 tif HB19K 17 43_635 tif 2 3e8 A 1 42 5 RP11 8 4 i2 16 RP11 1 4 12 A7 RP11 8 22 4 3 42 48 RP11 1 i n2 9 RP11 8 15 2 4e8 i 12 o RP11 1 L 12 1 RP11 8 15 i 12 2 RP11 1
39. control type only 1 8 3 Save Display Image Tool ImaGene s Save Display Image Tool provides the capability to save a screen capture of the overlaid 1 e composite images The saved image includes only the overlay as it appears within the main image panel ImaGene saves the image as a high quality 24 bit tiff format The image can later be recalled for reference for use in publications 1 8 3 1 Saving the Display Image Perform the following steps to save the current composite overlay to a tiff image file 95 1 From the menu bar select File then Save Display Image Alternatively click the Save Display Image icon from the toolbar 2 From the Save As Dialog that appears browse to a location 3 Specify a file name ImaGene will automatically add the tif file extension to the end of the name 4 The image is now saved and available to be viewed in virtually any graphics program such as Microsoft Imaging or Adobe Photoshop 1 8 4 Zoom Tool The Zoom Tool a allows the zooming of a specific region of the image The zoom methods that ImaGene currently supports include e After selecting the Zoom Tool left click with the mouse and drag a rectangle around the section of interest from within the main image panel e After selecting the Zoom Tool left click with the mouse and drag a rectangle around the section of interest from within the Image Map View e Hold the lt alt gt key on the keyboard left click the mouse and drag a
40. days after such oral disclosure to be either proprietary trademarked registered copyrighted confidential and or trade secret in nature shall impose a duty upon Licensee not to disclose to any third party such information disclosed by Licensor to Licensee either in writing or orally without the express written consent of Licensor The obligations of this section 7 shall not extend to any information which is lawfully known to Licensee prior to receipt from Licensor or its distributor or enters the public domain through no wrongful act or breach of this Agreement by Licensee or is received by Licensee from a third party having a legal right to disclose such information 8 LIMITED WARRANTY Licensor warrants for a period of 90 days from the date of commencement of this Agreement Warranty Period that during the Warranty Period the Software shall operate substantially in accordance with the functional specifications in the User s Manual LICENSOR FURTHER WARRANTS THAT DURING THE WARRANTY PERIOD THE MEDIA WHICH CONTAINS THE SOFTWARE SHALL BE FREE FROM DEFECTS IN MATERIAL AND WORKMANSHIP LICENSEE S SOLE AND EXCLUSIVE REMEDY AND LICENSOR S SOLE LIABILITY ARISING FROM BREACHES OF THE ABOVE WARRANTIES IS THE REPLACEMENT OF DEFECTIVE MEDIA OR IF LICENSEE SHALL SO REQUEST TO REFUND TO LICENSEE THE PURCHASE PRICE FOR THE DEFECTIVE SOFTWARE AND DOCUMENTATION PROVIDED THAT LICENSEE NOTIFIES LICENSOR IN WRITING OF SUCH DEFECT AND RETURNS TO LICENSOR T
41. disclose the Software and any accompanying documentation to any third party Licensee further acknowledges and agrees that any written documentation provided by Licensor to Licensee which contains a legend upon such documentation whether or not 196 such legend be a single legend affixed upon a multiple page document which legend identifies such document to be either proprietary trademarked registered copyrighted confidential and or trade secret shall impose a duty upon Licensee not to disclose to any third party the documentation or any information contained within such documentation either in writing or orally without the express written consent of Licensor Notwithstanding the foregoing provision Licensor may notify Licensee in writing within twenty 20 days after disclosure to Licensee of documents which do not contain a legend identifying such documents to be either proprietary confidential and or trade secret that such documents disclosed were either proprietary trademarked registered copyrighted confidential and or trade secret in nature Such notice shall impose a duty upon the Licensee not to disclose to any third party such documentation or any information contained with such documentation either in writing or orally without the express written consent of Licensor Licensee further acknowledges that any oral information provided by Licensor to Licensee which information is identified or summarized in writing within twenty 20
42. e Field Lists the field that the selected spot belongs to This name is specified when the grid was first constructed e Metarow Identifies the row within the metagrid where the selected spot is located e Metacolumn Identifies the column within the metagrid where the selected spot is located 30 Row Identifies the row within the subgrid where the selected spot is located Column Identifies the column within the subgrid where this spot is located Diameter Lists the diameter measured in pixels of the selected spot The diameter is determined during spot finding when various spot sizes are attempted Note Segmentation involves the partitioning of a microarray image into a set of regions that convey a specified meaning For microarrays the purpose of segmentation is to decompose a scanned optical image into regions that are meaningful in terms of spot signal and spot background The following visual spot information is available via the Segmentation Preview Name Lists the name of the source image file for the spot Original Displays the spot and its surrounding background without any segmentation information Segmented Displays an image of the segmentation or determination of background and signal that will be performed during quantification The red pixels represent signal values and the green pixels represent background values Black means the pixel is ignored 31 Composite Displays an overla
43. each other on the genome to create a moving average value You can see the result of a moving average as a yellow line in the figure below We could use the moving average to make calls However this approach is sensitive to the window size and outliers in the data BioDiscovery s approach is to use a statistically based algorithm similar in spirit to the Circular Binary Segmentation CBS algorithm developed by Adam Olshen at Sloan Kettering Institute We have modified the CBS algorithm and significantly improved the processing speed by using a Normal distribution function for testing for change points as oppose to the non parametric permutation based statistics used in the original CBS algorithm The result of the algorithm is to segment the genome into clusters of uniform ratios The cluster values are plotted as the magenta line in the figure below The calling algorithm then uses the cluster values and the user defined thresholds to establish regions of copy number variations 133 Chromosome 5 w mn 0 2 5e7 5 0e7 7 587 Pi 1 00e8 Ea 1 25e8 Ba Ea 1 50e8 zi Reasonable parameter values for the CGH algorithm Please note that the recommendation here will not be appropriate for every image and the selection of parameters should be done by the user based on their own images and knowledge of the underlying biology The following recommendations should be used as a starting point Significance Threshold
44. form any other machine readable materials including but not limited to libraries source files header files and data files and any user manuals programming guides and other documentation provided to you by Sun under this Agreement License to Use Sun grants to you a non exclusive non transferable and limited license to download install and use the Licensed Software by the number of users and the class of computer hardware for which the corresponding fee if any has been paid No license is granted to you for any other purpose You may not sell rent loan or otherwise encumber or transfer the Licensed Software in whole or in part to any third party License Restrictions The following restrictions apply to your license The Licensed Software is confidential and copyrighted You must take appropriate steps to protect the Licensed Software from unauthorized disclosure or use Title to the Licensed Software and all associated intellectual property rights is retained by Sun and or its licensors e Except as specifically authorized in this Agreement or any supplemental license terms you may not make copies of the Licensed Software other than a single copy of the Licensed Software for archival purposes You agree to reproduce any copyright and other proprietary right notices on any such copy e Except as otherwise provided by law for purposes of decompilation of the Licensed Software solely for purposes of inter operability you may not
45. grid in batch mode through loading a manually created XML batch file of a specific format described in section 5 1 5 File Format s to Export for Batch Processing ImaGene allows exporting the results of batch image processing into text MAGE ML and GEML formats The options for these various file format 87 export are provided in the batch editor window By default the results would only be exported in text file format The users can optionally choose to export results in any or all of these formats by checking their option boxes MAGE ML and GEML file formats would be saved in their respective destination folder using the same name as the text file ae Formats Text LJ MAGE ML L GEML 1 6 2 Create Batch Entry Window The Create Batch Entry Window allows entries to be added to a batch The window appears as a result of clicking either the Add Batch Entries Button or the Edit Selected Entry Button This window requires four parameters to be entered in order to batch a set of Images All images belonging to a common set can be added to the batch as a group For example if 5 images were generated with Molecular Dynamics hardware then all five images may be added here since they contain the same settings and grid within ImaGene Thus only a single settings and template file will be required for several similar images Be Create Batch Entry Mages C TEMPBatchiSamples tit TEMP Batch Sample tit TEMP Batch Samples tit T
46. images selected or highlighted from the images panel are removed or unloaded from analysis e Select Color Select the color to be used for the selected image when seen under the Composite Tab The default colors for the first two images are red and green however these can be changed to any color desired Changing the colors here in no way affects the resulting quantified values e Invert Values This will invert all pixel intensities within the selected image When the image is loaded ImaGene automatically determines which end of the grayscale spectrum is the high value This information is typically available within the image file itself However in rare cases this information is not present within the file and as a result ImaGene requires this information to be manually set Should it be determined that the expected values for the signal and the background measurement to be opposite to what is expected selecting Invert Values will solve the problem 18 e Hide The selected image will no longer be visible under the Composite Tab While not visible if quantified data will be generated for the image e Rotate Rotates the image 90 180 or 270 degrees around the top left of the image e Image Info Provides information about the image read directly from the image file such as the file name directory location width height type and resolution Grid Panel The Grid Panel lists all fields that have been created and
47. is the most common all the possible selected are Rectangle 3335 Staggered Left Grid sat Staggered Right Grid wes Staggered Up Grid z Staggered Down Grid 3322 Create MetaGrid This selection allows subgrids to be used to form a metagrid structure Before creating a metagrid at least one subgrid must be created The Create MetaGrid window requires the following parameters to be specified o Metarows The number of rows of subgrids o Metacolumns The number of columns of subgrids Load Template This will launch the template file browser panel 22 e Delete Selected Fields The current highlighted field will be removed e Clear Grid All fields that have been created or loaded will be removed e Convert to Single Subgrid If a metagrid structure exists and has been used to grid the image selecting this option will convert the metagrid structure to a simpler subgrid structure For example if we have a 2x2 metagrid with 15x10 subgrids after selecting this conversion the resulting subgrid size will be 30x20 During this conversion nothing changes except for the how the individual spot locations are represented This feature is optional and may be used if required to construct a multi level metagrid for example Properties This option opens a window displaying the parameters for the current selected field Only certain options can be modified while the remainder requires the grid to be deleted th
48. m mm i ai mu i gt EP er hs E eeeeaeaeaeaaee fefed Lo E ARE OEE e eeeeeeeeaeaeeaee eeeceeeaeeaeee gy eeeeaeaaaeae ee eeeeeaeeaaeeee O eeeeeeaeeaeaee eeeececeaeece eeeeeeeeeae 20 The bounding box represents the field eccececeeeee Wl 2 Right clicking on the Grid Panel provides B1gK_c Gene ID Grid Like other components of ImaGene the Grid Panel fully supports context sensitive menus options Create Grid The option launches the Grid Creation Window where the essential information about the design of the grid must be entered Remember that within ImaGene the grid subgrid is the most elemental structure followed by the metagrid and finally field The following parameters must be specified Create Grid E i x Field Marne la Rows Zz Columns hi Min Diameter ho Max Diameter hzo G Rectangular Grid i Staggered Lett Grid i Staggered Right Grid Staggered Up Grid a Staggered Down Grid Place Grid cancel Field Name Specify the name of the field here The name may be any name desired By default ImaGene provides the letter A to the first fields B to the next field and so on If a gene ID file is being used in conjunction with the image to provide gene names or accession numbers within the text output file of ImaGene the fields name if used must match between what is specified here and that is contained within the Gene ID file For exampl
49. modify or create derivative works of the Licensed Software decompile disassemble or 203 otherwise reverse engineer the binary portions of the Licensed Software or otherwise attempt to derive the source code from such portions e The Licensed Software is not designed or licensed for use in the design construction operation or maintenance of any nuclear facility e You may not publish or provide the results of any benchmark or comparison tests run on the Licensed Software to any third party without the prior written consent of Sun e Noright title or interest in or to the Licensed Software any trademark service mark logo or trade name of Sun or its licensors is granted under this Agreement Sun Sun Microsystems the Sun logo and Sun Ray are trademarks or registered trademarks of Sun Microsystems Inc in the U S and other countries Limited Warranty Sun warrants to you that for a period of ninety 90 days from the date of purchase as evidenced by a copy of the receipt the media on which Licensed Software is furnished if any will be free of defects in materials and workmanship under normal use Except for the foregoing THE LICENSED SOFTWARE IS PROVIDED AS IS YOUR EXCLUSIVE REMEDY AND SUN S ENTIRE LIABILITY UNDER THIS LIMITED WARRANTY WILL BE AT SUN S OPTION TO REPLACE THE LICENSED SOFTWARE MEDIA OR REFUND THE FEE PAID FOR THE LICENSED SOFTWARE UNLESS SPECIFIED IN THIS AGREEMENT ALL EXPRESS OR IMPLIED CONDITIO
50. of the table Go through the spots one by one analyzing spot images To normalize and analyze the data you need to set the appropriate paramters Go to the Settings tool from the File menu 111 2 ImaGene Parameter Settings yy _ ol xj Soot Findina Segmentation Quality Flags Measurements Normalization Analysis Controls and Alerts Image Preferences Background Correction Correction Local Measure Eckor Mean Sliding Window Window length 5 0 Normalization Algorithm Normalization by pe JLowess Z Scope Global ka Using control spots use all spots ka Smoothing o z e Take Log Load Save 4s Close 12 Select the Normalization tab and from the different drop down options choose the parameters for background correction and normalization 13 Select the Analysis tab and select either Expression or CGH analysis Based on this selection provide desired parameter settings 112 Normalized Signal Mean 7 oI fumes 14 When Normalization and Analysis settings are chosen the Analyze tool can be run using this button in the menu bar Ba a slem ge ele 15 The normalized data will be displayed in a new tab Normalized Data in the right panel 113 Composite 17hrs1 gel 17hrs2 gel 17hrs1 gel seg 17hrs2 gel seg Plots Normalized Data Analysis Fo me mc Genep Flag N Signaime N SignalMme N Signal Medi N 5 A 1 ff _Genetot e
51. placed on the image Typically only a single field will be required however depending on the design of the array being quantified several fields may be required As a new field is created it is added to the list Before explaining how and why to create a Field let us explore some of the definitions and structures of a field A field is the largest design element within a slide A field typically consists of the arraying done by a single print head on the slide For example if the arrayer has a print head with 8 pins in a 2 x 4 configuration the region of the slide containing the resulting printing is a field The metagrid in this example would be 2 x 4 as the resulting printing would generate 2 rows by 4 columns of subgrids The subgrid is not defined here but would be whatever rows and columns of spots that are printed by a single pin 12x12 for example The accompanying diagram demonstrates the relationship between the three levels of structure 19 The MetaGrid is 2 x 4 the following eeeaeeceaaeaaeae aS eeeeaeaaaaee cn eeeeaaaaaee O eeeeaeaaaaaee a a eeecaeaeaaaaee eeeeaeaeaaaeae nm Li eeeeeaaeaeaee eeeeaeaaaaee Lb wi eeeaeeaaeaaeaece eeeeaeaaaeaacec a Tw 0 a Th eeee eeeee 8 amp ee ee ee4 eet 8es amp o _ CL mi a e e eeeeeee amp 85ed amp e eeeeeeeete se La I p u ae in e ee eeeeeeeee eee eeeeee eaee i mi I ie Ae ay ese Seite Ree aD a m g Wi eaae it
52. previous procedure Additionally one can check combine signal test and background tests and two confidence numbers will be added together with equal weights 0 5 and compared to the background contamination threshold Flag for high ignored percentage T Ignored percentage As mentioned before some pixels may not be assigned either to the signal or to the background in the presence of local contamination Such contamination will be excluded from the measurements and called an ignored region The ignored percentage flagging works in the following manner It computes the area of ignored regions directly neighboring touching the signal area If it is denoted as a 1 then the following ratio is computed x 100 R I S is equal to the signal area If for some particular spot R is higher than the pre set threshold this spot gets flagged for low quality Flag for high open perimeter percentage i i v Open perimeter percentage This tool was designed to capture a specific type of spot deformation It computes the percentage of the signal perimeter that touches the border of rectangular snip Such a deformation can appear if the spot is significantly shifted from its expected position or if its shape has an abnormal form Whenever 72 the percentage crosses the pre set threshold the spot is flagged as low quality 5 Flag for abnormal shape regularity This algorithm computes shape reg
53. quantified you need to follow these steps 1 Make sure that you save quantification results from current image click Save button at the bottom of quantification table 2 Click GeneSight Lite button on the toolbar Lite GUI pops up 3 GeneSight Lite will start with a DataSet Builder window You can either use its Wizard or construct the data set using Builder window directly We will explain how to use the latter First find the result files saved by ImaGene in the file browser window on the left hand side Drag and drop the control data file under the Control panel and the experimental data file under the Experiment panel and wait until GeneSight DataSet Builder 15 x File Tools Help y o e 2 Cancel Done Save Sot All Remove Selected Preview Help Select files on the leftto add to the DataSet on the right Note This version of GS will give correct results only ifthere are no duplicate files in a DataSet Make copies if you intend to use that file multiple times CA c Experiment 4 Control Experiment Info File Era i E 1205_r bt i E 1205_g tet O Browsen jE LicenseAgreementtd El 2 Sample Data ob 6 1205 No handling Files are consistent v till i pE 1205_gbe CE a gt Pair Data Perform Ratio GG 17hr i B A 17hr_new ah Perform Ratio amp Add as Replicate i v HEM s ee Con i gt El Repeated Experimental Condi
54. regions of just 1 deletion are called This is expected to be a negative value 1 Amplification gt This is the log ratio threshold for which regions of more than one copy are called This is expected to be a positive value 2 Amplifications gt This is the log ratio threshold for which regions of two or more copies are called This is expected to be a positive value Default Chromosome Plot Bounds These are the minimum and maximum log ratio values displayed on the CGH plots Note that any value falling outside this range will be represented as a small x mark at the left or right edge of the plot 46 area These values have no effect on the call algorithm and are exclusively for display purposes only Note Please refer to the CGH Tutorial later in the manual for additional details and usage examples CGH Analysis Mode Settings Panel When the CGH Analysis is complete six new tabs appear in the image display panel The tabs containing plots allow users to print or save the information The tabs containing numerical data can be saved to tab delimited text files Normalized Data Overview Whole Genome Chromosome CGH Report Summary The Normalized Data panel is produced when the analysis is run and is identical to that produced in the Expression mode but includes the Base Pair and Chromosome annotations along with any custom annotation fields 47 Composite CGHSamole j CGHSampleAcys tif CGHSampleAcy3 tif seg
55. shaped segmentation does not appear for any spot A segmentation is considered to be donut shaped if there are ignored pixels completely surrounded by signal pixels and their median intensity is lower then signal s This option is available for auto segmentation only e Signal Percentages These parameters are a percentage of all intensity levels within the signal region The intensity ranges are raw values that do not include any statistical measurements such as mean and median The high percentage value can be set as high as 100 since the sample should contain the pixel with the highest intensity value If you want to filter out possible noise sources such as a speck of dust set this percentage at a smaller value like 95 This way the pixels associated 68 with a dust particle assuming they will be fluorescing at a high intensity level will be filtered out e Background Percentages These parameters are a percentage of all intensity levels within the background region Set them the same way as described for the signal percentages Press the Home key to set a slider to 0 Press the End key to set a slider to 100 Note An important difference between automatic and manual segmentation is the fact that under manual the parameters and their corresponding values are applied uniformly across all spots of the image Under automatic segmentation each spot is calculated independently usually generating a more accurate segmentation
56. signal values are present and the grid flexibility is set with a high number of pixels ImaGene will tend to find pixels with high expression values For full automation use this tool in conjunction with auto grid placement tool Note If this button is utilized several times spot searching will start from the position where the previous attempt left it Be careful multiple auto adjustment can dramatically deform grids on low quality images Wrangle The wrangle feature of ImaGene applies new stricter constraints to the results of spot localization without requiring further spot finding Essentially this allows users to reduce the spot search radius without re performing the spot finding The benefit of this feature is to assist processing for those with either slower computer hardware or for those with numerous spots A sample application would be to perform spot finding for grid geometry on an array image with a local flexibility set to a large number of pixels After spot finding if the resulting circle placement has high variability the Local Flexibility can be reduced After reducing Local Flexibility click the 101 Wrangle Button to apply the new setting without waiting for spot finding to be performed again 102 Part 2 ImaGene Tutorials 2 1 Basic Analysis This tutorial demonstrates performing basic quantification using ImaGene The tutorial covers the essential steps from loading the initial array images until a re
57. support The following Lessons will be covered e Image Selection Describes how to load an image into ImaGene and use the controls on the Image tab to enhance the on screen display of the image e Template Selection Demonstrates the loading and placement of BioDiscovery pre made templates 120 Quantifying Pre Processing Pre Analyzing and Saving the Data Describes the quantification and subsequent output of the pre processed and pre analyzed data Loading GAL GEML or MAGE templates Image Selection l 2 Launch ImaGene from the desktop icon or from the ImaGene folder within the Windows Start Menu Click on Load Images icon under the Main tab ale Images Grid Gene Info Process Browse to the Samples folder within the ImaGene folder The location is typically C ImaGene SampleData Select Rat_Agilent tif from the Agilent folder Click the Open Button to complete the image loading A progress bar displays time remaining 121 File Fak ef Sadin Ato Hararo Teoh Hele hk 5 com fe 9a e omen tains preie Composite 0 Fest Agient tf 1_flat_Aglent ti ajajajaja oe ae Enea Fic Chores Bradt Preana mages o Pa en Le saai Brighhness _ _ _ _ _ oooO Conbrast S D heves Depi Colors ij Select both the images in the Main tab and click on the Auto align images button above I z alja r al d y A l aL Unga Align Im
58. text file Such file can contain a column of multiple gene IDs one gene ID per line Once loaded these gene IDs will be assigned to their corresponding control type This feature is useful for bringing a list of selected genes from some alien program GeneSight for instance and analyze corresponding spots on microarray images There is a maximum limit of 500 unique gene names for a given control type To highlight all the spots on the image belonging to the specified control types click Update Visual This action will also show the control violations If the image fails the controls test a corresponding warning box will pop up Control type s for their corresponding spots will be shown in the results table in a column 79 Note Control spots play important role in automated grid finding procedure By specifying all spots that are not meant to contain any signal negative controls within a control type named BLANK or EMPTY you can help ImaGene place grid automatically with higher success rate Also if you want ImaGene to base its grid placing procedure on bright control spots only you can create a control type BRIGHT and auto grid finding will look for brightest spots on the image to match them to the dedicated bright control spots from BRIGHT type Note Control spots can be used during background correction and normalization During background correction you can use dedicated negative control sp
59. the following are valid formats for the Gene ID File Header information MR MC SR SC Gene ID Annotation1 Annotatio2 Note You can optionally add Annotation columns after the Gene ID column The number of Annotation columns is not limited 183 Gene ID Format 2 ImaGene Format The second format follows the same rules as the first except for the addition of a field column Columns Field Metarow Number MR Metacolumn Number MC Subgrid Row Number SR Subgrid Column Number SC Gene ID Annotation Annotation2 Header Information Field MR MC SR SC Gene ID Annotation Annotatio2 Note You can add optional Annotation columns after the Gene ID column The number of Annotation columns is not limited 5 1 2 Gene ID File CGH and Expression Module The format and content for this Gene ID file is required for CGH analysis but may be used for Expression analysis The new format is a also a tab delimited text file that may look as follows Metarow Metacol Row col To Chromosome Base Pair 1 1 RPLI 1Le4H12 41561116 J 2 RFP11 185m20 2 15197943 1 1 L 5 RPLI 40N8 5 fe f4446 1 1 E 4 RPLI f4e3 20 45337121 It must contain a Header Row with the following required text strings headers in any order Metarow Metacol Row Col ID Chromosome and Base Pair Optional headers include Field as in earlier versions of ImaGene and the new column Color wh
60. v1 0 xml http www rosettabio com tech geml default htm e MAGE ML xml format http www mged org e Export template as GAL GEML or MAGE Displays the Export Template dialog box This is utilized to save a displayed template to a template file in Array Lists gal GEML v1 0 xml or MAGE ML xml format Selection e Adjust Metagrid Utilized to select and move an entire metagrid e Adjust Subgrid Utilized to select and move an individual portion of the metagrid subgrid e Adjust Spot Utilized to select and move one spot in a metagrid e Lasso Adjust Utilized to select and move a specific free form area of the metagrid e Rectangle Adjust Utilized to select and move a specific rectangular area of the metagrid 10 Auto Auto Align Images Aligns the displayed images automatically if they are misaligned Auto Adjust Grids Automatically places the displayed grids globally onto the image spots Auto Adjust Spots Automatically adjusts each spot to better align them with the corresponding image Wrangle Enforces new local spot flexibility parameters This essentially reduces the distance used in spot finding without requiring spot finding to be reapplied Measure Tools Make Measurements Quantifies the image spots to generate the intensity values for signals and backgrounds as well as quality control results such as flags Analyze Applies data analysis and runs the ch
61. 09 494979954 eeeeceteceteaece ses 098066608 000868008686066 eeceeteeesecesetars O8esCeeqgtetgageeageas Seaceseesonggeesese Seteceeseseoegag gee J Setegaeteqeseseeqgeges FPS Rg Qrgraggs 24454 Teter emesis bi ahha eseCedecesestceqegas 88000 64009060 CLOT I eteeoteceteaeatecace ELLELE ELLI LLIE T T L LEL LLILLLCLCLL ICE E TIL DOCOCOCOS 8668665660065 Dii E ts 299492954554 geteteta ELLE E E LLLE IS Sseovetetececeeosece eteqgnesgaeeeceeeeece eeeettaetaegeecesescese tel tele tel Clete Le tata Reg eet 222 844949429 Rgeer en eh fs gee ge Hees T i 2242542055 45884 222424592492 2945954 26088490669 0459860000 eteeegteqseeseggges q 24 gee eae ng ee LLT ede LI LLE LL a9299900682009994 e5 oooo Cobo CoOSeetbaebeseea SeeCeseese Cesgaeeeeseeg 992056299 i a i k a ak e E eL LE LLIE SSsetceteeceaesastaestet 20264 6994966260956 6656 8666945664646 ee06eC88 666666600 6996609966 9429074922456 8059446244 bebi LL E LE LILL LI E LL akini Jo ini i io i i E io E Di iai elaia bonlat bola boaimiia 1i im To Eo ia Ei 9024 424 44945944949 44 wkk hi 45208457445 24424 SF 4441S S FH OHA ESS 2070900 6662484e2224424e Oooc0cococ 10L LLT TTILE ieis oldn eii E het eRe pE LLLE r TLL LLCLET 262206909 486e00 89688 9962069608096 96 e000 eeneqgqeeteanrtesaaateare ese eC esses Ceseeseesn 6666860860066 660865 0666666660 66666 0006 S e enCee6Gte FE Be SSseeaecetesee8et e46 X Normalized Signal Mean Note any selection made usin
62. 1205_config_with_norm xml lt Configuration gt 190 lt Destination gt C ImaGene Sample Data 1205 Out_batch_norm lt Destination gt lt Channel gt 0 lt Channel gt lt ChannelName gt lt ChannelName gt lt SubstituteGridImage gt null lt SubstituteGridImage gt lt SubstituteGridChannel gt 0 lt SubstituteGridChannel gt lt AdjustGrid gt true lt AdjustGrid gt lt AdjustSpots gt true lt AdjustSpots gt lt AlignImages gt false lt AlignImages gt lt Normalize gt true lt Normalize gt lt Analyze gt false lt Analyze gt lt Entry gt lt Batch gt The file above contains options that you can recognize from working with Batch Editor like Image Channel Configuration Template etc where you specify locations of all resources needed for processing an image Also you can notice that some other features were added in ImaGene 6 0 O lt AlignImages gt specify whether to align images if a batch entry contains two images parameters Image2 and Channel2 should be utilized for this purpose see below Default value true lt AdjustGrid gt specify whether to automatically adjust grid for the current image Default value true lt AdjustSpots gt specify whether to automatically adjust spots for the current image Default value true lt SubstituteGridImage gt specify an image name here from which to take adjusted grid and put it on top of the current image The image which will provide the subst
63. 2217 0 04580457 _ UNCHANGED peti 44762 D49133 5 77912520 28778958 UNCHANGED RP11 _628743 10 1463 0 78819480 93449837 UNCHANGED RPI1 7 f44390 0 43217 fI 79744851 3652742 RP11 14_ 344048 10 5366 0 66396 0 12732777 UNCHANGED RP11 f07639 0 3137 0 51408 0 20036542 UNCHANGED RP11 11 _ 815047 0 53213 D 5807904 0 04865569 UNCHANGED RPI1 161802 0 42773 0 16081 0 58654544 UNCHANGED _ Expression Analysis Results Panel 45 ae ImaGene Parameter Settings ioj x Spot Finding Seamentation Quality Flags Measurements Normalization Analysis Controls and Alerts Image Preferences Measure Normalized Signal Mean Omit Flagged Spots Reference Channel Channel 4 f Channel 2 Organism Human ar Analysis type os Expression ie CGH CIGH Parameters Significance Threshold o 000 2 Deletions 1 3 Detaut Chromosome Plot Bounds 1 Deletion 1 D 2 0 eX lt 20 1 Ampliticatian jos 2 Amplitications fi All Load Save 4s Close The required CGH Parameters include the following Significance Threshold This is a p value below 1 related to the probability that a region is an amplification or deletion based on the levels chosen 2 Deletions lt This is the log ratio threshold for which regions of two deletions are called This is expected to be a negative value 1 Deletion lt This is the log ratio threshold for which
64. 22323107719421387 00 RP11 16P4 34053907 4997291564941406 0 4997291564941 406 0O RPW 1128h 38412432 0 2555885314941 406 0 2555665314941406 0O RP11 1146G4 51044501 0 6169955730438252 0 6169955730438232 00 RP41 747H31 54718902 0 29855295995971 68 0 2965529899597168 00 RP11 24605 56884053 0 24956512451171875 02495651 2451171875 00 RP11 69P12 A 99238716 0 095231294631 95801 0 095231 29463195601 00 RP11 613L21 02517234 25427746772766113 0 25427746772766113 DO RP11 24J14 07639467 0 07250690460205078 0 07250690460205078 00 RP11 355G24 07852158 3029269245605469 0 3029269245605469 DO RP11 252A16 08159981 0 27843761444091797 0 27643761444091797 00 RP11 60513 54325812 0 7407321929931 641 0740732192993164 0 0 RP 11064 N23 63025013 3 4547557830610547 2 0 RP11 1402D8 63548394 0 0229845046997 0703 0 072986450469970703 00 RP11 S1Ng 62246372 0 37342333793640137 0 37342333793640137 00 RP11 80M24 95477228 1 35842486706543 1 0 RP11 288613 202146767 0 383756160736084 0 363756160736084 00 RP11 1123E2 204192811 1 4044792652130127 1 0 RP11 142G24 216148774 0 9657568740844727 D RP 41 1146 216258282 085987091 06445312 0 085987091 06445312 D0 RP11 42C8 216557598 0 6951 8947604 31836 0 6951694760131636 0 0 RP11 1 24 24 217175606 1678229187561035 1 1678225187561035 20 RP41 556M16 218473004 0 34867191314697 266 0 34867191314697266 00 RP441 76K24 219911336 2375087 7380371094 C 237S087 7380371094 1 0 RP11 52F17 4 224523598 L0 07935738563537598 0 0793573856353759
65. 54451 SPS pox Aa A n fo 2 RPeMAaakKg fs 5959354 0 9 382862 9 4900 A Mo f BR RPtt85M20 2 f5197943 84S A A n 2 2 RPN 26M6 f4 64404822 D 9247893 9 242 A M A BR B RP1NAS7N24 9028135 0 9 020368 8 983 A n n 2 4 Reit te2ci9 6 mieezsoea D 9306953 C8 A Ao A BR 6 RP184BG B 7724919 8834726 8 793 A of of 2 B RPemaBeerS B 13307084 O 10176981 0 04 A Mo A BR p RPmaBsB11 6 f50687195 8 282149 8 6137 A A A BR B RPN 7Fe fO 92575444 D 9860801 9 937 A M f BR B RPt1 186B12 0 rz A Mo f R fo RP1 252c8 f2 f02561524 BIDE A Ao A BR m RPB 5 p3621470 D B97145432 Peme Af n R f2 Remeg BO B51mees O 6787525 8 8067 a A A BR f3 RPN 212P7 PF f27674031 O 0 067266 0 051 A Mo a R f4 RP1 2901 0 p4613e0 O 6788827 8 966 A A A R fs RPm 21He f3 602119277 O 6433919 5 081 R RP11 15RMQ n 70311918 _ RRRORAT R71 mM lt Mm gt Meta Row T The Overview panel displays the organism s chromosomes depending on the organism selected from the drop down list in the Analysis tab with amplification deletion calls marked as shown Amplifications are marked by a green line to the right of the ideogram and deletions are shown as red marks on the left side of the ideogram By clicking on any of the ideograms the chromosome will be 48 selected and the display will change to the single Chromosome view described later in this section Composite HB19K 17 43_532 tif HB19K 17
66. 7 956 6 633 659 6 674 882 6 990 221 6 731 781 6 990 221 and 7 099 502 and additional pending applications IMAGENE ose hie ceckceicd cu ciesi cect doe cies ccclcisd E NA E E EA clei 1 COPY RIGEEE NOTICE coiii E 2 TRADEMARK Secre e AEE EEE R 2 PATENI S coina a ee ee as 2 INTRODUCTION TO IMAGENE oe cpososks cosas teccscottete secatecevaicseeeusticecchcntescouseatecssanesyeessbiveccees 6 OUTOKS TAR PG UDR riiin Na A OA EOE EAE aS 7 PART 1 LAB USE RSG UBD E iesccecccccccscedsstesvadecovsssevsccevccssssavessesuecots SEERP cosstessecsavssvansasuseecetes 8 1 1 IMAGENE MAIN WINDOW OVERVIEW ssissestavsdevatecsnncsienssatasesennentanddaradeganneaientialanssunnnaiets 8 I ZAC ON TROL ABS ss csctaccnsatcneaevacuatzateddae neuen a dabindennetanlansanvebagues selanteabinuntdaus 15 De Del Mah TAD as costes era ctaues E saute ereedsd E T edad cotedad sateke N 15 122 PP PEVICW TIROW Data 14b seieren r ia e a EA AE A AET 29 RAS Normal zaton Tab enra sienen E A A E tents 39 1 2 4 Analysis Tab Expression and CGH Modules c cccccccccccccccssseseeseeceeeeanesesseceeeeaas 43 T IMAGE BE 21 y We 5 repent een eer tar Re er rn ne 55 1 4 IMAGENE PARAMETER SETTINGS WINDOW sseeeeecceececeeeceeeeeseeeeennnaaeeeeeeeeeeeeees 64 LAIMA GENE WIZARD oroni tate E E deel aul weawiducaeneh gua E EAEE 82 1 6 BATCH EDITOR AND BATCH PROCESSING Tara aeo aa E E 84 1 04 The Main Baich Editor Wind Wsurarirern a A ROAR 84 160 2 Create Batch Enty WINGOW renier
67. 8 DO 4 alue 0 29855298999597168 Save Report GH Report Summary all The Summary panel shows a view of all the chromosomes individually shown in the Chromosome Panel 53 1 3 Image Display Panel This panel displays loaded images A tab appears along the top of the panel for all current loaded images Click on a tab to display the corresponding image The Composite tab displays a false color overlay for all loaded images You can use this tab to overlay multiple images prior to analysis The number of images that ImaGene can load is limited based upon the computer hardware specifications Composite Samplea Ait SampleB tit All image manipulation tools such as Zoom and Rotate can be applied within the image panel Once zoomed into a region of the image scroll bars become available along the sides of the panel Note There is a Zoom Slide bar located at the bottom of the panel Move it to the left to zoom out of the image or move it to the right to zoom into an image 55 Segmentation Tabs Once the image s have been quantified additional tabs one for each image plus one for plots will appear along the top of the image display panel The segmentation tabs with the name of the image file name followed by seg displays the segmentation as it had been performed for the given image From this view you have the ability to see the macro view of the image and analyze any large defects an
68. 912 Gerei02 i4014 O Geeii2p 14016 0 6 GeretU2 0 16 42 GenetU2 d 16 04 Gerei02 1 16 06 Genet 2 4 18 02 Geret02 d 18 04 Geret02 1 18 08 Selected rows 1 Right click to freeze selection 17910421 bo 2237 1a BTA Ds 04252 Save Epoo ciose Also located within the data output directory is a file called log txt This file contains start and stop times for any or all the batches It also contains any errors that may have occurred Begin Batch Fri Aug 20 16 20 02 PDT 2004 Begin Run Info Info Info Info Info End Run Begin Run Info Info Info Info Info End Run End Batch Fri Aug 20 16 21 41 PDT 2004 current image C TEMP Batch SampleA tif channel name of Empty Spots 0 of Poor Spots 51 of Negative Spots 7 current image C TEMP Batch SampleB tif channel name of Empty Spots 0 of Poor Spots 60 of Negative Spots 24 151 Batch processing is an easy and flexible way to provide image analysis in a high throughput environment or walk away convenience for overnight processing The previous example demonstrates the steps required to perform a batch The overall success of the batch depends on a number of factors including the quality of the images the accuracy of the settings and the uniformity of the images to one another 152 Part 3 GeneSight Lite 3 1 What is GeneSight Lite After intensity measurements are extracted from
69. A BATCH PROCESSING accusa a E E T 144 24 4 Preparing for batch Processing sisisi iena ie i i 144 DAZ Addn FEniries tog Bathie EEA E A E E 146 DAD POS Balch PLOCCS SIO orien eE AE EA A 149 PARES GENE SIGH Da TE neiaa O aO AAE a 153 Sak WHATS OGENESICGHI LITE aeri r a AATE E A A E OE 153 32 HOW TO USE GENE SIGHT ATE areari r A A A TEA Na 153 PART 4 INSTALLATION AND LICENSING esseeeeecccsssecccccccsccccccccccssecccccccsseccecccsssceceee 159 Al IMAGENE INSTALLATION aiea E E E E 159 Lld WARAOWS aaeain e a EE O 159 gba AGRAG CHO TIPO ALE oai e ET a E ES 159 4 3 Un Instalaton TNSTUCUONS riena a a a E Res 161 LTM O OSN Sa ches Gute asta ten ie Teak sacs E A Genome une stanton N E A 161 LES 6 7 6 ae eae ON OCR RAEN ORY 8 eT RTE TNT ERT SPRINT we RT Rr PT Ree ER Oe RT eRe T 161 A LICENSE MANAGEMEN T iarann aee ee Ven sessed AONNE A teal ONO 162 ALA TAC CNS CO M ONI TE aE E T EAEE NR TO ENOTNE 162 ALa Nde LOCKE TACENSES rrene e RAA O E T EEN OA ONEA 162 BD IT OOUE LACCNSOS irais e KEA EE NOE E OENE 165 4 3 FLEXNET NODELOCKED INSTALLATION GUIDE FOR WINDOWS sssecsscccscerrserreerrseeees 167 4 4 FLEXNET SERVER INSTALLATION GUIDE FOR WINDOWS scccceseceeseceeeceeeeceeeees 174 4 5 FLEXNET CLIENT INSTALLATION GUIDE ALL PLATFORMS ccccssecccesseceeesecsseeeeeees 180 PARIS APPENDICES aiora EON 182 gt TEE SPECIBICA TIONS S araa E A A EAA E 182 led GeneID FEE Pres S onModnle rennen a E E A E EOK 182 5 1 2 Gene ID File CGH and
70. DMENTS No amendments or other modifications to this Agreement may be made except by a writing signed by Licensor and Licensee 18 U S GOVERNMENT RIGHTS If Licensee is the U S Government or if Licensee is a contractor or subcontractor at any tier of the U S Government and is licensing the Software for use by the U S Government or in connection with any contract or 199 other transaction with the U S Government Licensee acknowledges that by accepting delivery of the Software the U S Government agrees that the Software qualifies as commercial computer software and that the accompanying documentation qualifies as commercial computer software documentation within the meaning of the acquisition regulations and contract clauses applicable to this procurement The terms and conditions of this Agreement are fully and exclusively applicable to the Government s use and disclosure of the Software and accompany documentation and shall supersede any conflicting terms or conditions No license of any kind is granted in the case of acquisitions which contain or are subject to the clauses FAR 52 227 19 COMMERCIAL COMPUTER SOFTWARE RESTRICTED RIGHTS JUNE 1987 or DFARS 252 227 7013 RIGHTS IN TECHNICAL DATA AND COMPUTER SOFTWARE OCT 1988 or any other clause which purports to grant to the U S Government rights greater than or additional to those set forth in this Agreement or which purports to impose additional requirements upon Licensor to make thi
71. EMP BatchiSampleB2 tit Grid CATEMP Batch template tpl Gene ID CTEMP Batch template tpl Configuration CATEMP BatchiSample AB xml Output Directory CATEMP The required parameters include 88 Images The images to be included with the batch and to be associated with the Grid file Typically while the hybridization information about the images may be unique the geometry of the arrays 1s the same If this is the case then multiple images can be loaded here Grid The grid file that matches the images to be batched A template file may also be used as this includes a grid If a template file is used then a gene ID file does not need to be specified Note A template file contains the grid as well as the corresponding gene ID info The grid file does not contain gene IDs Gene ID The Gene ID file is utilized to track information about the genetic material spotted at each location within the array This information will be saved along with the quantified values in the output text file and visualization tools Please see section 5 1 for the proper file specification for an ImaGene gene ID file Configuration The configuration file contains information about the parameters and their corresponding values used during analysis within ImaGene Common examples of stored parameters include local flexibility for spot finding and the desired quality measures for the output text file The file itself is an XML file that can be sa
72. GRID gt true lt ENFORCE_GRID gt lt STANDARD_CHECK3 gt false lt STANDARD_CHECK gt lt CONTROL_TYPE_VARTHRESH_0 gt 10 0 lt CONTROL_TYPE_VARTHRESH_0O gt lt CONTROL_TYPE_ID_2_1 gt HOT lt CONTROL_TYPE_ID_2_1 gt lt STANDARD_HIGH gt 10000 0 lt STANDARD_HIGH gt lt PERCENT gt 50 median lt PERCENT gt lt MEASURE_MEDIAN gt true lt MEASURE_MEDIAN gt lt SLIDING_WINDOW3false lt SLIDING_WINDOW gt lt BACKGROUND_THRESH gt 0 9995 lt BACKGROUND_THRESH gt lt CHANNELI gt false lt CHANNELI gt lt PERIM_TO_AREA gt true lt PERIM_TO_AREA gt lt BLANK_HIGH gt 1000 0 lt BLANK_HIGH gt lt CONTROL_SPOT_LIST gt No control spots defined lt CONTROL_SPOT_LIST gt lt DEFAULT_RESOLUTION gt 10 0 lt DEFAULT_RESOLUTION gt lt GENES_ALL gt false lt GENES_ALL gt lt CONTROL_TYPE_VARTHRESH_1 gt 10 0 lt CONTROL_TYPE_VARTHRESH_1 gt lt PERCENTILE_CONTROL_SPOT_LIST gt No control spots defined lt PERCENTILE_CONTROL_SPOT_LIST gt lt LOWESS_CONTROL_SPOT_LIST gt No control spots defined lt LOWESS_CONTROL_SPOT_LIST gt lt CONTROL_TYPE_N gt 0 lt CONTROL_TYPE_N gt lt REGULARITY_FLAG gt true lt REGULARITY_FLAG gt lt REGULARITY_THRESH gt 0 6 lt REGULARITY_THRESH gt lt BKGD gt Local lt BKGD gt lt CONTROL_TYPE_ID_N_0 gt 1 lt CONTROL_TYPE_ID_N_0 gt lt OFFSET_THRESH gt 60 0 lt OFFSET_THRESH gt lt STANDARD_CV gt 5 0 lt STANDARD_CV gt lt CONTROL_TYPE_LOWALERTTHRESH_0 gt 0 0 lt CONTROL_TYPE_LOWALERTTHRESH_0O gt lt SCOPE gt SubGrid lt SC
73. HE DEFECTIVE MEDIA CONTAINING THE SOFTWARE AND THE DOCUMENTATION DURING THE ABOVE WARRANTY PERIOD EXCEPT AND TO THE EXTENT EXPRESSLY PROVIDED ABOVE THE SOFTWARE AND DOCUMENTATION WHICH ARE THE SUBJECT OF THIS AGREEMENT ARE PROVIDED ON AN AS IS BASIS WITHOUT ANY WARRANTIES OF ANY KIND INCLUDING ANY AND ALL IMPLIED WARRANTIES OR CONDITIONS OF TITLE NONINFRINGEMENT MERCHANTABILITY OR FITNESS OR SUITABILITY FOR 197 ANY PARTICULAR PURPOSE WHETHER ALLEGED TO ARISE BY LAW BY REASON OF CUSTOM OR USAGE IN THE TRADE OR BY COURSE OF DEALING IN ADDITION LICENSOR EXPRESSLY DISCLAIMS ANY WARRANTY OR REPRESENTATION TO ANY PERSON OTHER THAN LICENSEE WITH RESPECT TO THE SOFTWARE AND DOCUMENTATION WHICH ARE THE SUBJECT OF THIS AGREEMENT LICENSEE ASSUMES THE ENTIRE LIABILITY FOR THE SELECTION AND USE OF THE SOFTWARE AND DOCUMENTATION AND LICENSOR SHALL HAVE NO LIABILITY FOR ANY ERRORS MALFUNCTIONS DEFECTS LOSS OF DATA OR ECONOMIC LOSS RESULTING FROM OR RELATED TO THE USE OF SOFTWARE AND OR DOCUMENTATION 9 LIMITATION OF LIABILITY Notwithstanding any other provision of this Agreement the cumulative liability of Licensor and or Licensor s suppliers distributors and or agents to Licensee or any other party for any loss or damages resulting from any claims demands or actions arising out of or relating to the Software the accompanying documentation and or this Agreement shall not exceed that license fee paid to Licensor by Licensee for the u
74. ImaGene User Manual Version 7 BioDiscovery Inc Copyright Notice 1997 2006 BioDiscovery Inc All Rights Reserved The ImaGene Users Manual was written at BioDiscovery Inc 2121 Rosecrans Ave Suite 3315 El Segundo CA 90245 Printed in the United States of America The software described in this book is furnished under a license agreement and may be used only in accordance with the terms of the agreement Every effort has been made to ensure the accuracy of this manual However BioDiscovery makes no warranties with respect to this documentation and disclaims any implied warranties of merchantability and fitness for a particular purpose BioDiscovery shall not be liable for any errors or for any incidental or consequential damages in connection with the furnishing performance or use of this manual or the examples herein The information within this manual is subject to change Trademarks GeneSight ImaGene GeneSight Lite GenePie GeneDirector and CloneTracker are registered trademarks of BioDiscovery Inc Windows Wordpad and Excel are either registered trademarks or trademarks of Microsoft Corporation in the United States and or other countries Other product names mentioned in this manual may be trademarks or registered trademarks of their respective companies and are the sole property of their respective manufacturers Patents This product is protected by U S Patent Nos 6 349 144 6 57
75. Licenses obtained by Licensee for the Software 2 RESTRICTIONS Licensee agrees that it will not assign sell sublicense transfer pledge lease rent or share the Software the accompanying documentation or Licensee s rights under this Agreement nor delegate any of Licensee s obligations under this Agreement Any attempted assignment sale sublicense transfer pledge lease rental or delegation shall be null and void Licensee shall not utilize the Software to provide image processing services directly to third parties for any compensation without first obtaining the express written consent of the Licensor Licensee shall not nor attempt to reverse engineer decompile disassemble modify reproduce reverse assemble reverse compile or otherwise translate the Software or any part thereof 3 LICENSOR S RIGHTS Licensee agrees and acknowledges that the Software and the accompanying documentation which are the subject of this Agreement are proprietary confidential and trade secret products of Licensor and or Licensor s suppliers and that Licensee shall undertake all necessary steps and efforts to prevent unlawful or illegal distribution of such proprietary confidential and trade secret information Licensee further acknowledges and agrees that all right title and interest in and to the Software and the accompanying documentation including associated intellectual property rights are and shall 195 remain with Licensor and or Licenso
76. ML and MAGE ML file A template is a grid file which contains gene ID information Abr Load Gene IDs Displays the Load Gene ID File dialog box Use this interface to select and open a gene ID txt file Auto Adjust Grid Automatically places the displayed grids globally onto the image spots Auto Adjust Spots Automatically adjusts each spot to better align them with the corresponding image Make Measurements Quantifies the image spots to generate the intensity values for signals and backgrounds as well as quality control results such as flags dtp ll aP Analyze Is activated upon generating measurements and results This will run the normalization and Analysis methods and configurations specified in the Settings Panel tabs of Normalization and Analysis Images Panel The images panel displays the names of images currently loaded within ImaGene Each loaded image is listed here as well as its corresponding Composite color The Composite Color is the color the image is displayed as when seen within the Composite Tab of the Main Image Panel 17 Images WS Sarmplea tif The Images Panel fully supports context menus and as a result provides the following menu choices Load Images De Remove Selected Images E Select Color O Invert Values L Hide CJ Flip Horizontal Rotate Image Info e Load Images Selection of images from the file system to load into ImaGene e Remove Selected Images The
77. Make sure that you select the option to install the FlexNet Service component during the software install Qlone Tracker 2 Setup j i X Select Optional Components To instal Penna eck ihe check box ers F the check box is clear that component all mot be metalled 167 c Remember the directory you ve selected for FlexNet during the install Note that C FlexNet is the default Select FLEXIm Folder x Please choose the installation Folder Directories Hs Local Disk C Sy 2125 pswek fy Agilent BioAnalyzer E H E BandLink H E Documents and Settings n GCOSJava off GD_script 2 6 H GeneDirector cei 3 Do you already have a license file called license lic a If yes then skip to step 6 b If no then proceed to step 4 4 Determine the server s Ethernet Address and HostName a Start the LMTools program by clicking on Start gt Program Files gt Product gt LMTools Click on the System Settings tab c You will see the following screen 168 LMTOOLS by Globetrotter Software http www globetrotter com lel xj Fie Edt Mode Help Sennce Licence File System Settings Unies Statt Steg Fieresd Server Status Server Diag Config Services Bonrerwing Time Sethngs System Tere zont Pacii Siandud Time GMT Time Tue Ape OS 1 Sd 2002 Difference Fron LACT s0 MSDOS Time fio 34 47 Local Time 10373657 FLEXID F Windows Directory CAWINNT
78. NS REPRESENTATIONS AND WARRANTIES INCLUDING ANY IMPLIED WARRANTY OF MERCHANTABILITY FITNESS FOR A PARTICULAR PURPOSE OR NON INFRINGEMENT ARE DISCLAIMED EXCEPT TO THE EXTENT THAT THESE DISCLAIMERS ARE HELD TO BE LEGALLY INVALID Limitation of Liability TO THE EXTENT NOT PROHIBITED BY APPLICABLE LAW IN NO EVENT WILL SUN OR ITS LICENSORS BE LIABLE FOR ANY LOST REVENUE PROFIT OR DATA OR FOR SPECIAL INDIRECT CONSEQUENTIAL INCIDENTAL OR PUNITIVE DAMAGES HOWEVER CAUSED AND REGARDLESS OF THE THEORY OF LIABILITY ARISING OUT OF OR RELATED TO THE USE OF OR INABILITY TO USE THE LICENSED SOFTWARE EVEN IF SUN HAS BEEN ADVISED OF THE POSSIBILITY OF SUCH DAMAGES In no event will Sun s liability to you whether in contract tort including negligence or otherwise exceed the amount paid by you for the Licensed Software under this Agreement The foregoing limitations will apply even if the above stated warranty fails of its essential purpose Termination This Agreement is effective until terminated You may terminate this Agreement at any time by destroying all copies of the Licensed Software This Agreement will terminate immediately without notice from Sun if you fail to comply with any provision of this Agreement Upon termination you must destroy all copies of the Licensed Software Rights and obligations under this Agreement that by their nature should survive will remain in effect after termination or 204 10 11
79. OPE gt lt COMBINE_REPL gt true lt COMBINE_REPL gt lt SUBGRID gt false lt SUBGRID gt lt CONTROL_TYPE_ID_1_1 gt STANDARD lt CONTROL_TYPE_ID_1_1 gt lt IGNORED_MEDIANS gt true lt IGNORED_MEDIAN gt 188 lt CONTROL_TYPE_LOWALERTTHRESH_2 gt 0 0 lt CONTROL_TYPE_LOWALERTTHRESH_2 gt lt FOLD gt 1 0 lt FOLD gt lt CONTROL_TYPE_IFCVALERT_1 gt false lt CONTROL_TYPE_IFCVALERT_1 gt lt CONTROL_TYPE_LOWALERTTHRESH_1 gt 0 0 lt CONTROL_TYPE_LOWALERTTHRESH_1 gt lt HOT_LOW gt 10000 0 lt HOT_LOW gt lt EMPTY_FLAG gt true lt EMPTY_FLAG gt lt CONTROL_TYPE_ID_2 0 gt _Pro lt CONTROL_TYPE_ID_2 0 gt lt CONTROL_TYPE_IFLOWALERT_2 gt false lt CONTROL_TYPE_IFLOWALERT_2 gt lt CONTROL_TYPE_IFUPALERT_0 gt false lt CONTROL_TYPE_ IFUPALERT_0 gt lt CONTROL_TYPE_IFCVALERT_0 gt false lt CONTROL_TYPE_IFCVALERT_0 gt lt CONTROL_TYPE_UPALERTTHRESH_2 gt 0 0 lt CONTROL_TYPE UPALERTTHRESH_2 gt lt POOR_THRESH gt 0 995 lt POOR_THRESH gt lt CONTROL_TYPE NAME 2 gt HOT lt CONTROL_TYPE_NAME_2 gt lt CONTROL_TYPE_ID_1_0 gt 1999 lt CONTROL_TYPE_ID_1_0 gt lt PERIMETER FLAG gt true lt PERIMETER FLAG gt lt BACKGROUND_WIDTH gt 5 0 lt BACKGROUND_WIDTH gt lt OFFSET gt true lt OFFSET gt lt CONTROL_TYPE_VARTHRESH_2 gt 0 0 lt CONTROL_TYPE_VARTHRESH_2 gt lt BKGD_MEASURE gt Bckgr Mean lt BKGD_MEASURE gt lt MEASURE_AREA gt true lt MEASURE_AREA gt lt LOG_ENABLED gt true lt LOG_ENABLED gt lt MEASURE_STDEV gt true lt MEASURE_STDEV gt l
80. SH_1 gt 500 0 lt CONTROL_TYPE_UPTHRESH_1 gt lt CONTROL_TYPE_LOWTHRESH_1 gt 1 0 lt CONTROL_TYPE_LOWTHRESH_1 gt lt CONTROL_TYPE COLOR_1 gt 16724992 lt CONTROL_TYPE COLOR_1 gt lt DONUT_FLAG gt true lt DONUT_FLAG gt lt EMPTY_THRESH gt 2 0 lt EMPTY_THRESH gt lt HOT_CHECK gt false lt HOT_CHECK gt lt FLEXIBILITY gt 50 0 lt FLEXIBILITY gt lt BLANK_LOW30 0 lt BLANK_LOW gt lt BACKGROUND_FLAG gt true lt BACKGROUND_FLAG gt lt CONTROL_TYPE_IFLOWALERT_1 gt false lt CONTROL_TYPE_IFLOWALERT_1 gt lt TYPE FOLD gt false lt TYPE_FOLD gt lt SIGNAL_HIGH gt 0 98 lt SIGNAL_HIGH gt lt MEASURE_MODE gt true lt MEASURE_ MODE gt lt NEGATIVE_ FLAG gt false lt NEGATIVE_FLAG gt lt IGNORED_AREA gt true lt IGNORED_AREA gt lt POOR_FLAG gt false lt POOR_FLAG gt lt PERIMETERTOAREA_ THRESH gt 0 7 lt PERIMETERTOAREA THRESH gt lt CONTROL_TYPE_UPTHRESH_0 gt 100000 0 lt CONTROL_TYPE_UPTHRESH_0 gt lt CONTROL_TYPE_COLOR_2 gt 65536 lt CONTROL_TYPE_COLOR_2 gt lt CONTROL_TYPE_UPALERTTHRESH_0 gt 0 0 lt CONTROL_TYPE_ UPALERTTHRESH_0 gt lt IGNORED_FLAG gt true lt IGNORED_FLAG gt lt TYPE_ERROR gt false lt TYPE_ERROR gt lt CONTROL_TYPE_UPALERTTHRESH_1 gt 0 0 lt CONTROL_TYPE_ UPALERTTHRESH_1 gt lt POOR_AND gt false lt POOR_AND gt lt CONTROL_TYPE_IFCVALERT_2 gt false lt CONTROL_TYPE_IFCVALERT_2 gt lt BACKGROUND_HIGH gt 1 0 lt BACKGROUND_HIGH gt lt CONTROL_TYPE_ID_0_0 gt 2000 lt CONTROL_TYPE_ID_0_0 gt lt STANDARD_LOW gt
81. SubGrid Using control spots se all spots T For Lowess normalization not only a scope of Global or Subgrid has to be set a smoothing factor has to be specified as well A smoothing factor determines how many neighboring data points will be used for the normalization of each data point The data is Normalized when the Analysis button is selected in the tool bar or Measurement Menu After the analysis process is complete a Normalized Data tab appears in the Image Display panel along with the Analysis Results Panel and for CGH analysis the respective CGH report and result plots The normalized data can be saved as a text file by clicking on the Save Report icon at the bottom 41 pepe pelure em fa E E ce E e E E a a T E E 1 R e RPIi te5m20 2 f5197943 7591S pssszas 1o B RPMs 5 B72744465 O 6844778 9800584 A Af hw _Fett 2raes S e a T 0 071046 ao n Ao fo o 6 Remas FSBOs 0 f1oosozre f13 927979 ao of no fao B Renaz fe pasom D 9 071795 ao no o no o fno RPt79G7_ B po560442 O 897433 0 227888 A of Ao fo B RPn59215 fo poesrae D 6896872 9132032 a A A fo B Rema 22287436 45288 15 655639 a A fA tO RP11 663P13 Wo p9593395 D fl4407039 15 235259 so A Ao o poo omo RPA 5 743871 2 B86874645 8702667 ao a Ao ofo oaa Reeg oo faas2630 D fh1 098706 12 369519 so n Ao fo A RPA 5 O proest O 8424718 9031169 a a Ao fo fa Rensscs to fpiesosose A f12 647262
82. T E E z Normalized Signal Mean ch1 log x axis Y axis Signal Mean ch1 x Normalized Signal Mean ch2 x Normalized If multiple images are loaded the user can plot not only two measurements within one channel but also two measurements belonging to different channels For instance plot of signal means can show the difference between fluorescence characteristics in two channels or even 59 can give a rough idea about regulation of the genes Selection of points on the scatter plot will result in the selection of corresponding rows in the results table and highlighting of selected spots on the image Note More spots can be added to the current selection by simply continuing to select other groups of spots To start a new Selection right click on the plot to clear the previous selection M A Plot M A plots can show the intensity dependent ratio of raw microarray data The plot uses M as the y axis and A as the x axis where M Log R G and A 2 Log R G This plot is utilized to check how good the normalization method is Ideally if the normalized data is intensity independent M A plot will show the data points distributed along the horizontal zero line Blue color For this reason the Normalized box should be checked Choose the plot type M A Plot v M A Plot 60 Box Plot This type of plot is utilized to visualize the measurement s distribution between different categories of spots belongi
83. a failed 0 if the spot passed perimeter to area test 1 if it did not see section 1 4 Offset failed O if the spot passed offset test 1 if it did not see section 1 4 Empty spot 1 if the spot was qualified as empty O if it was not see section 1 4 Negative spot 1 if the spot was qualified as negative O if it was not see section 1 4 Saturated spot 1 if saturation was detected 0 if no saturation was found for the spot see section 1 4 36 Some of the measures can be excluded from or added to the table at any moment through Measurements panel of Settings dialog box Individual spots can be selected for review either by selecting the row from within the Quantification Table or by selecting the spot within the image If the spot is selected from the image the Quantification table will automatically scroll to the proper location and the corresponding spot row will be highlighted Notice also that the Segmentation Preview automatically updates and displays the segmentation information for the selected spot Save Once the data has been quantified and results are satisfactory the final step is to Save the data ImaGene will save the data to common tab delimited text files which can easily be opened in other programs such as Microsoft Excel or text editors ImaGene will save each image s data to a separate file and will automatically name the file based upon the image name Export to XML Before saving the d
84. ading and Manipulation Describes how to load an image into ImaGene and use the controls on the Image tab to enhance the on screen display of the image e Grid Placement and Manipulation Describes how to create a grid and adjust it to fit the image properly e Quantifying Normalizing Image or Image Pairs Analyzing paired images and Saving the Data Describes the quantification analysis and subsequent output of the CGH data CGH Overview Array CGH is a powerful method for detection of regions of chromosomal gain or amplification and loss homozygot and hetrozygot deletion The 131 basic principal behind array CGH is to label genomic DNA from a sample under study and hybridize this onto an array Typically a different label is used for a control DNA sample The labeled extracts are then competitively hybridized on an array where each spot on the array each probe represents a particular region on the chromosome The probes can be either BACs or oligos ImaGene can process either platform The CGH analysis is different from expression analysis in one crucial aspect The location of the probes on the genome is an important aspect of the analysis and is therefore taken into consideration during the analysis The figure below depicts how probes are mapped to specific locations on the genome In ImaGene 7 you specify this mapping information in the GeneID file As described in Section 5 1 2 each row of he GeneID file correspon
85. ager service every time Below is an example of a node locked computer being both a license server and a client This diagram is also a good reference since it shows the file names for each part of the license 163 FlexNet Node Locked Dataflow Server Candi Daemon License Daemon Report File biod exe Log report log license lic License Manager Daemon imgrd exe Send Rev Licensed Client Application ImaGene Read Only Local License File local lic 164 4 2 3 Floating Licenses A floating license allows BioDiscovery products to run on a set number of different computers with the limitation that no more than a certain number of computers can run the software simultaneously For example a 5 seat floating license server would allow up to 5 computers access to a particular product The user may install the software on as many computers as she wishes but only 5 can run the software at one given time FlexNet provides distinct advantages over other licensing systems in that it can track license usage at the current moment or over an extended period of time This allows system administrators to track who is checking out licenses most often and if additional licenses need to be purchased While less common the floating license scheme is actually easier to set up than the node locked scheme since only one server needs to be set up Each of the client computers only needs to be told where th
86. ages 2 Main Preview Normalization Anatysis Composite Red_Fl_Agdentif Green_Rat_Agilenbi als GS es led Ra Agihan it Green_Fai_Agilanttit Gane lD Not Salactad Pevaires Display Cols pig Display Control Cheer Al Acted idl Adius oat 122 Images Grid Gene Info Process Template Selection 1 Click on the Load Template button in the Main tab See ll i eacaraea Images Grid Gene Info Process 2 Browse to the Agilent folder and select the template tp template Pile dt Grid Selection Ants Mewoe Took Bek 3 3 ra Fo 33a fe lt a r Ne ts unos Pat Prive E E bf 1_fut_Aghert nf Genel Dilhi D Gee hack BeLa sat 3 From the File menu select Settings 4 In the settings window click on Load and select open the settings xml file from the Agilent folder Close the settings window 123 NOTE I maGene version 7 runs Orange packing segmentation algorithm automatically when staggered array designs are used This can be seen from the preview panel where the selected feature is diamond shaped In ImaGene 6 if the array is a high density one e g Agilent 44k array make sure to check the box for Orange Packing Segmentation in the Segmentation tab in the settings window Be ImaGene Parameter Settings oO x Image Preferences Spot Finding Segmentation Quality Flags Measurements Fa 7 Backgrou
87. alf of spot pixels have their intensity at the level of maximum image intensity Note The user can use any combination of the above quality measures with arbitrary thresholds However the quality parameters dialog has a Default button for a default configuration for the thresholds Whenever at least one 73 of selected criteria fails the spot is highlighted with mark on image and receives flag value 3 in the results table Note Multi Channel Flagging button can be utilized to change the logic for combining automated flags between multiple channels For all three types of automated flags Empty Poor and Negative a flag can be assigned to a spot if it is flagged in at least one channel or in all channels e Manual flagging Utilized to manually flag spots for the following reasons 1 No particular reason code 1 X mark 2 Empty spot code 5 X mark 3 Poor spot code 6 mark 4 Negative spot code 7 mark The manual flag can be placed using a Tag Spots tool from the tool bar 4 Click on the Tag Spots button then right click on the image to select a type of flag Place flags by clicking on the corresponding spots The manual flag marks will be red in color Flagging Logic The unusual spots are flagged with the following logic x Flag the spot if channels are Empty Flag Allflagged At least one flagged Foor Flag Allflagged f Atleast one flagged Negative F
88. alue threshold adjuster sliders located beneath the image panel the scanned slide images can be lightened and darkened in the main panel views The tool is designed solely to enhance viewing of the image and does not affect the quantified values generated by ImaGene When adjustments are made to these sliders they are applied only to the Image s selected within the Images Panel Contrast SS The specific elements of the Display Control and their roles are defined below Brightness e Brightness Adjuster Top Slider Sets the Maximum intensity value threshold to be displayed Moving the slider to the right will decrease the threshold and increase the saturated appearance This does not alter the true pixel values of the selected image s e Contrast Adjuster Bottom slider Moving the bar all the way to the left corresponds to a linear brightness scale e g if pixel 1 has value 100 and pixel 2 has value 1000 pixel 2 will be 10 times brighter up to the saturation point As the slider bar is moved to the right the brightness scale changes nonlinearly faster for smaller values slower for larger values so a pixel value of 100 may have brightness of 80 and a pixel value of 1000 might have a brightness of 110 for example Bu Auto Adjust Contrast Brightness Clicking on this icon automatically adjusts the contrast brightness of the images to have the optimal settings for display Zi Map View Display The Map Vi
89. anted reasonable attorney fees and costs 14 SEVERABILITY If any provision of this Agreement shall be held illegal unenforceable or in conflict with any law of a federal state or local government having jurisdiction over this Agreement the validity of the remaining portions or provisions hereof shall not be affected thereby 15 NO WAIVER The failure of either party to enforce any rights granted hereunder or to take action against the other party in the event of any breach hereunder shall not be deemed a waiver by that party as to subsequent enforcement of rights or subsequent actions in the event of future breaches 16 ENTIRE AGREEMENT Licensee acknowledges that it has read this Agreement understands it and agrees to be bound by its terms This Agreement and any modifications made pursuant to it constitutes the complete and exclusive written expression of all terms of the Agreement between Licensor and Licensee and supersedes all prior or contemporaneous proposals understandings representations conditions warranties covenants and all other communications between Licensor and Licensee relating to the subject matter of this Agreement whether oral or written This Agreement may not in any way be explained or supplemented by a prior or existing course of dealing between Licensor and Licensee by any usage of trade or custom or by any prior performance between Licensor and Licensee pursuant to this Agreement or otherwise 17 AMEN
90. arch and non diagnostic use and solely in accordance with the terms and conditions of this Agreement Licensor and its suppliers reserve all other rights and the Software may not be used in any manner other than as provided in this Agreement 21 UPDATING Licensee acknowledges and agrees that the Software may communicate via the Internet or other communications systems with Licensor s computer systems for the purpose of checking the status of the License checking the status of maintenance for the Software and or determining if any fixes updates and or upgrades to the Software are available Licensee agrees and consents to any such communications between the Software and Licensor s computer systems and the transfer of data between the Software and 200 Licensor s computer system Licensee also agrees and consents to if applicable remote configuration of the Software on Licensee s computer systems Licensee acknowledges and agrees that the Software permits downloading of Software fixes updates and upgrades and that such fixes updates and upgrades may occur without notice Licensee agrees to install and use any and all such fixes updates and upgrades and if applicable discontinue use of the previous version of the Software Licensee agrees that any and all such fixes updates and upgrades are and shall be governed by this Agreement unless superseded by an agreement associated with such a fix update and or upgrade 22 PRIVACY Li
91. at server is on a network or even over the Internet Below is an example of three clients communicating with a single license server This diagram is also a good reference since it shows the file names for each part of the license 165 FlexNet Floating Client Server Dataflow Server Gandar Daemon License Daemon Report File biod exe Log report log license lic Send Rey License Manager Daemon imgrd exe Send Rev Licensed Licensed Licensed Client Client Client Application Application Application CloneTracker ImaGene GeneSight Read Only Read Only Read Only Local License Local License Local License File local lic File local lic File local lic 166 4 3 FlexNet NodeLocked Installation Guide for Windows These instructions apply to all BioDiscovery products running on any supported platform In some places the tag Product will appear This represents the name of the BioDiscovery product being installed like CloneTracker for example The default install directory for FlexNet is C FlexNet In the following examples it is assumed that this is the directory being used If FlexNet is installed into another directory then make the appropriate modifications to the path 1 Install the BioDiscovery software with the FlexNet license manager service a Make sure that the account you are using has administrative privileges on this computer b
92. ata you also can export the measurements in GEML or MAGE ML format GEML v 1 0 format GEML profile export only four values per spot are available signal background value and standard deviation User can choose between mean median or mode for export as signal background value MAGE ML format XML the most complete format exported file will contain QuantificationType_package BioAssay_package and BioAssayData_package of MAGE standard All ImaGene measurements will be exported For more details go to http www mged org Close Close clears the quantified data values without saving them Once the quantification is cleared the image will need to be reprocessed before the data may be saved again Upon selecting Close you will be prompted to verify your action Clicking yes will then clear the data The Quantification table will no longer be visible 37 Context Menu You can select several spots at a time pressing Ctrl or Shift keys when making selection in the results table Right clicking the selection and the context menu will be displayed Raw Col Flag Sign i h h nnm Anu q961 555 Restore default column order O 425407 Freeze row selection Cp bs2 a6iss AAS R25 M Sare oeehe towe ttle 2B 1308672 Flag selected Flag 1 O E eao ernn H 74308 74308 Sel k E rra3o8 aoe een G8 SHER Iag a ho Rvt728 R11726 Negative SpotFlag font Raa pie 12142 oi
93. atch has been created the information about the batch is displayed The following information is contained within the table e Image The name of the image e Grid The name of the grid e Gene ID The name of the Gene ID file e Configuration The name of the configuration file e Output Directory The name of the output directory e Channel Name The Channel to be processed when using composite images Contigur Qutput Di cample Templat Templat Cantig xml Gesktap Sample Templat Templat Config xml Desktop sample Templat Templat Config xml Desktop Buttons From within the Batch Editor Window buttons exist to perform the following tasks Entries Available Unlimited Add Batch Entries Remove Selected Edit Selected Entry UO Show Full Path e Entries Available This field indicates the total number of images that are available to be processed For most users the field will indicate an unlimited number of images to be processed e Add Batch Entries Adding batch entries allow several images to be associated with settings and a grid file for processing For example if 10 images were added to be processed then the button only needs to be clicked once Specify the 10 images and all the images will be added to 85 the batch If an additional 13 images of a different type is to be added this process of adding batch entries needs to be repeated until all images are added
94. ation of this Agreement 6 TERM This Agreement is effective upon Licensee s opening of the package containing the Software or upon Licensee s acceptance of this Agreement This Agreement shall continue thereafter until terminated Licensee may terminate this Agreement at any time by returning the Software and all copies thereof and extracts therefrom to Licensor Licensor may terminate this Agreement and revoke any License granted hereunder upon the breach by Licensee of any term hereof If the License granted hereunder is terminated for any reason upon notice of such termination Licensee shall immediately de install the Software from the computer on which it is installed and shall certify to Licensor in writing under penalty of perjury of the laws of the United States of America that the Software is de installed and all copies thereof have either been destroyed or returned to Licensor Any confidential proprietary or trade secret information or material provided to Licensee in connection with the Software shall be immediately returned to Licensor unless otherwise specified by Licensor The provisions of sections 2 5 7 8 10 12 16 and 19 shall survive any termination of this Agreement 7 CONFIDENTIAL INFORMATION Licensee hereby acknowledges that the Software and any accompanying documentation contain confidential proprietary and or trade secret information belonging to Licensor Licensee further acknowledges and agrees that it shall not
95. btracted from 1 Ignored Area Area of ignored regions directly neighboring touching the signal area is computed Spot Area Signal Area plus Ignored Area pixels Ignored Median Median pixel intensity computed over the local ignored region Area To Perimeter This quality measure defines spot s circularity The area of a spot is divided by the square of spot perimeter and multiplied by 4r As a result this measure ranges from 0 highly non circular shape to 1 a perfect circle Open Perimeter Computes the proportion of signal perimeter that touches the border of rectangular snip around the spot XCoord X coordinate in pixels of grid circle corresponding to the spot YCoord Y coordinate in pixels of grid circle corresponding to the spot Diameter Diameter in pixels of grid circle corresponding to the spot Position Offset Offset in pixels of the center of the grid circle from the expected position in the grid Offset X X offset in pixels of the center of the grid circle from the expected position in the grid Offset Y Y offset in pixels of the center of the grid circle from the expected position in the grid Expected X X coordinate of expected position of the circle in the grid Expected position in the grid is computed fitting least square lines to circle centers in every row and column 35 Expected Y Y coordinate of expected position of the circle in the grid Expected posi
96. censor believes that the personal information Licensee provides to Licensor must be both kept private and used in a responsible fashion Licensor consistently puts its best efforts towards achieving both these objectives The updating feature of the Software provides information such as product serial number operating system information product language etc to Licensee for the purpose of checking the status of the License checking the status of maintenance for the Software and or determining if any fixes updates and or upgrades to the Software are available and Licensee agrees and consents that Licensor may store process and use such information for that purpose Licensee agrees that in the event that Licensor sells merges or otherwise re organizes all or part of its business with another entity the other entity may have access to and process store and use Licensee s information including the information collected through the updating feature of the Software on the same terms and conditions as set forth in this Agreement Licensee agrees that Licensor may process and or store Licensee s information in United States databases Licensee permits Licensor to share Licensee s information within Licensor and transfer it to countries in the world where Licensor does business Licensee permits Licensor to disclose Licensee s information when Licensor is required to do so by law 201 Packages cern colt cern jet Copyright 1999 CERN
97. d the corresponding segmentation Typical usage of the segmentation tabs is for detailed post processing quality assurance analysis Composit Sampleatif SampleBtif Samplesif seq SampleB tif seq Plots Unlike the Preview Panel the Segmentation tabs use lines to indicate the segmentation s signal and background regions All signal regions are surrounded by RED lines All ignored regions values not counted as signal or background are surrounded by YELLOW lines The remaining pixels within the image are all background regions The Segmentation Tab line colors e Red All pixels within the red lines are signal values e Yellow All pixels within the yellow lines are ignored pixels 56 Note The description and use of colors between the Preview Panel and the segmentation tab vary slightly The segmentation tab includes the use of yellow to indicate ignored pixels whereas the Preview Panel uses black to indicate ignored regions Also the segmentation tab does not use a color to indicate the background region however within the Preview Panel background values are indicated by a green color The differences between the two displays are accounted by the fact that per customer requests the segmentation tab is designed to prevent eyestrain during extensive visual inspection Plots Tab This tab appears only after image has been quantified It contains a set of useful data visualization tools that can aid the analysis process
98. displaying both the progress through the entire batch and the progress through the current image Upon completion of the batch this small window closes 2 4 3 Post Batch Processing To view the results of Batch Processing ImaGene includes two tools the results reviewer and the ImaGene log file 149 1 Launch the ImaGene software 2 From the File menu select Review Results sie Edit Grid Selection Auto E Load Images gt Remove Selected Images c Review Results Batch Editor kal Save Display Image Settings Exit 3 Browse to and select any one of the four snapshot files or files ending with a sst extension which were produced during the previous batch processing The file will be located within the data output directory specified Click the Open Button 4 ImaGene loads the images data quality measures and flagging as occurred though batch processing From here you are free to determine the quality of the batch processing and whether or not the image needs to be manually processed 150 ImaGere 6 1 Standard w Balch Edition 215 xj Oene ID Gene102 F 14 06 Field A Metarow 4 Metacolumn 1 Diameter 18 0 Fle Ean Gnd Selection Amo Measure Too HO ss sSCsS e a fa mm s e S ete 2 ss e ele ono m 17hes2 gelf 17hrsi gelf 17hrs gel segi 17hs1 gel seg Plots 10 Column 3 Geret 02 1 4 a Goret 02 14 04 Genet02 1 1406 Genet02 n 14 08 iGenet024 14 s10 iGenet024 14
99. ds to a spot on the array The columns with headers Chromosome and Base Pair are used to specify this genome location information for each probe Array O O O O O O O O O FOO SFOR O O FOO TONAR O O Chromosome CGH Analysis Details Once ImaGene quantifies the spot values for each channel it creates log base 2 ratios for each spot If there are multiple spots with the same GeneID ImaGene automatically combines the ratios from these spots by taking an average It will then arrange the ratios according to their position 132 along the chromosome see the figure below Here you can see each probe as a small blue circle along the length of a chromosome Please note that the color of each circle probe can be specified in the GeneID file using a column with header Color This can be a useful feature allowing you to color code control probes or those with questionable annotation In the figure below you can also see the two user specified calling thresholds as green and red vertical lines These lines specify the threshold for calling a certain region as a one copy gain multiple copy gain single loss or two copy loss The most trivial calling algorithm would be to simply use these thresholds and the probe locations This will cause a very noisy output marking any probe that exceeds these thresholds as a copy number change event To improve on this very basic approach we can use multiple probes that are relative neighbors of
100. e all GeneSight Lite tools can be used simultaneously and any gene sub selection in one tool will propagate into all other tools 158 Part 4 Installation and Licensing 4 1 ImaGene Installation 4 1 1 Windows Introduction Most users are familiar with the standard windows installation system and will not encounter any difficulties The ImaGene installation simply places the necessary files on the host computer and modifies the Start Menu ImaGene Installation l 2 3 If you are installing ImaGene on a Windows NT 2000 or XP operating system you MUST log in with an account that has Administrator access Launch the installation by double clicking on the exe file Review the version information on this screen and then click the Continue button to unpack the installation files When the Welcome to ImaGene Setup screen appears click the Next button Read the license agreement and then click the Yes to agree to the terms and conditions Read the setup notes and then click the Next button to continue Note the default name ImaGene of your program folder This will be the location of the ImaGene shortcuts on your Start Menu If you do not want to use this name enter another name in the Program Folders field Then click the Next button to begin the installation An installation progress bar appears so that you can monitor the installation When the installation is complete a message box appears Click the Finish but
101. e within the gene ID file if I have a column where I specify the field and that field has the name Top then I must enter Top for the field name within ImaGene Rows The number of rows of spots contained within the grid Typically this is simply counted visually Columns The number of columns of spots contained within the grid Typically this is simply counted visually Min Diameter This specifies the minimum expected diameter of the spot and is measured in pixels The size of the spot can best be 21 determined by use of the Ruler Tool Due to the variety of array types and the variability of individual arrays there is no set procedure for determining the minimum diameter here The most common rule of thumb is to specify the size of approximately 10 of the smallest spots of the array Depending on the type of array if the spots are highly uniform then the minimum diameter specified here would be close or equal to the maximum diameter specified next Max Diameter Similar to the Min Diameter specified above this parameter reflects the maximum anticipated spot size measured in pixels As with the Min Diameter using the top 10 of large spots and measuring the sizes with the Ruler Tool can approximate this value Spot Orientation Spot Orientation reflects how each row is located relative to the row that preceded it The selections here reflect how the row above the subsequent row is position While rectangular
102. e Make Measurements button under the Main Tab Images Grid Gene Info Process 2 After afew moments the quantified data is visible under the Raw Data tab 3 You can then run your CGH analysis by selecting the Analyze button under the Main panel This will run the Analysis using your chosen normalization and analysis settings Images Grid Gene Info Process 4 When finished running 6 new tabs will appear on the right side panel These include Normalized Data Overview opens first by default Whole Genome Chromosome CGH Report and Summary Composite CGHSampleAcyS tif CGHSampleBCy3 tif CGHSampleACy5 tif seg CGHSampleBCy3 tif seg Plots Normalized Data Overview whole Genome Chromosome CGHReport Summary 142 LS uw tb ol Oo J B wD a o a oe Mm ra ba gt i i pi pa 2 ra pa H j Ba pa pa LJ s gt lt H H H pa fi s P pa Ba i Hi pa t Hi 1 52 ji Ps Ho bd mS T H pa pa ra i p pa pa H ab ra H pa P Fi i pa pa pa H Hi ba T ra 14 15 16 17 18 19 20 ail ee x L IC Fa f ba Po J l z l pd q H s H s P lt pa BI pa X pa h i pa pa pa ji pa pa oO A P na a i pa pi H The plots allow users to drill down by selecting elements to obtain ever
103. ely relevant to the images being quantified Please see section 1 2 for additional information on the control tabs Main Preview 13 Image Display Panel Located at the right hand side of the window This panel displays loaded images and is where grid placement takes place Status Bar Located at the bottom of the window The status bar provides feedback to the user regarding loading and handling of images Also other tools such as the Ruler and Image Intensity display their information here Current active tool Ruler Jax 22 2857 dy 0 0 distance 22 2357 Current Active Tool Bar Located directly above the Status Bar at the bottom of the window Displays the tool currently being used in ImaGene Current active tool Zoom i distance 0 0 14 1 2 Control Tabs Located along the left hand side of the main ImaGene window are two tabs containing essential information about the images and quantification results Main Previews 1 2 1 Main Tab The Main Tab is utilized to perform the essential steps of microarray image analysis There are a group of icons on the top to streamline the image processing steps The workflow can be easily followed by selecting the icons from left to right Below them there are different sections for images grids and gene IDs as well as controls for image display and clearing Main Preview Images Grid Gene Info Process Images GBHB19K 17 43_532 tif
104. emain unchanged in their original location Do not confuse saving the batch file with saving the configuration files used to run the batch 86 e Run The batch and all its entries will be started Soon after clicking Run the Batch Editor Window will disappear and the Batch Progress Indicator becomes visible Channel Selector Located along the middle right side of the ImaGene Batch Editor Window is the Channel Selector The channel selector is designed for multi channel images also known as composite images Essentially a composite image is a single file which contains several images within This makes it possible to load a single file into ImaGene containing 2 4 or more images Several scanner manufacturers produce such file formats While most of the scanner software exports the images in a single one image per file format ImaGene is flexible enough to support both formats There may be times when only a single channel of a multi channel scan is of interest If this is the case then only processing the desired channel will save time Note Whenever multi channel images are processed in batch grid finding spot adjustment and measurement extraction are performed separately on each channel unlike processing of multi channel images in usual mode of ImaGene where you can align channels and use same grid for all channels at the same time However two channels or even two image files can be aligned and processed with a single
105. en re created as new The following parameters are displayed within the properties window ea MetaGrid Properti Field W Metarows Metacolumns Rows Columns Min Diameter 5 0 Max Diameter 5 0 Field The name of the field This value may be changed Metarows The number of metarows previously specified Metacolumns The number of metacolumns previously specified Rows The number of rows of spots within the subgrid that was previously specified o Columns The number of columns of spots within the subgrid that was previously specified O O O 23 o Min Diameter The minimum expected diameter of the spots to be used during spot finding This value may be changed o Max Diameter The maximum expected diameter of the spots to be used during spot finding This value may be changed How to Create Grid Perform the following steps to create a subgrid 1 Load the desired images into ImaGene Right click with the mouse on the Grid Panel and select Create Grid 3 Specify the parameters within the create grid window If a Gene ID file is being used then the field name must match the name of the field within the gene ID file The min and max diameter can be calculated with the Ruler Tool 4 Click the Place Grid Button and click on the four corners of a subgrid If you make a mistake right click with the mouse to remove the last placement Perform the following steps to create a metagrid 1 Clic
106. ere color determines how this probe will display in the results plots Additional custom headers can include User defined annotations which will also appear in the ImaGene results files 184 5 1 3 Output File ImaGene exports standard tab delimited text files when saving data The information below is intended to describe the general format of the test file and explain particular fields therein ImaGene saves the quantified data from each image processed into individual text files Due to the multi image processing capability ImaGene no longer exports two channel ratio data Researchers are required to either generate any desired ratio information themselves or import the raw data into a data analysis program such as the GeneSight software tool The data file is divided into two main parts The first part is header information about the parameters set for analysis The second part of the file includes the data extracted from the images Note Due to the nature of tab delimited text files wrapping of columns is common To easily view information BioDiscovery recommends importing the data files into a spreadsheet program such as Microsoft Excel Begin Header version 6 0 0 Date Mon Sep 06 16 40 28 GMT 08 00 2004 Image File C ImaGene Sample Data CDD1 CCD1_cy3 tif Page 0 Page Name Inverted false Begin Field Dimensions Field MetarowsMetacols Rows Cols A 2 2 21 22 End Field Dimensions Begin Measurement parameters Segmentation
107. ew lower left display on Main Panel provides a comprehensive and unabridged view of the image while indicating exactly where within the image the primary image display panel is zoomed The Map View displays the entire image exactly as it appears along the main image display s composite view Any corrections to contrast or rotation will likewise be visible within the Map Window The Map View allows users with large arrays to more easily scan the image and zoom in for easier gridding and spot adjustments Clear All The part of the image currently being displayed within the main image display is bound by a yellow rectangle within the Map View You may also zoom using the Map View by left clicking and dragging with the mouse to select the desired region To zoom completely out and display the entire image within the main image panel double right click with the mouse 28 Clear All Clears everything currently loaded in ImaGene including images grids gene IDs etc 1 2 2 Preview Raw Data Tab The Preview Raw Data Tab displays information about the image both prior to Preview Tab and after Raw Data Tab quantification Numerical values and segmentation can easily be viewed and reviewed to determine that optimum settings are established The Preview Raw Data Tab is divided into two primary parts Segmentation Preview Quantification Preview Main Raw Data Gene ID RP11 591L21 Field A Metarow 1 Row 16
108. fig Services tab c You will see the following screen LMTOOLS by Macrovision Corporation http waww macrovision com 5 ioj x File Edit Mode Help Save Service service Mame FLEXIm Service 1 Remove Service Configure Service Browse Path to the Imgrd exe file JC FlexNet Imgrd exe itd Path to the license file JC FlexN ethlicense lic Browse Path to the debug log file C FlesNet debua log Browse View Loa Lose Log jw Start Server at Power Up M Use Services d You can leave the Service Name as its default FlexNet Service 1 e Enter the Path to the Imgrd exe file or click the Browse button to the right In this example it is C FlexNet Imerd exe f Enter the Path to the license file or click the Browse button to the right In this example it is C FlexNet license lic g Enter the Path to the debug log file or click the Browse button to the right In this example it is C FlexNet debug log h Make sure you place a check mark in both Start Server at Power Up and Use Services Note that the Use Services checkbox may be disabled under Windows 95 98 ME 1 Click on the Save Service button 170 j Answer Yes on the dialog that appears to confirm these settings 8 Select the license services a Click on the Service License File tab b The following screen will appear LMTOOLS by Macrovision Corporation http waw macrovision com l s olx File Edit Mode Hel
109. g one of the plots will be common for all other plots results table and image tab The one exception is if a group of spots is 62 selected using Scatter plot Box plot or Results table the selection will not be visible on Histogram plot because of its specifics But if the selection is made using the Histogram plot it can be viewed in all other tools Note Mouse over any of the data elements on any plot and see a tool tip containing short info about gene ID and measurement values 63 1 4 IlmaGene Parameter Settings Window ImaGene encapsulates most of its parameters and settings within one common interface the ImaGene Parameter Settings Window Within this window are tabs which control virtually all aspects of the array image analysis From initial spot finding settings to complicated auto reporting of alert values the ImaGene Parameter Settings window provides complete user control Selecting File then Settings may open the window Edit Grid Selection Auto E Load Images a Remove Selected Images ese Review Results Batch Editor hd Save Display Image Settings The window is organized into several tabs which group related parameters together The tabs available and their parameters include Spot Finding Spot finding involves the localization of the array signal as printed on the array medium Due to the warping of the medium or potentially bent pins during printing spots and sometimes entire sub grids require s
110. he images need to be overlaid Not all images will require this action but due to variances in scanning some will To overlay the Images Automatically 4 click on the Auto Align Images button under the Main tab Images rid Gene Info Process In additional to auto alignment ImaGene offers manual tools for rotation and translation of images 104 File Edit Grid Selection Auto Measure Tools Help a Main Preview ta ui He ts U Undo Remove Images a Composite 17hrsi gel 17hrs2 gel ABE i Process E4 E Images Grid Gene Info Images 17hrsi gel 17hrs2 gel Brightness o Contrast Hl Reverse Display Colors Grid Active tool Adjust Spot Clear All Grid Placement and Manipulation 1 From the menu bar select Tools followed by Zoom 2 Drag a rectangle around the top sub grid of the array to zoom into this region of the image Cy 3 Select Tools then Ruler from the menu bar to measure the approximate sizes of the spots within this array This information will be used later 105 4 With the ruler tool click and drag from one side of a spot to the other side of the spot The distance is displayed in a status bar located beneath the main image panel This distance is measured in pixels 5 Next count the number of rows and columns of spots within this sub grid 6 Click on the Create Grid button under the Main tab
111. i Condition 1Mean locBGC 2 Condition 1 icontrohMean locBGC LJ Show Gene IDs Regression Shif 0 082 Slope 0 893 R2 0 917 Create Partition and Make Active Create Partition Use Full Gene Set 13 Enter Hot Genes for the name of this sub selection 14 Close the Scatter Plot Window 15 From the menu bar of GeneSight Lite select Utilities then Generate Report 16 Click Save Report to save the selected genes to a text file on the file system The file is a common tab delimited text file which can be opened by programs such as Microsoft WordPad or Excel 119 amp Select the look of the Report k E ioj x Plot Reports DefaultDS gs Show Only Selected Genes Select the reports from the available plots to add to your report Scatterplot Gene101 a 0 4922771 0 3698183 0 1224587 1 5524668 15 Gene101 a 0 1210529 0 2999103 0 1788574 0 2740591 Gene101 a 0 3781497 0 9496428 1 3277926 1 1615517 5 15 38 Gene101 b 0 3550547 0 6067184 0 2516637 ig Jeenetob 07650074 paret7r7osaes2 T 49 Gene101 b 0 2833074 0 3791 777 0 6624852 63 Gene101 c 0 1934036 0 2906671 0 4840707 gt m 76 Gene101 d 0 1772153 0 2246307 0 4018460 1 1062391 1 x Condition 1 control Me 84 Gene101 d 0 1341477 0 628501 7 0 7626494 ho olm
112. ical Support at support biodiscovery com before removing any version of ImaGene from your computer as un installation may affect your licensing ImaGene Un Installation 1 Click Start gt Settings gt Control Panel gt Add Remove Programs 2 Click on ImaGene to select this program from the Currently Installed Programs list 3 Click the Change Remove button to initiate the un installation process and display the ImaGene confirmation dialog box 4 Click the Yes button uninstall ImaGene A second ImaGene confirmation dialog box displays when the un installation completes Click the OK button to acknowledge that ImaGene has been removed your computer 4 1 4 Macintosh Please see the release notes provided with your ImaGene software distribution 4 1 5 Unix Please see the release notes provided with your ImaGene software distribution 161 4 2 License Management 4 2 1 License Manager The License Manager is a small software service daemon that controls which computers can run a particular BioDiscovery product and what features are available in that product The main purpose of the license manager is to prevent software piracy but there are additional benefits as well such as user license tracking time limited demos and advanced user network permission settings BioDiscovery has switched to GlobeTrotter s FlexNet license manager This popular license manager provides an excellent balance of features flexibil
113. id two 2 business days following delivery by overnight courier and two 2 business days following confirmation of transmittal by facsimile Any notices provided under this Agreement shall be given at the address and or facsimile number for the parties as set forth upon the sales document for this License unless change of such address and or facsimile number has been provided previously in writing 198 12 GOVERNING LAW AND VENUE This Agreement shall be construed and governed in accordance with the laws of the State of California without giving effect to any choice or conflict of law provision or rule Licensor and Licensee consent and agree that personal jurisdiction over them with respect to any dispute arising as to this Agreement shall rest solely with the state or federal courts of the State of California Licensor and Licensee hereby expressly waive the right to bring an action in any state or federal court other than the California state or federal courts located within the County of Los Angeles This Agreement shall not be governed by the United Nations Convention on Contracts for the International Sale of Goods the application of which is expressly excluded 13 ATTORNEYS FEES If any action is brought by either party to this Agreement against the other party in an effort to enforce or effect any provision or language contained within this Agreement the prevailing party shall be entitled to recover in addition to any other relief gr
114. ification Setting the proper buffer size helps to ensure accurate results The desired size is dependent on several factors including the spot size density image quality and spot shape e Background Width kground width 5 0 The measurement in pixels to determine how far background measurements will extend from the buffer region In other words the measurement will extend X pixels from the end of the buffer Remember that the background should include enough pixels to provide a sufficient sampling ImaGene should not be used with no background values as these values are required in numerous quality measurements Note The background width will not extend beyond the snip or rectangular boundary around each spot Even though the background can be set to an extremely high value the background measurements will stop at this boundary and not include signal values from surrounding spots e Auto Segmentation 2 is Selecting this option will turn on BioDiscovery s patented automatic segmentation algorithm Under most circumstances this should be selected Under Auto Segmentation the only parameter that ImaGene will use is the Background Buffer The typical value should be slightly less than 1 2 the radius Under Auto Segmentation the slider bars for the signal and background percentages become disabled e Do not allow donut shapes X Do not allow donut shapes When this option is selected ImaGene will make sure that a donut
115. image size itself The snapshot file contains the following components within it Grid Gene ID Segmentation Quantified Data Quality Measures Flag Values 92 While the sst file contains almost all the required information to reload and review the data it does not contain the actual images To review results ImaGene must have access to the original images By default ImaGene will attempt to load the images from the location specified within the sst file However if the images have been moved and ImaGene is not able to load them ImaGene will prompt the user to browse to and select the appropriate images 1 7 2 Loading Results to Review Loading data for subsequent review can be accomplished by performing the following steps l 2 3 From the ImaGene menu bar select File followed by Review Results The Review Results icon located along the toolbar may also be used With the Open dialog browse to and select the snapshot file Depending on the file ImaGene may give a prompt to specify the location of the original images The information and data should now be visible within the main ImaGene user interface If reprocessing the images is desired click the Cancel button along the bottom of the Results tab and use Quantify button of the main panel 93 1 8 lmaGene Tools ImaGene includes a number of tools designed to help facilitate array analysis These tolls are designed to allow manipulation quantification and analys
116. is of array images 1 8 1 Auto Alignment Tool The Auto Alignment tool tiid automatically overlays several images on top of one another If two or more images are loaded activating this tool will attempt to overlay the spots from each image While this tool is designed largely for transposing of images or left right and up down movement ImaGene can handle up to 10 degrees of rotation as well The Auto Alignment tool is intended to provide quick and highly accurate process to solve a common problem of array image analysis arrays which have been scanned slightly out of alignment If the images have a high degree of rotation or another extreme problem then the manual manipulation tools of transposing and rotation may be required Note The Auto Alignment tool is intended to account for shifts of the array in scanning and not for warping of the medium being printed upon 1 8 2 Auto Grid Placement Tool Fj aa ImaGene s Auto Grid Placement Tool FA automatically repositions a grid structure to the proper location within the image Either a grid or a template file is required to be loaded before this tool can be utilized If the geometry of an array stays the same through and between experiments the use of a common template for all experiments would be beneficial However due to the differences in arraying and scanning the location of the array within the image may vary across experiments and time The solution is to use the Auto G
117. itioned based on this information b Place in Saved Position Select this option to place the grid in the identical position of the original grid Use this option if the size and resolution of the images has not changed 4 Finally click the Open Button to place the grid Gene ID The Gene ID file is utilized to track information about the genetic material spotted at each location within the array This information will be saved along with the quantified values in the text output file and visualization tools Note that for CGH analysis the Gene ID format requires more information as described in the appendix 5 1 2 If a Gene ID file was selected the name of the corresponding file is displayed here GeneID Sample ABGenelDs tt The Gene ID also supports right click context menus with the following options Gene ID Sample_ABGenelDs te fl Load Gene IDs O Reverse Display Colors ra Clear Gene IDs 29 e Load Gene IDs Selecting this option will open a window allowing for the selection of the gene ID file to be used Note that while advantageous for a number of reasons loading a Gene ID file is not mandatory Please see the section of Gene IDs later within this manual for additional information on Gene IDs as well as appropriate file format e Clear Gene IDs If a gene ID has already been selected this option will remove the selected file from use Template You can load and save a template file that will contai
118. itute grid should be processed before current image This feature is useful if you are sure that some images are perfectly aligned with each other Default value null 1 e no substitution will be made lt SubstituteGridChannel gt designates a channel in substitution image file from the previous option Default value 0 lt Normalize gt determines whether to perform normalization on the output data Settings for normalization will be taken from the configuration file Normalized data will be outputted into a TXT file normalized will be added to the name of this file Lowess normalization is possible only if an entry contains two images see option lt Image2 gt Default value false 191 lt Analyze gt determines whether to perform ratio analysis on the normalized data Settings for analysis will be taken from the configuration file Analysis data will be outputted into a TXT file analyzed will be added to the name of this file Analysis is possible only if a batch entry contains two images see two following options Default value false lt Image2 gt allows including another image in the same batch entry This way ImaGene will load both images at the same time Image and Image2 auto align them if lt AlignImages gt parameter has value true load just one grid for both images adjust grid and spots based on composite image and process both images in one quantification giving an opp
119. ity and easy of use It is purely software based so there is no need for any sort of hardware like a parallel port dongle and it can handle licenses from multiple companies If your institution business is already running FlexNet based products the addition of BioDiscovery licenses will be very easy Many specific FlexNet questions are answered in this online FAQ http www globetrotter com FlexNet Imfaq shtml It is not necessary to review this FlexNet FAQ since the basic setup of FlexNet is covered in the accompanying text The FlexNet license manager can be configured in two possible ways Node Locked and Floating 4 2 2 Node Locked Licenses A node locked license allows BioDiscovery products to run on a single specific computer The license is hard coded to that specific computer s ID and will not run on any other computers Node Locked is the more common of the two possible configurations In order to run in the Node Locked mode the computer must be both a FlexNet license server and it s own client This requires the user to setup a server on the computer in addition to the regular installation of the software 162 The FlexNet license manager service will run in the background at all times regardless of whether or not a BioDiscovery product is also running This may seem inefficient but the overhead of the license manager is minimal and it would be inconvenient for the user to have to manually start the license man
120. k Guidelines at http java sun com trademarks html Not do anything harmful to or inconsistent with Sun s rights in the Java Marks and e Assist Sun in protecting those rights including assigning to Sun any rights acquired by Licensee in any Java Mark For inquiries please contact Sun Microsystems Inc 901 San Antonio Road Palo Alto California 94303 207
121. k on and select the desired subgrid to use as a basis for the metagrid This can be done by using a left click on the grid within the Grid Panel 2 Right click and select Create MetaGrid from the menu 3 Inthe Metagrid Parameters Window specify the number of rows and columns of subgrids contained within the metagrid 4 Click Place Metagrid and click on the top left spot in each of the corner subgrids Typically this process will require four clicks How to Save a Grid Once a grid has been created the following steps will save the grid for later use 1 From the menu bar select Grid followed by Save Grid 2 In the Save Dialog browse to the location where you wish to save the grid file 3 Specify a file name ImaGene will automatically add the grd file extension to the end of the name 24 How to Load a Grid To load a previously created grid perform the following steps 1 From the menu bar select Grid followed by Load Grid 2 From the Load Grid Dialog browse to and select the desired grid file 3 Click on one of the following radio buttons to select a grid placement method a Place Manually Select this option to set the location and size of the saved grid manually This is useful when images have been scanned at different resolutions or the overall position of the array shifted between scans To place the grid manually left click on the four corners of the entire array structure The grid will then be resized and pos
122. lag i All flagged At least one flagged ox All flagged In order for a spot to be flagged it has to be found flagged in all the images 74 At least one flagged If a spot is found flagged in any one of the images it will be flagged in all the images quantified together Measurements Various spot measurements can be computed by ImaGene and outputted to a tab delimited text file To select measurements that you are interested in you can use the Measurements panel of Settings dialog eat ImaGene Parameter Settings _ o Normalization Analysis Controls and Alerts Image Preferences Spot Finding Segmentation Quality Flags Measurements W Median J Mode J Total J Standard Deviation J Area J Ignored 4rea J Spot 4rea J Ignored Median W Area to Perimiter Ratia J Offset From Expected Position J Shape Regularity J Open Perimeter Select all Unselect all Load Save As Close e Mean signal and background mean intensity e Median signal and background median intensity e Mode signal and background mode intensity mode corresponds to the peak of corresponding histogram e Total signal and background total intensity all pixel intensity summed up 75 Standard deviation standard deviation of signal and background intensity distribution Area number of pixels segmented as signal and background Ignored area number of pixels in the areas segmented as ignored
123. le from the Menu Bar File Edit Grid Selection Auto Measure Tools Help 8 File Edit Grid Load Images Allows selection of images to be loaded from the file system Remove Selected Image The image which is selected or highlighted from within the images panel is removed or unloaded from analysis Multiple images can be removed by highlighting several images then selecting Remove Selected Images Review Results Allows selection of a snapshot or sst file which will allow for the results of previous analysis to be reviewed or reexamined Simply browse to and select the desired sst file and ImaGene will load previously created settings data segmentation and flagging information along with the images processed Batch Editor Allows creation of batches to automatically process data Selecting this option will open the Batch Editor Window allowing for entries to be added to a batch Save Display Image Allows a 24 bit tiff image of the composite overlay to be created and saved to the file system The image can be useful in post processing or future generation of publications Settings The Settings option opens the ImaGene Parameter Settings Window This window and the tabs within the window contain all the parameters within ImaGene for Quality Measures Auto Flagging Spot Finding and Alert Logging Exit Closes the ImaGene program Undo Cancels the last executed command For example if you moved a spo
124. locations 109 17 18 19 20 21 22 Using the scroll bar along the right of the main image panel scroll up so that the second to last sub grid is visible Click on the Adjust Subgrid button under the main tab Click on and drag this sub grid slightly to the left to provide a better fit Click the Adjust MetaGrid Button from the toolbar and click on any sub grid Alternatively click on the Auto Adjust Grid button under the Main tab and the sub grids will be adjusted automatically Images rid Gene Info Process Click next on the Auto Adjust Spots button under the Main tab to perform automatic spot adjustment 110 Images rid Gene Info Process Quantifying Pre Processing Pre Analyzing and Saving the Data l oe Oe 10 11 Click on the Make Measurements button under the Main Tab aal Images Grid Gene Info Process After a few moments the quantified data is visible under the Raw Data tab Select Plots tab on the Image Display panel Select Histogram on the Plots panel Select Signal Mean Ratio from the list of measurements Make sure that log option is not selected for the Histogram Select 2 fold regulated genes by specifying 2 in the Upper bound text box Freeze the selection using the Mark Selected Rows option by a right click on the selected rows Sort the data by column Selected clicking on its header All selected genes will be in the bottom
125. lone locations Templates can also be imported as GAL GEML or MAGE format In order to do so follow the steps below 1 From the Grid menu select the option Import template as GAL GEML or MAGE 129 tn Import template as GAL GEML or MAGE GAL In the next window select the appropriate option among GAL GEML or MAGE for the file types GAL is the default option here om Import Template eee Gl tees Bla fa MWG Pan Yeast ID 1 correct posistion last1 6mod gal fa MVYG gal Array Lists gal D All Files Array Lists gal GEML v1 0 format xml X cj MAGE ML format xml Browse and select your template file and click on Open oO Import Template MWG Pan Yeast ID 1 correct posistion last1 6mod gal Array Lists Cga 0000 130 4 The template will be displayed in the Grid and Gene ID panels in the Main tab Grid Block 18 x18 Block 18 x 18 Blocks 18 x18 Block4 18 x18 Blocks 18 x 18 Block 18 x 18 Gene lO an YeastiDO 1 correct posistion last bmod gal 2 3 CGH Tutorial This tutorial demonstrates performing basic CGH quantification using ImaGene The tutorial covers the essential steps from loading the initial array images until the final CGH report is saved to disk For users experienced with standard ImaGene expression analysis many of these topics will be familiar The following lessons will be presented within this tutorial e Image Lo
126. lumn Number of column in the subgrid where the spot is located GeneID Gene ID information for the spot Optional Probe Annotation Fields for the spot Flag Numeric code of the flag for the spot 0 no flag flag codes 1 7 Signal Mean Pixel intensity averaged over the local signal region Background Mean Pixel intensity averaged over the local background region Signal Median Median pixel intensity computed over the local signal region Background Median Median pixel intensity computed over the local background region Signal Mode Mode pixel intensity computed over the local signal region mode corresponds to the peak location in intensity distribution 34 Background Mode Mode pixel intensity computed over the local background region Signal Area Number of pixels in the local signal region Background Area Number of pixels in the local background region Signal Total Total pixel intensity summed over the local signal region Background Total Total pixel intensity summed over the local background region Signal Stdev Standard deviation of pixel intensities over the local signal region Background Stdev Standard deviation of pixel intensities over the local background region Shape Regularity First the signal area of a spot is inscribed into a circle Then the number of non signal pixels that fall within this circle 1s computed and divided by the circle s area This ratio is su
127. m the list d Place a checkmark next to Always use this program to open these files e Click the OK button The text file will contain a line that looks similar to this SERVER HOSTNAME 00065b1bb4ee 172 vq mh h Make sure that HOSTNAME is your computer s Hostname from step 4 i Make sure that the following Ethernet Address matches your computer s Ethernet address from step 4 If it still doesn t start then email the C FlexNet debug log file to support biodiscovery com 11 Run the License Wizard configure the local client a Start the License Wizard by clicking on Start gt Program Files gt Product gt License Wizard b Click on the NodeLocked tab c You will see the following screen Ea BioDiscovery License Management Wizard Ioj x NodeLocked Floating Computer Mame Localhost 127 0 0 1 User Namne Joe Green Institution Name Green Corp Serijal MurmberlBDH zad Save HodeLocked Cancel Enter your User Name Company Name and Serial Number e Itis important that you enter your actual Serial Number since this information is used to determine eligibility for upgrades using the BioDiscovery Auto Update feature f The serial number can be found on the case of the CD shipped to you or in the accompanying User Registration form g Click the Save NodeLocked button to save this information and exit 12 Start the BioDiscovery software If the license server is running
128. med within ImaGene Change Skins Launches a dialog for choosing between different skins for ImaGene interface When a specific skin is selected the change will take effect next time you launch ImaGene Auto Update Utilized to automatically update ImaGene with the newest version available for download from BioDiscovery Customer Support Center Launches a Web Browser with the customer support page for ImaGene The latest technical documentation requests for new features and obtaining sample images or templates etc are all accessible through this site About ImaGene Displays the About ImaGene dialog box This interface contains information license number mode etc about ImaGene Toolbar Located directly beneath the menu bar This region is composed of multiple buttons that provide a one click method for executing program commands Context Menu Displayed by right clicking on the various elements within the ImaGene graphical user interface GUI Not all elements support context menus Elements that support this feature are listed below Images Panel Grid Panel Gene ID Selector Flagging Tool 12 e Results panel in Raw Data tab Images E Sarnplea tif Sample tit AY Load Images a Remove Selected Images BB select Color ans O Hide i A g e e 20x16 LJ Flip Horizontal Rotate Image Info Control Tabs Located at the left hand side of the window The control tabs provide information immediat
129. mmit the changes or cancel to return to the Batch Editor without saving any changes 1 6 7 Normalization and analysis in batch mode When batch procedure is designed through Batch Editor no normalization or analysis is performed on extracted measurements However normalized data or analysis results can be produced in batch through usage of batch XML file of a specific format described in section 5 1 5 91 1 7 Reviewing Results One of the most powerful features of ImaGene is the ability to review results from the previously processed data ImaGene was designed to load and display data exactly as it was processed irrespective of time The capability to review the results provides the following benefits Compare old data with recently processed data Review the results of batch processing Establish post image analysis quality controls and screening Reload and take screen captures for publication 1 7 1 The Snapshot File sst file The snapshot file is the result of quantification within ImaGene Upon saving the quantified data to the file system ImaGene also saves another file which contains a snapshot of the data after quantification This snapshot file ending with an sst file extension is utilized by ImaGene to load and allows the results to be reviewed The snapshot file itself is a proprietary binary file format Due to the large amount of information contained within this file its size can grow large often surpassing the
130. n both grid structure and corresponding gene IDs For this purpose you can use BioDiscovery s serialized data format tpl or you can import and export the template using several well known formats GAL tab delimited text file containing location and structure of every subgrid called Block within this format and gene IDs http www moleculardevices com pages software gn_genepix_file_formats html gal GEML v 1 0 XML standard representing a pattern that cannot fully describe a grid structure but rather provides location and gene ID info for every spot http www rosettabio com tech geml default htm MAGE ML the most complete XML format imported exported file will contain DesignElement package and ArrayDesign package of MAGE standard This format can support multiple subgrids and metagrids http www mged org Reverse Display Colors Mark this check box to reverse the displayed colors of all loaded raw images e g changing intensity scale from black to white to white to black This feature has no effect on the Composite tab display Changing the appearance of an image does not affect the pixel intensity of the original data This is utilized to visualize dim spots in the image display LJ Reverse Display Colors 26 Display Control The Display Control allows manual adjustment of how the images are displayed By moving the Brightness Max pixel value threshold adjuster and Contrast Minimum pixel v
131. nd buffer bo B Auto Segmentation A po LJ Do not allow donut shapes Erse ote ttiot leia ay Orange packing Segmentation ImaGene version 6 eat ImaGene Parameter Settings iol x Measurements Normalization Analysis Controls and Alerts Image Preferences Spot Finding Segmentation Quality Flags Background buffer 2 0 V Auto Segmentation Background width jio o fi Do not allow donut shapes Signal percentages Reckgrouind percentages Low High Low High 10 90 10 90 1 00 100 100 Load Save As Close ImaGene version 7 124 To verify that Orange Packing segmentation is being applied view a selected spot in the Preview panel Mame Original Segmented Composite Histogram O_US225026 1 _US2e5026 Grid Spot adjustment 1 Click on the Auto adjust grid button in the Main tab 2 Click on the Auto adjust spots button in the Main tab Quantifying Normalizing Image or Image Pairs Analyzing paired images and Saving the Data 1 Enter the Normalization and Analysis parameters in the two Settings Tool panels These panels are described above in sections 1 2 3 and 1 2 4 125 ImaGene Parameter Settings Tiol E Analysis Controls and Alerts Image Preferences ua a Bdo men E oms awi Use all spots kd M Take Log Normalization Settings 126 ae ImaGene Parameter Settings ol x S
132. neto ett 0 h2e2221 hioesso h2378552 1203 ao ho ho b ho Genetore 1 0 mize morsso2 h1esssss h20 1 657233 1 624554 1 625214 1 72 T gt o A 5 1 uo Save Report 16 The Analyzed data will be displayed in a new tab Analysis in the right panel 114 Composite 17hrs1 gel 17hrs2 gel 17hrsi gel seq 17hrs2 gel seq Plots Normalized Data Analysis __ GeneID Refe _ Experiment Difference Significance ene101 c 1 a15 0159287 6 949533 3 9336047 ene101 c 1 0310 656332 10 849881 10 1935494 UNCHANGED ene101 e 1 a510 621387511 796173 11747859 1 833193 0 34870243 UNCHANGED fi1 057189 0 68903995 UNCHANGED 10 525988 1061625934 UNCHANGED 0 8132596 UNCHANGED 11053488 53859925 UNCHANGED 12808828 i 3076272 0 222038 5 753296 48764113 0 63222384 UNCHANGED NCHANGED h1 56387 NCHANGED NCHANGED 11 763578 NCHANGED NCHANGED 11 0385475 NCHANGED 9 652801 10 483963 NCHANGED 10 949913 11 885287 NCHANGED NCHANGED hos01059 10 45383215 UNCHANGED 0 588286 11615666 A 0273798 11 080713 NCHANGED NCHANGED 11 808783 NCHANGED 10 4799385 10 40886 d NCHANGED 11 336381 A 1 758776 NCHANGED 13 2154708 12 26371 wee AMA ew A A PAPA A MAPA M AMMA OA TREO Ea RO SIGNIFICANT UP Save Report 17 Click the Save Bu
133. ng to different subgrids different rows having different flags etc The different categories may be used for X axis and different measurements for Y axis and with or without normalization The box visualizes lower and upper percentiles of distribution These percentiles can be changed using scroll bars on the left hand side Box plot also visualizes distribution outliers This is utilized when looking for abnormalities in the data due to special or other categorization Note for more detailed description of Box plot refer to GeneSight manual provided with ImaGene Choose the plot type Box Plot F f Box plot 10 25 7 8 9 40 414 412 44 15 16 Percentiles Condition Names 0 iag Y axis 61 GenePie This plot displays the spot s expression values for each channel as portions of a circle The colors within a pie correspond to the individual channels The most common use of the GenePie chart is to plot differential expression patterns between channels Different measurements may be used for GenePie plot with or without normalization The values on X and Y axes represent the spot coordinates in pixels The empty circles represent the spots where the expression value is not available NaN for either or all the channels usually after taking log ratios or normalization 7 co f B TL a i ee a uw co mm a ke m 1 D m senePie ONocco o oosesecee9g 0008 60600002082
134. null 0 28161 3613 647 7194 15924 0 636 0 64460 6015 631 3684 83 0 278 0 2337393 0 180066 0 27675 3652 41 7804 0 7232 0 0 83 0 0 0 0 9577 0 0 37 7295 36 0467 9 0 0 5892 0 4163 0 4169 38 1458 35 6297 39 3132 34 1084 1 9176 1 1674 1 5212 9 0587 11 9599 0 0 0 0 0 0 0 0 0 0 0 0 A 1 1 1 2 null null 0 7143 6123 645 0494 3064 0 630 0 15128 1542 624 7039 98 0 263 0 700074 0 169648 0 7305 3032 46 2776 0 649 0 0 98 0 0 0 0 8995 0 1351 55 8644 35 9673 9 0 0 5331 0 3766 0 3772 56 241 35 59 57 9795 33 7959 2 4982 1 7385 1 7941 9 4711 13 5513 0 0 0 0 0 0 0 0 0 0 0 0 5 1 4 Configuration File The configuration file stores information about virtually all the parameters within ImaGene This file is an XML file format which is easily saved and 186 loaded from the ImaGene Parameter Settings Window While the contents of this file are not important to most users be aware that this file is required for batch processing Advanced users integrating ImaGene into high throughput environments may also find the information useful Listed below is a sample ImaGene configuration file lt xml version 1 0 encoding UTF 8 gt lt ImaGene_Parameters gt lt SPOT_AREA gt true lt SPOT_AREA gt lt MEASURE gt Normalized Signal Median lt MEASURE gt lt CONNECT_FLAG gt false lt CONNECT_FLAG gt lt CUT_OFF gt 0 0 lt CUT_OFF gt lt SIGNAL_LOW30 0 lt SIGNAL_LOW gt lt CONTROL_TYPE_LOWTHRESH_2 gt 0 0 lt CONTROL_TYPE_LOWTHRESH_2 gt lt CONTROL_TYPE_UPTHRE
135. or both If Subgrid based is checked the median of the background values of all the spots in a subgrid is used for background correction If Using signal from control spots is checked the median of the signal values of all the control spots is used see detailed explanations about control spots in Section 1 4 Controls and Alerts subsection When both boxes are checked the median of signal values of the control spots within the same subgrid of the spot being background corrected will be used Log Transformation Log transformation with bases of 2 can be applied to the raw or background corrected data Normalization There are four options for normalization type None Divide by percentile Subtract percentile and Lowess When either Divide by percentile or Subtract percentile is selected a percentile value has to be set to 50 median 25 or 75 A scope of Global or Subgrid also has to be chosen for normalization In addition user has to select whether all spots should be used for computing normalization or utilize dedicated control spots see detailed explanations about control spots in Controls and Alerts of 1 4 ImaGene Parameter Settings Window Normalization coefficients can be computed based on o All spots o Exclude Control Spots for Lowess normalization o Use Control spots for normalizations 40 Mormalizatian Normalization type Divide by percentile Percentile Scope Global hi Global K
136. ors Grid Active tock None Coar Ad 8 Open the CGH Settings tool and select the Load button Then browse and select the CGH settings file cghdemo settings xml 138 ImaGene Parameter Settings 9 From the Settings tool go to the Normalization tab and select desired normalization parameters 139 ImaGene Parameter Settings 10 From the Settings tool go to the Analysis tab and select desired Analysis parameters see section 1 2 4 for full descriptions 140 5i ImaGene Parameter Settings Oj x Spot Finding Seamentation Quality Flags Measurements Normalization Analysis Controls and Alerts Image Preferences Measure Normalized Signal Mean Omit Flagged Spots Reference Channel Channel 1 Channel 2 Organism Human v Analysis type Expression CGH EGH Parameters Significance Threshold 0 05 2 Deletions lt fas oo Default Chromosome Plot Bounds 1 Deletion lt 1 0 2 0 o 1 Amplification gt os 2 Amplifications gt ho 11 Click on the Auto Adjust Grid button under the Main tab and the sub grids will be adjusted automatically aal Images Grid Gene Info Process 12 Click next on the Auto Adjust Spots button under the Main tab to perform automatic spot adjustment aal Images Grid Gene Info Process 141 Quantifying Normalizing CGH Image Pairs Analyzing CGH copy number and Saving the Data 1 Click on th
137. ortunity to perform Lowess normalization or two channel ratio analysis This option will also be useful if one of the channels of the same array is too dim to provide reliable grid finding By default not included in entries lt Channel2 gt serves in combination with lt Image2 gt option By default not included in entries lt ChannelName2 gt serves in combination with lt Image2 gt option Used to name output TXT file If lt Channel2 gt exists but lt ChannelName2 gt does not or is empty prefix with channel number will be used By default not included in entries 192 5 2 Technical Support BioDiscovery is available to answer any questions about ImaGene Your questions will be addressed promptly so you can focus on what is most important your research Your ImaGene serial number will be requested when you contact technical support using any of the following methods E mail support biodiscovery com Phone 310 414 8100 United States Fax 310 414 8111 Mail 2121 Rosecrans Ave Suite 3315 El Segundo CA 90245 USA Note Free technical support is available for one year from your date of purchase 5 3 Warranty Information BioDiscovery guarantees ImaGene to be free from defects up to 30 days from the date of purchase BioDiscovery will promptly address any problems you may have through either technical support or by sending a replacement copy of ImaGene For warranty information review the license agreemen
138. osen normalization procedures configured using the settings panel to determine for 1 the Gene Expression Module whether the genes are up down regulated or have no change against a pre determined fold threshold Or 2 the CGH Module whether there is regional copy number variations or amplifications and deletions Zoom Turns the cursor into a magnifying glass Utilized to adjust the on screen display size of images Scroll Turns the cursor into a hand Utilized to scroll all images and grids at the same time Translate Images Turns the cursor into a cross with arrows Utilized to move selected images Rotate Images Turns the cursor into a circle Utilized to move selected images in a circular path based on a manually set anchor point in the image Ruler Turns the cursor into a small ruler Utilized to measure the size of spots or distances between them Image Intensities Turns the cursor into a small light bulb Utilized to measure the intensity of spots 11 Tag spots Turns the cursor into the letter X Utilized to manually flag spots Right click with this tool to see a list of available flagging options Tag spots with lasso Similar to Tag spots but can manually flag a group of spots at a time by drawing a boundary ImaGene Help Displays the ImaGene Help documentation Wizard On Off Turns on and off the ImaGene Wizard The wizard provides step by step guidance on the proper steps to be perfor
139. ots to estimate on feature background from their signal values During normalization you can compute normalization coefficient based on a normalization control type make sure you create one before normalization or you can perform normalization based on all spots excluding controls 80 Image Preferences When ImaGene loads an image it needs to obtain the resolution of the image in order to load array templates correctly applies to GAL GEML and MAGE ML templates Normally it reads the resolution information from the header of the image file However some scanners do not store this type of information in the image files In these cases ImaGene has to apply a default resolution to the images The Image Preferences tab allows users to enter the image resolution 10 0 micron pixel by default It can also prompt users when the set resolution is being used Note Only when there is no resolution information in the image file should the resolution setting in the Image Preference tab be used The resolution in the image file will make ImaGene ignore the setting in this tab Si ImaGene Parameter Settings 3 zy ol x Spot Finding Segmentation Guality Flags MAAS Ferment Normalization Analysis Controls and Alerts Image Preferences Default Image Resolution in micronsipixellt 0 0 Load Save As Close 81 1 5 ImaGene Wizard The ImaGene Wizard was designed to assist the users to better understand ImaGene tools and
140. p Services allow FLEXnet Servers to run in the background Server List Configuration using License File f Configuration using Services FLExIm Service 1 c Select the Configuration using Services radio button d Select the Service Name you used from step 7 9 Start the License Server a Click on the Start Stop Reread tab b The following screen will appear 171 LMTOOLS by Macrovision Corporation http waww macrovision com i iol x File Edit Mode Help FLEXnet license services installed on this computer Stop Server ReRead License File T Force Server Shutdown _ Advanced stings gt gt NOTE This bos must be checked to shut down a license server when licenses are borrowed Using License File C FlexsNetslicense lic c Double check that the Service Name selected is the same as the one you configured in step 7 Click on the Start Server button e The text at the bottom of the window should change from Using License File to Server Start Successful 9 Did the license server start successfully a If yes then you re almost done Skip to step 11 b If no then you need to do some troubleshooting Proceed to step 10 10 Troubleshooting the FlexNet license server a Double check that you entered the correct file paths in step 7 b View the license file by right clicking on the license lic in the FlexNet directory and selecting Open With c Select Notepad fro
141. port is generated via GeneSight Lite The following lessons will be presented within this tutorial e Image Loading and Manipulation Describes how to load an image into ImaGene and use the controls on the Image tab to enhance the on screen display of the image e Grid Placement and Manipulation Describes how to create a grid and adjust it to fit the image properly e Quantifying Normalizing Image or Image Pairs Analyzing paired images and Saving the Data Describes the quantification and subsequent output of the data e Generating a Report via GeneSight Lite Describes report generation using ImaGene built in data analysis module GeneSight Lite Image Loading and Manipulation 1 Launch ImaGene from the desktop button or from the ImaGene folder within the Windows Start Menu 2 Click on Load Images icon under the Main tab Images Grid Gene Info Process 103 a Browse to the Samples folder within the ImaGene folder The location is typically C ImaGene SampleData Select 17hrs1 gel and 17hts2 gel images Multiple selections can be performed by holding the lt ctrl gt key on the keyboard and left click xi Look in 17 hrnew File name MM hrst gel 1 hrs gelr Files oftype Image files tif gel int Click the Open Button to complete the image loading Once the images are loaded and are visible within the Main image display located along the right hand side of the main ImaGene window t
142. pot Finding Seamentation Quality Flags Measurements Normalization Analysis Controls and Alerts Image Preferences Measure Normalized Signal Mean M Omit Flagged Spots Reference Channel i Channel 1 i Channel 2 Organism Human nalysis type Expression Parameters Fold threshold e o Load Save As Close Analysis Settings Click on the Make measurements button in the Main Tab Once the quantification completes the Analyze tool is activated However the settings for Normalization and Analysis must be chosen from the Settings Tool see sections 1 2 3 and 1 2 4 for a full description a 127 Composite Red Rat Agilenttit Green Rat Agilent ti Red_Rat_Agilenttifseg Green_Rat_Agilenttif seg Plats Normalized Data Analysis Annotati Difference KPro25G 03 00S5 cox1 6329507 144029455 6 073436 A_43_P21252 Q0075 COX1 8 090106 6 048014 p0 042092323 UNCHANGED 43 PF22195 _90110 MRNA 643 P11302 _q0250 cytoc brOie r ino 7 062393 F S18510F 02541175 UNCHANGED arO02c a p2 6 4552083 7 995527 5443444 42 P5122322 fydrOS4o r E2 9 964594 0 130186 1 145792 MCHAMGED 4 To Save the Raw quantification table results click on the Save button located under the Results Tab Selected rows 0 Export XML Right click to mark rows M Specify a file name and location to save the data file 6 Using Windows Explorer or My Computer na
143. pot finding in order to properly determine the location of the signal value Depending on the type and characteristics of the array if spot finding is not performed the resulting quantification may be inaccurate 64 ImaGene Parameter Settings ol x Measurements Normalization Analysis Controls and Alerts Image Preferences Spot Finding Segmentation Quality Flags J Find Negative Spots V Enforce Grid Constraints Local Flexibility 3 0 pixels Global Flexibility Rigid Flexible Load Save As Close The following parameters affect how ImaGene performs spot finding Find Negative Spots While not a problem for most arrays negative spots can potentially cause problems for ImaGene spot finding If the image may contain negative spots BioDiscovery recommends leaving this option checked When Find Negative Spots is enabled ImaGene will look for negative spots while also looking for regular spots Note lf all the signal values within the image are negative due to specific scanner settings then it may be a necessity to invert value for the images Occasionally the scanner software does not save information about which values are high and low within the tiff image The result is that ImaGene does not know whether white or black pixels are the high values Ifall signal values are negative which can be deduced through quantification or use of the intensity tool then select Invert Values and re quantify
144. r later analysis Like wise results can be reviewed to determine processing quality 2 4 1 Preparing for batch Processing Prior to performing batch processing a configuration file and either a grid or a template file must exist To generate these files perform the followings l Launch ImaGene if the software is not already started Load a sample image that is indicative of the group of images to be batch processed The next step is to setup the configurations to be used To do this open the ImaGene Parameter Settings Window by selecting File followed by settings from the menu bar For this example be certain AutoSegmentation is checked located under the segmentation tab and that all the measurements are checked located under the measurements tab Go to Quality Flags tab check Empty Spots set empty spot threshold to 1 Check Poor Spots and click Change Parameters button and click Default on Quality Parameters dialog close dialog Finally click the Save As button to save the 144 configuration file to the file system to a location you choose Remember this location as we will recall it shortly ImaGene Parameter Settings Spot Finding Click the Close Button to exit the ImaGene Parameters Settings Window The next step is to perform grid placement To do so right click on the Grid Panel and select Create Grid Enter the parameters for creating the grid Click the Place Grid Button and click on the four corner spot
145. r s suppliers This Agreement does not convey to Licensee any interest or rights in or to the Software and the accompanying documentation except only a limited revocable right of use in accordance with the terms of this Agreement All rights not expressly granted are reserved by Licensor and its suppliers Licensor may assign this Agreement to a third party at any time 4 RESTRICTED RIGHTS Licensee will comply with all applicable laws regulations treaties or other agreements in connection with its use of the Software and accompanying documentation Without limiting the foregoing Licensee hereby covenants that neither the Software and the accompanying documentation nor any information or know how embodied in such Software and accompanying documentation will be directly or indirectly provided transported or removed or authorized to be provided transported or removed in contravention of any export laws regulations or decrees of the U S Government or any agency thereof This Agreement is subject to termination by Licensor in the event Licensee fails to comply with any such laws regulations or decrees 5 LICENSE FEES Subject only to section 8 of this Agreement the license fees paid by Licensee in consideration of the License granted under this Agreement are non refundable and shall not be returned or credited to Licensee under any circumstance including but not limited to any request for a pro rata refund by Licensee or any reason for termin
146. rable internal end user license the License to use except for any commercial diagnostic purpose or use the basic software product entitled ImaGene tm version 7 and those software modules expressly authorized in writing by Licensor if any the Software and the accompanying documentation in the form delivered to Licensee If Licensee would like to use the Software and accompanying documentation for any commercial diagnostic purpose or use Licensee will need to purchase a separate and additional license from Licensor Unless Licensee has requested and expressly obtained written permission from Licensor and until such time that Licensee has paid a multiple licensee fee for the concurrent use of the Software the Software is licensed as a single product and notwithstanding the fact that the Software itself does execute and or access multiple central processing units CPUs concurrently Licensee shall not separate execute or access the Software for use on more than one CPU at any one given time Subject to Licensee s purchase of more than one License the License granted hereunder is for use only upon a single stand alone computer and only one instance of the Software may be executed and or accessed at any one time 194 where such computer upon which the Software is executed and or accessed is owned leased or otherwise substantially controlled by Licensee Subject to Licensee s purchase of more than one License neither concurrent u
147. rid placement tool once the image and the grid have been loaded The tool will automatically move the grid to the proper location 94 Because the Auto Grid Placement Tool is based on the same grid placement algorithm that Batch Processing uses the tool is also beneficial to test grid placement on a set of images prior to setting up a batch For example instead of wasting time trying to process a poor quality image through the batch processing module first try the Auto Grid placement on the image If the Auto Grid Placement cannot locate the proper array geometry then the mage will probably need to be processed manually Note It is important that at least 60 of the spots in each row and each column of every sub grid are visually resolvable in order for the grid placement algorithm to find the correct grid location In case the image has some spots that are a priori blank or empty please assign the gene IDs starting with BLANK or EMPTY or include their IDs using the Paramater Settings panel as a control type called BLANK or EMPTY This way ImaGene will take possible absence of the signal in those spots into account and produce reliable results Note If image contains bright control spots designed to provide reliable grid placement and that are brighter than any other spots throughout the image assign corresponding gene IDs to a control type named BRIGHT and ImaGene will base auto grid placement on spots from that
148. rt for the Northern District of California Severability If any provision of this Agreement is held to be unenforceable this Agreement will remain in effect with the provision omitted unless omission of the provision would frustrate the intent of the parties in which case this Agreement will immediately terminate Integration This Agreement is the entire agreement between you and Sun relating to its subject matter It supersedes all prior or contemporaneous oral or written communications proposals representations and warranties and prevails over any conflicting or additional terms of any quote order acknowledgment or other communication between the parties relating to its subject matter during the term of this Agreement No modification of this Agreement will be binding unless in writing and signed by an authorized representative of each party Remedies It is understood and agreed that notwithstanding any other provision of this Agreement your breach of the provisions of Section 3 of this Agreement will cause Sun irreparable damage for which recovery of money damages would be inadequate and that Sun will therefore be entitled to seek timely injunctive relief to protect Sun s rights under this Agreement in addition to an and all remedies available at law Nonassignment Neither party may assign or otherwise transfer any of its rights or obligations under this Agreement without the prior written consent of the other party except that S
149. s Agreement effective unless Licensor specifically so consents by separate written agreement Failing such agreement and if this Agreement fails to meet the U S Government s stated needs or is inconsistent in any respect with federal law the U S Government agrees to return the Software and accompanying documentation unused to Licensor The Contractor Licensor Manufacturer is BioDiscovery Inc 100 North Sepulveda Blvd Suite 1230 El Segundo CA 90245 19 INDEMNITY Licensee acknowledges that Licensor has no knowledge of or control over the uses of the Software and accompany documentation made by Licensee Licensee agrees to defend indemnify and hold Licensor harmless from and against any and all losses liabilities damages costs and expenses including but not limited to reasonable attorneys fees arising out of or related to any suit claim or proceeding relating to the use of the Software and accompanying documentation including without limitation any loss related to Licensee s failure to conform to the requirements of section 4 of this Agreement 20 PATENTS This product is covered by one or more of U S Patent Nos 6 349 144 6 577 956 6 633 659 6 674 882 6 990 221 6 731 781 6 990 221 and 7 099 502 Purchase of the License to the Software conveys to Licensee only the nonexclusive non transferable revocable internal right under the applicable aforementioned patent s to use by Licensee only the Software only for rese
150. s for each of the spots prior to being saved All measurements previously selected to be quantified including all quality measures are indicated here Additional information regarding flagging and spot location information 1s provided Each section of the data as presented by a grouping of columns is color coded to facilitate easy review of the analysis The following color codes describe the table Red Quality flag code Cyan Quantified intensity values mean median quality measures etc Purple Spot location related information Yellow Quality criteria failures and controls warnings 33 3435 7 339593333 1 8 47 2265157094 55 5615 763546 66 2060301507 41 796875 47 3009350315 149 97 T2 4493392070 680995215311 51 0000000000 24 026H6GGG 116 180602006 J Note By default these colors are grouped together but you can drag and drop the column headers to change this order Should the default order of the columns change you can restore the columns to the default order by using Restore default column order button in the bottom of the table The default measurement order in the table is as follows Field Name of a field where the spot is located Metarow Number of metarow in the metagrid where the spot is located Metacolumn Number of metacolumn in the metagrid where the spot is located Row Number of row in the subgrid where the spot is located Co
151. s of the array Specify a gene ID file to be used with this array To do so select Grid from the menu bar followed by Load Gene IDs Locate and select the study Gene ID file within the ImaGene samples directory Note that the Gene ID while helpful is not required for batch processing The final step is to save a template that can be used when setting up the batch To save a template select Grid followed by Save Template from the menu bar Specify a name and save this to the location of your choice 145 Selection Auto Measure Tools He Load Grid oka Save Grid 5 Clear Gene IDs Load Template fi Save Template Importtemplate as GAL GEML or MAGE Export template as GAL GEML or MAGE 2 4 2 Adding Entries to a Batch Once the configuration file and the template files exist these can be combined with one or more images to create a batch To add entries to a batch 1 From the menu bar select File followed by Batch Editor els Edit Grid Selection Auto EN Load Images ee Remove Selected Images lest Review Results Fd Save Display Image Settings Exit 2 Click the Add Batch Entries Button on the Batch Editor Window 3 The Create Batch Entry window appears allowing us to specify the files previously created as well as other information First click the Images button and browse to the ImaGene samples directory Select all image files that you plan to batch process Holding the lt ctrl gt key while
152. se of the Software and accompanying documentation In no event shall Licensor and or Licensor s suppliers distributors and or agents be liable for any indirect incidental punitive consequential special or exemplary damages for any lost profits or savings or for any loss of data or loss of use of equipment even if Licensor and or Licensor s suppliers distributors and or agents have been advised of the possibility of such damages and regardless of the form of action whether in tort contract or otherwise SOME STATES DO NOT ALLOW THE LIMITATION OR EXCLUSION OF LIABILITY FOR INCIDENTAL OR CONSEQUENTIAL DAMAGES SO THE ABOVE LIMITATION OR EXCLUSION MAY NOT APPLY TO LICENSEE 10 TRADEMARK ImaGene tm GeneSight tm and GenePie tm are registered trademarks of Licensor No right license or interest to such trademarks is granted hereunder and Licensee agrees that no such right license or interest shall be asserted by Licensee with respect to such trademarks 11 NOTICE All notices required or provided under the terms of this Agreement shall be given in writing to all parties and may be delivered by First Class U S Mail postage prepaid U S Registered Air Mail postage prepaid overnight air courier courier charges prepaid or facsimile Notices shall be effective as follows five 5 calendar days following mailing by First Class U S Mail postage prepaid seven 7 calendar days following mailing by U S Registered Mail postage prepa
153. se on two or more computers nor use in a local area network or other network is permitted Upon having purchased and obtained written consent from Licensor to hold more than one License to the Software Licensee may concurrently load use or install the Software upon the number of computers or CPU s for which Licensee expressly holds a License Upon loading the Software Licensee may retain the Software for backup purposes only In addition Licensee may make one copy of the Software on a second set of diskettes or on compact disc or cassette tape for the purpose of backup in the event the original diskette s or compact disc s for the Software is damaged or destroyed Licensee may make one copy of the User s Manual for backup purposes only Any such copies of the Software or the User s Manual shall include Licensor s copyright and other proprietary notices Except as authorized under this paragraph no copies of the Software the accompanying documentation or any portions thereof may be made by Licensee or any person under Licensee s control or authority Licensee agrees that it will not use the Software for any purpose that is illegal or in any way that might result in any loss of its or any third party s property or information Licensee agrees that it shall have the sole responsibility for protecting its data used in connection with the Software The terms and conditions of this Agreement shall apply to all additional subsequent or multiple
154. selecting the images allows several files to be selected at once 146 Create Batch Entry fx m Next click the Gird Button Either a grid or a template may be specified here Browse to and select the template we created previously Notice the Gene ID selection below the Grid is now not enabled This is because we previously specified a gene ID and associated it with this template Hence there is no need to do so again Click the Configuration button and specify the configuration file previously created The last parameter to specify is the Output Directory ImaGene needs to know where to store the quantified data files as well as the sst snapshot files Click the OK Button to add the entries to the batch Notice within the main Batch Editor window there are four entries corresponding to the four images previously specified If another set of images is to be batch processed this process of adding entries can be repeated as many times as needed 147 Imagene Batch Editor 9 Click the Save Batch button to save this batch for possible reuse later From the Save As window specify a location and file name for the batch file 10 In the File Format s panel select any or all of the formats that you want the batch results to be saved in by checking their option boxes 148 Imagene Batch Editor Load yaaa a Cave Batrh Run 11 Finally click the Run Button to begin the batch A small Batch Processing Window appears
155. sis Quick Start Guide ImaGene was designed to be the fastest and easiest software solution for the quantification of microarray images The description below outlines the essential steps required to set up ImaGene For additional information please view the remaining sections of this user manual Setting Up ImaGene Install ImaGene Submit registration information and request a license from BioDiscovery Start ImaGene Load an image Establish Settings Perform Grid Placement Quantify the Image Run the Analysis oe ae ee Part 1 Lab Users Guide 1 1 ImaGene Main Window Overview The ImaGene Main window is the primary program interface It serves as the focal point of all work within ImaGene This window is utilized to among other things load images place grids produce quantified data and review results This chapter identifies the components of this window and explains how each fits into the array analysis and feature extraction process Fie Edt Grid Selection Auto Mean Toot Sjel alelelalalalelrie goals ECE Man Preview ceposte Sampie tit Sorgenti alajsjajajs Elsa rega imagas m Sangin __ Cirera MlM Fiinta Dept Colors Grid Menu Bar Located along the top of the window Click on one of the menus File Edit Grid Selection Auto Measure Tools or Help to view the options available on the selected menu The following options are availab
156. so a Ao o foo As RNAS 5 p69os167 O 8047106 8 989404 Af Ao fo A6 RPMASAM3 A2 M3754451 R B93619064 6 949245 so n Ao pR fA ReMaasma 5827305 BATE 115629 ao A A pR R Rma OB fomsos293 D 929753 9683414 a n A pR B Remans B o parsoe59 O 6717261 9 940787 a A of pR Remoa g pgs D 259812 9459071 Af Ao R 6 RPnSes3 Mi 787025 D SOI f10 896739 Af A o pR B RM55 Ko faeasairo D 9156521 9589938 a n A R e RME a priere D NSIS 8 131852 A A of 2 B RP1 662H24 B f43160905 pM fi0 505361 Ah 4 2 8 Remaoswae penses p basoss hos7o025 RAJ so n A R fo Ren7esoir Ar poea2663 B 64156911 8441132 a A Ao o BR mo o RPNA23E2 f 204192811 2 499459 6254031 aeo A fo pR f2 RPn oa6 3 fo3z82901 SBS 12743311 a an A pR m Rascas ko oo p2874378 D 9999847 67277 Af no o BR of RP atta po fnasasoosa NB fl1 220811 so A A R f5 RP 11 26M6 fa p4so4se22 O 6854649 9 20664 ao A Ao R f6 RPn 24m4 oh pozeasae7 PSS 6733376 e h p B p peaa pm psw p P S EELT GS E 42 1 2 4 Analysis Tab Expression and CGH Modules Starting with ImaGene 7 the normalization settings have been moved to the Analysis tab of the ImaGene Parameter Settings window Depending on the type of ImaGene license purchased the Analysis tab will either indicate Expression or CGH settings For users licensed for both Expression and CGH modules an radio buttons allow easy switching between the 2 types of analysis The Analysis tool in Expression mode will select up and down reg
157. spects of array image analysis Proper segmentation yields more robust data free from contamination and other adverse effects 66 Be ImaGene Parameter Settings B x Measurements Normalization I Analysis a Controls and Alerts Image Preferences Spat Finding E Flee NCES os cunwor Quality Flags Background buffer 3 0 M Auto Segmentation Background width fis o JY Do mot allow donut shapes Signal percentages Background percentages Low High Low High Os 100 Os 100 100 100 Load Save As Clase ImaGene supports two primary types of segmentation manual and automatic With Manual Segmentation the appropriate values must be specified for ImaGene to use when analyzing the image Often these proper values can be arrived at through experimentation or experience Alternatively through a robust patented statistical approach ImaGene can calculate the appropriate segmentation parameters automatically Under normal circumstances BioDiscovery recommends use of the Automatic Segmentation due to its superior contamination removal capability Regardless of the segmentation used both methods assist is in providing the highest quality data available Listed under the Segmentation Tab are the following parameters e Background Buffer Backgrouncg hunter 20 The distance in pixels between the signal and the background measurement regions 67 Within this region all pixel values are ignored during quant
158. sured in pixels 1 8 7 Intensity Tool The Intensity Tool displays the raw pixel intensities within the Status Bar located under the Main Image Panel After selecting the tool simply position the cursor over the pixel of interest and the pixel intensity for each image will be displayed Note The values provided here are raw intensities meaning they are not mean or median values but rather single pixel values 1 8 8 Flagging Tool 97 m ne The Flagging Tool EJ provides a method of manually flagging spots within ImaGene While spots may be flagged or marked for a variety of reasons the most common reason is due to a defect within the spot or array Once a spot has been flagged a numerical value indicating the type of flag is then denoted in the output along with the quantified data The flag value can then be used in any data analysis software such as BioDiscovery s GeneSight or can be used in post analysis quality control measures In addition to the numerical value a symbol is also placed on the spot to make the flag visually unique Each symbol corresponds to a different flag ImaGene supports a variety of automatic and manual flagging types To change the type of flag right click the mouse and select from the context sensitive menu the desired manual flag The following is a list of possible flags and the corresponding values Flag Empty Spot Flag O Poor Spot Flag O Negative Spot Flag O Remove Flag
159. t you could select Edit gt Undo to cancel this action and in effect deselect the spot Redo Restores the last canceled command For example if you used the Undo command to cancel a spot movement you could select Edit gt Redo to move the spot again Create Grid Launches the Create Grid window where the essential information about the design of the grid must be entered Load Grid Displays the Load Grid dialog box This is utilized to select and open a previously saved grid grd file 9 e Save Grid Displays the Save Grid dialog box This is utilized to save the displayed grid to a grid grd file e Clear Grid Removes all displayed grids from the Grid Display Panel e Load Gene IDs Displays the Load Gene ID File dialog box This is utilized to select and open a gene ID txt file e Clear Gene IDs Removes the displayed gene ID information e Load Template Displays the Load Template dialog box This is utilized to select and open a template tpl file A template is a grid file which contains gene ID information e Save Template Displays the Save Template dialog box This is utilized to save the displayed template to a template tpl file e Import template as GAL GEML or MAGE Displays the Import Template dialog box This is utilized to select and open a template file as e Array Lists gal http www moleculardevices com pages software gn_genep ix_file_formats html gal e GEML
160. t CONTROL_SPOT gt false lt CONTROL_SPOT gt lt BLANK_CHECK gt false lt BLANK_CHECK gt lt BACKGROUND_BUFFER gt 2 0 lt BACKGROUND_BUFFER gt lt CONTROL_TYPE_LOWTHRESH_0 gt 150 0 lt CONTROL_TYPE_LOWTHRESH_0 gt lt SATURATION_FLAG gt false lt SATURATION_FLAG gt lt AUTO_SEG gt true lt AUTO_SEG gt lt OPEN_PERIMETER gt true lt OPEN_PERIMETER gt lt BLANK_CV gt 5 0 lt BLANK_CV gt lt CONTROL_TYPE_ID_N_1 gt l1 lt CONTROL_TYPE_ID_N_1 gt lt NORM gt Subtract percentile lt NORM gt lt CONTROL_TYPE_ID_N_2 gt 2 lt CONTROL_TYPE_ID_N_2 gt lt CONTROL_TYPE IFUPALERT_2 gt false lt CONTROL_ TYPE _IFUPALERT_2 gt lt CONTROL_TYPE_IFLOWALERT_0 gt true lt CONTROL_TYPE_ IFLOWALERT_0 gt lt NEGATIVE_AND gt true lt NEGATIVE_AND gt lt SIGNAL_FLAG gt false lt SIGNAL_FLAG gt lt WINDOW_LENGTH gt 5 0 lt WINDOW_LENGTH gt lt ImaGene_Parameters gt 5 1 5 Batch File The ImaGene batch file is an XML file containing links to all the configuration files required to batch process images The file is created saved and loaded from within the Batch Editor Window Similar to ImaGene configuration files the contents are only relevant for advanced users and those who need to create a batch file from scratch As ImaGene can also be launched from the command line creating a batch manually and 189 passing in this information as a command line argument provides a viable alternative than simply running the software from the Batch Editor Listed
161. t D SNS aff B beeoret b hosssorz hesr hosesoos h1s4 a ho ho h k Genetor gt 0 mazza hzsoz8 h1325308 2 05 aff amp Genetoritag gt h2oseer2 h13a2288 hiesssmn 1133 a ho h h 6 Genetores 0 moso hosess2s h1219604 0 46 ah NF eom homses h21as7i3 h0284388 218 a ho ho h e _Genetor ot 0 h2sraeoe homnsos h2303039 0 73 ahh B Geneto es 0 honz hisimer hossz352 M153 ao ho ho h ho _Genetot c 3 0 maeeere B73ass7 hisoseos beas aff h Genetot es 0 b263853 118067768 b285154 h20 af ho p h Genetot es 0 ass2o6 b1essss Worses B322 ah bb Genetores 0 bross hiaoosa Beori hiss ao h h 2 b Genetores 0 106493 fhi7sosos ho720339 163 aoho ho b k Geneto gs 0 missas hi7erees h1483683 1183 a ho h b 6 beemoisab mamos hizetsr3 h1434829 1 80 aff b b Genetores 0 masse hiiromi h1414387 EE Ah h o b p Genetotms gt m nan meere hiaz harm af 2B Genetot os 0 mes homes hiess hoso aff 2 b _Genetot av 0 hoz s9 Mirar ho432823 1 85 af 2 m beoreo baseras 114sosa3 bBaeasoe h156 Po ho h b h beoreo b hon hen hn Non aff b p _Genetotes 0 masorss hioraszs h13195e8 1 08 af BB Genetores 0 hoseznz hiam2os foaoeses _ 50 af ho b k Genetorgs p hosssea fi296208 hosssess 223 aff b b Genetorigag gt marar hiees29 h140281 1685 aff b b bemos 0 masesa hizerser hisossaz hiye ah BF beomo b maros horra hisroraa hzo aff 8 b Genetoog 0 mesmo h2rsssrs hisas 2516 o h ho b b Ge
162. t or contact us via E mail info biodiscovery com Phone 310 414 8100 United States Mail 2121 Rosecrans Ave Suite 3315 El Segundo CA 90245 USA 193 Software License Agreement and Limited Warranty THIS SOFTWARE LICENSE AGREEMENT AND LIMITED WARRANTY AGREEMENT IS ENTERED INTO BY AND BETWEEN BIODISCOVERY INC LICENSOR AND YOU WHETHER YOU ARE AN INDIVIDUAL OR AN ENTITY LICENSEE READ THE FOLLOWING TERMS AND CONDITIONS CAREFULLY BEFORE OPENING THIS SEALED PACKAGE CONTAINING THE ENCLOSED SOFTWARE OR BEFORE PROCEEDING FURTHER WITH THE USE OR INSTALLATION OF THIS SOFTWARE BY YOUR OPENING OF THE PACKAGE CONTAINING THIS SOFTWARE OR BY INSTALLING OR UTILIZING THE INSTANT SOFTWARE YOU AGREE TO BE BOUND BY THE TERMS AND CONDITIONS SET FORTH HEREIN IF YOU DO NOT AGREE TO BE BOUND BY THE TERMS AND CONDITIONS YOU MUST RETURN THIS PACKAGE AND THE SOFTWARE WHICH IT CONTAINS TO YOUR PLACE OF PURCHASE NO LATER THAN 10 DAYS FROM YOUR RECEIPT OF THE SOFTWARE UPON RECEIPT OF THE UNOPENED PACKAGE YOUR PURCHASE PRICE WILL BE REFUNDED THIS SOFTWARE PRODUCT IS PROTECTED BY COPYRIGHT LAWS AND INTERNATIONAL COPYRIGHT TREATIES AS WELL AS OTHER INTELLECTUAL PROPERTY LAWS AND TREATIES AND THIS AGREEMENT THE SOFTWARE PRODUCT WHICH IS THE SUBJECT OF THIS AGREEMENT IS LICENSED UNDER THIS AGREEMENT NOT SOLD 1 LICENSE GRANT For consideration promised and or received Licensor hereby grants to Licensee one nonexclusive nontransfe
163. tab the raw data can be transformed by taking background correction log transformation and or normalization There are a variety of data transformation options lt ImaGene Parameter Settings Ioj x Spot Findinn Seamentation Quality Flaqs Measurements Normalization Analysis Controls and Alerts Image Preferences Background Correction Correction Local x Measure Bckgr Mean Sliding Window Window length p 0 Normalization Algorithm Normalization type ETIE Scope Global Using control spots fuse all spots hi Smoothing 0 2 V Take Log Load Save As Close Background Correction Background mean median or mode can be used in background correction After selecting the measure background correction can be applied locally globally or not at all Local Global or None When applying the background correction locally a check box Sliding Window is displayed When it is not checked the local background value of each spot is used for correction If the Sliding Window box is 39 checked a value for the Window length must be specified The window length identifies a small square region of spots with the spot being background corrected as the center spot The median of the background values of all spots is used for background correction When applying the background correction globally users can select between Subgrid based Using signal from control spots
164. th the license lic file can be anywhere on your computer but we put it in the C FlexNet directory to make it easier to keep track of 6 Configure the license services a Start the LMTools program by double clicking on Imtools exe in the FlexNet directory Click on the Config Services tab c You will see the following screen LMTOOLS by Macrovision Corporation http waww macrovision com oj x File Edit Mode Help Save Service service Mame FLEXIm Service 1 Remove Service Configure Service Browse Path to the Imgrd exe file JC FlexNet Imgrd ene es ets Path to the license file JC FlexN etlicense lic Browse Path to the debug log file C FlexNet debug log Browse View Loa Eose Log v Start Server at Power Up IW Use Services d You can leave the Service Name as its default FlexNet Service 1 e Enter the Path to the Imgrd exe file or click the Browse button to the right In this example it is C FlexNet Imgrd exe f Enter the Path to the license file or click the Browse button to the right In this example it is C FlexNet license lic 176 g Enter the Path to the debug log file or click the Browse button to the right In this example it is C FlexNet debug log h Make sure you place a check mark in both Start Server at Power Up and Use Services Note that the Use Services checkbox may be disabled under Windows 95 98 ME 1 Click on the Save Service button j Ans
165. th the quantified values in the text output file and visualization tools For example if a spot is selected from a scatter plot within GeneSight Lite the specific Gene ID associated with the selected spot in addition to all the other detailed quantification information will be displayed To accomplish this ImaGene needs the Gene ID file to associate a reference ID to each spot in the array of spots in the image ImaGene supports two formats for the Gene ID file The first format will be familiar to existing ImaGene users since the basic structure is the same across versions The second format is based upon the new multi level grid structure available in ImaGene This new format takes advantage of the use of fields or unique grid meta grid structures To deal with the unlimited number of possible fields and grid structures an additional column needs to be added to the Gene ID file Note Choose the Gene ID format based on your array structure and preferences However when creating the Gene ID file do not combine the two formats in the same file as ImaGene will not be able to process the hybrid file format Note In ImaGene 6 0 you can add unlimited number of annotation columns after gene ID column in gene ID file and these are displayed in the results as Annotationl N For N such columns 182 Gene ID Format 1 Traditional Format Listed below is a brief description of the various parts of the Gene ID file as well as the precise s
166. thew Austern and Alexander Stepanov Check the JAL home page http reality sgi com austern_mti java for more info Copyright 1996 Silicon Graphics Inc Permission to use copy modify distribute and sell this software and its documentation for any purpose is hereby granted without fee provided that the above copyright notice appear in all copies and that both that copyright notice and this permission notice appear in supporting documentation Silicon Graphics makes no representations about the suitability of this software for any purpose It is provided as is without expressed or implied warranty 202 Java 3D 1 2 1 01 Binary Code License Agreement SUN MICROSYSTEMS INC SUN IS WILLING TO LICENSE JAVA 3D 1 2 1_01 TO YOU ONLY UPON THE CONDITION THAT YOU ACCEPT ALL OF THE TERMS CONTAINED IN THIS LICENSE AGREEMENT AGREEMENT PLEASE READ THE TERMS AND CONDITIONS OF THIS AGREEMENT CAREFULLY BY CLICKING ACCEPT BELOW OPENING THE PACKAGE DOWNLOADING THE SOFTWARE INSTALLING THE SOFTWARE OR USING THE SOFTWARE YOU ACCEPT THE TERMS AND CONDITIONS OF THIS AGREEMENT IF YOU ARE NOT WILLING TO BE BOUND BY ITS TERMS SELECT THE DO NOT ACCEPT BUTTON AT THE BOTTOM OF THIS PAGE AND THE INSTALLATION PROCESS WILL NOT CONTINUE RETURN THE UNOPENED SOFTWARE TO THE PLACE OF PURCHASE FOR A REFUND OR DO NOT DOWNLOAD THE SOFTWARE Licensed Software Licensed Software means the JAVA 3D 1 2 1_01 software in binary
167. tion in the grid is computed fitting least square lines to circle centers in every row and column CM X X coordinate of the center of the mass of spot s signal region CM Y Y coordinate of the center of the mass of spot s signal region CM Offset Offset in pixels of the spot s center of the mass from the expected position in the grid CM Offset X X offset in pixels of the spot s center of the mass from the expected position in the grid CM Offset Y Y offset in pixels of the spot s center of the mass from the expected position in the grid Min Diam Diameter of the circle inscribed into the spot s signal region Max Diam Diameter of the circle inscribed into the spot s signal region Control Name of a control type for current spot See Settings Controls Alerts panel Failed Control 0 if the control passed all tests 1 if at least one of the tests failed Background contamination present 0 if the spot passed background contamination test 1 1f it did not see section 1 4 Signal contamination present O if the spot passed signal contamination test 1 if it did not see section 1 4 Ignored failed O if the spot passed ignored percentage test 1 if it did not see section 1 4 Open perimeter failed 0 if the spot open perimeter test 1 if it did not see section 1 4 Shape regularity failed 0 if the spot passed shape regularity test 1 if it did not see section 1 4 Perim to are
168. tions er Add to Data Set P Add as Experiment P Add as Control C m O P Add as Replicate Add Repeated Experimental Conditions to Data Set Note if you have only one file with measurements drag this file directly into the right side panel of DataSet Builder and hit Done 4 Click on Pair Data Perform Ratio button the new data set will show up in the panel on the right hand side Click Done button on Builder s toolbar Builder will ask you to enter such information as experiment date and user name to tag the data set 154 EA Condition 1 Enter Experiment Information Condition 1 8 1 0 04 Specify the information and click Ok Additional dialog box will pop up asking you to select measurements that you want to import into GeneSight Lite E 1205_r txt Select Experiment Columns Select measurements and click OK New data set will be created and corresponding measurements will be shown on the upper left side of the GeneSight Lite window For more information on how to build a new data set please refer to GeneSight manual Chapter 6 155 Bo Gene Sight 4 1 Like fw Shak Foitian A Diuna gs Condition 1 pair E Mean clin E 00 Mean ratio E Conditi 1 Maa E mean iE FE mean i i Joea U E GI BG Weari 1 FGenei a EA Conamon 1 cartralp PR E Mein a E siGene3 0 fad ae me GT T Gened 2 Genes f 5
169. to its second mode In this mode Wizard will just prompt the user what tool is being used and how to use it Using ImaGene Wizard the user has an opportunity to learn how to use the software in an interactive fashion 83 1 6 Batch Editor and Batch Processing The ability to batch process images is an optional component that can be added to the basic ImaGene A batch is simply a series of similar images with the same settings to be processed automatically With this module ImaGene can be used in high throughput environments where large numbers of arrays are processed everyday The same data are exported from ImaGene whether performed in batch mode or not During batch mode ImaGene display a simple intuitive progress window indicating information about the current image and state of the batch 1 6 1 The Main Batch Editor Window The ImaGene Batch Editor Window allows batches to be created saved and loaded Batches can be created and saved for later use or as a template that may be modified at a later date This window allows for the selection of channels of multi channel image composite for processing Es Imagene Batch Editor E z ojx Entries Available Unlimited Sample B SampleAB Add Batch Entries Sample B Sample B Remove Selected Edit Selected Entry CJ Show Full Path E Channel 1 File Formatts Text LJ MAGE ML LJ GEML Save Batch 84 Batch Table Once a b
170. ton to complete the installation and close the install program Prior to use please review the next section on licensing ImaGene 4 1 2 ImaGene Update 159 If computer is connected to the Internet ImaGene automatically checks for any update available from BioDiscovery at the start up User can control frequency of update checks through menu item Help gt Update ImaGene Help Wizard On Change skins a Customer support center About lmaGene Five different frequencies are available from checking at every start up to not checking at all 5i ImaGene 6 1 0 x Last on line update session Besseseseeseseereselssessresseesssossesssele Daily Weekly Monthly When Prompted Only Check for Updates Now When ImaGene detects a new version available for download the following message will be shown fe4 Update Proceed with on line update Yes Ho To proceed with the update press Yes Your ImaGene will be updated and your old files will be backed up inside ImaGene installation directory When update is complete it will prompt you to restart the software 160 4 1 3 Un Installation Instructions Introduction This section explains how to remove ImaGene from your computer For example if you are no longer using an older version of ImaGene you can use this procedure to remove it from your computer Note If you have a licensed version of ImaGene please contact BioDiscovery Techn
171. tton to save the Quantified data and snapshot file to an output directory of your choice To save the normalized and Analysis Reports you must select the Save Report button from their result panels 115 ImaGene 6 1 Standard w Batch Edition isis Glsisis a sle cie aT alm lm Scatter plot 1fhrs1 gel 17hrs1 gel 4 7hrs2 gel K b 9 J HEE BA Ya _Gene101 d 9 a4 B peram _Gene101 t 9 a6_ 6 2919622 015921 _Gene101 h 9 a8_ 6 Saos pr 690092 _Gene101 j 3 a10 0 Genetoi 19 612 0 ESOS EECEESEA Gene101 d 11 a4 EATA _Gene101 b 13 a2_ 0 _Gene101 d 13 a4 0 sets aroser Gene101 4 13 06 0 pors prer Gene101 j 13 a10_6 1088 5753 bereans m i A m Signal Mean ch1 x lo Signal Mean ch2 Generating a Report Via GeneSight Lite L 2 If further analysis is needed click the GeneSight Lite icon from the toolbar After a brief moment GeneSight Lite and the Dataset Builder will appear For complete information on GeneSight Lite please see the GeneSight User s Manual With the file system window along the left of the Dataset Builder browse to the data file previously generated within ImaGene 116 2 DataSet Builder Experiment 4 Control i E 17hrs1 bt i E 17hrs2 bt El 17hr_GenelDs bt H El 17hrs1 bt i E 17hrs2 bt Perform Ratio amp AQQ p E Repeated Experimental Conditions Select and drag 17hrs1
172. txt to the Experiment Panel Select and Drag 17hrs2 txt to the Control Panel Click the Pair Data Perform Ratio Button Click Done from the Dataset Builder Toolbar a a a 7 Select User Defaults for all Experiments and click the OK Button f Condition 1 Enter Experiment Information Condition 1 a1 ofo4 ES 8 Click OK on the next window to use all experimental conditions available within the data files 117 So 17hrs1 txt Select Experiment Columns xj Select the fields to load MetaRow MetaCal Row Cal O coord Y coord Diameter Flag Wean O Mode O median O Total O Area O Standard Deviation 9 Click on while holding the lt ctrl gt key Mean listed under Condition 1 control first and then Condition 1 in the GeneSight Lite main window Be certain both Mean labels are highlighted DefautDS gs Ff Condition 1 pair pE Mean docBGC difference Le ET Mean docBGC difference cy I iia Condition 1 ft EF EE ene ste be EP Mean locBGC Cy iia Condition 1 ai E S hiean ocho ET Mean locBGC cy 10 Click the Scatter Plot Button from the toolbar Scatterplot 11 Drag with the mouse to select all the genes located along the top right of the plot or those genes with high intensity values 118 Scatterplot o x File Edit Sub Selected Genes Color Scheme Choose URL Help Reference ER Q ma PNI Lasso Zoom Print Find Gene Goto web Probe Drill Down Cluster G
173. ual button is pressed 78 7 Pass Fail image for maximum value whenever the number of spots belonging to this type that violate the maximum expression criterion become higher than the specified percentage the warning box will pop up after the quantification procedure is completed or Update Visual button is pressed 8 Pass Fail image for inter spot CV whenever the inter spot CV of signal means within this type violates the maximum CV criterion the warning box will pop up after the quantification procedure is completed or Update Visual button is pressed Whenever the new type is added its name shows up in the type list Next step would be to add gene IDs that belong to this type a Select a type from the list click on Add gene ID button b A new dialog box will pop up If the gene ID file was previously loaded all of those gene IDs will be listed on the right hand side of the dialog box A selection is made by highlighting the gene ID and clicking Add Alternatively a text field may be used to enter a gene ID and click Add c This field can also be utilized to designate a key for assigning gene IDs The key is created using a symbol For example by adding a key BLANK all gene IDs that start with BLANK are assigned to the current control type d When finished adding gene IDs click Close Alternatively the PO AAUE NE oe button may be utilized to load a list of gene IDs from a
174. ularity the measure that characterizes the closeness of a spot s border to a circular shape The first step of this algorithm inscribes a signal area of a spot into a circle Then the number of non signal pixels that fall within this circle is computed and divided by the area of the circle This ratio is then subtracted from value of shape regularity This variable will range from O highly non circular shape to 1 a perfect circle Whenever this ratio falls below the pre set threshold spot is flagged as low quality 6 Flag for low Area to Perimeter ratio The measure of the spot s quality plays a role similar to shape regularity mentioned in Item 5 above The area of a spot is divided by a square of spot perimeter and multiplied by 47 As a result this measure ranges from 0 highly non circular shape to a perfect circle Whenever this ratio falls below the pre set threshold the spot is flagged as low quality 7 Flag for significant offset from expected position ri Offset Expected position of a spot in the current grid 1s computed by fitting least square lines to columns and rows of the grid Whenever the offset becomes larger than pre set percentage of the distance between grid nodes the spot is flagged as low quality 8 Flag for saturation One may want to flag spots that have significant number of pixels saturated at the level of maximum scanner sensitivity ImaGene flags a spot for saturation if more than h
175. ulated genes according to a fold threshold set by the user CGH mode calculates insertion deletion clustering based on user defined thresholds A reference channel is selected and the user chooses normalized results e g Normalized Signal Mean Median or Mode Spot replicates on the slide are combined their mean value is used and flagged spots can be disregarded in the analysis Omit flagged spots To begin the Analysis click the Analysis button along the processing toolbar or select Measurements gt Analyze Images Grid Gene Info Process Note Channel I and 2 refer to the order of the loaded images For example the first image in the image panel corresponds to Channel 1 43 5 x ImaGene Parameter Settings Spot Finding Seamentation Quality Flags Measurements Normalization Analysis Controls and Alerts Image Preferences Measure Normalized Signal Mean l omt Flagged Spots Reference Channel Channel 1 i Channel 2 Organism Human ai Analysis type Expression Parameters Fold threshold 20 Load Save 4s close Expression Analysis Mode Settings Panel When the Expression Analysis complete an Analysis tab appears in the image display panel with labels of genes for up red color and down green color regulations The results can be saved in a text file by clicking on the Save Report button at the bottom of the panel 44 S Geom De fet Ep l onee ana 515611
176. un may assign this Agreement to an affiliated company 205 Java 3D TM Software Version 1 2 1 01 Supplemental License Terms These supplemental license terms Supplement add to or modify the terms of the Binary Code License Agreement collectively the Agreement Capitalized terms not defined in this Supplement shall have the same meanings ascribed to them in the Agreement These Supplement terms shall supersede any inconsistent or conflicting terms in the Agreement or in any license contained within the Software License to Distribute Sun grants to Licensee a non exclusive non transferable royalty free limited license to reproduce and distribute the binary code form of the Licensed Software provided that Licensee L Distributes the Licensed Software complete and unmodified except for the specific files identified as optional in the Licensed Software README file only as part of and for the sole purpose of running Licensee s Java compatible applet or application Program into which the Licensed Software is incorporated Does not distribute additional software intended to replace any component s of the Licensed Software Agrees to incorporate the most current version of the Licensed Software that was available 180 days prior to each production release of the Program Does not remove or alter any proprietary legends or notices contained in the Licensed Software Includes the provisions of Sections 2 3 4 5 6
177. use a slider on Quality Flags panel of Settings dialog box or type the threshold value directly in the text field 70 Negative spots This tool will automatically flag any spot with the signal mean lower than background mean A green symbol will be displayed on this spot on the image and corresponding flag in the results table will have value of 4 Poor spots ImaGene has a complex tool for detecting low quality spots It 1s designed to accommodate various possible interpretations of spot quality This tool includes 7 different criteria for spot fail pass test that can be used in combination as well as separately To change configuration of this tool click Change Parameters button Change Parameters on Quality Flags panel of Settings dialog box Once you click it the new dialog box will pop up and you will be able to select any combination of the following flagging tools l Flag for background being contaminated Test against whole image Y Background 0 5 06 07 08 09 1 0 Test against the subgrid only 9 9995 The top most check box on quality parameters dialog corresponds to the flagging method based on background abnormality detection The background level of each individual spot 1s compared to the local background level distribution over the image through a t test The resulting p value is subtracted from and is called confidence in contamination presence This threshold can be set at an
178. ved and loaded to the ImaGene Parameter Settings Window Output Directory ImaGene requires the location where to place subsequent output quantification text files after image processing Click browse and select the output directory The location can be either be local or on a network drive OK Once all the required parameters have been specified the OK Button becomes enabled and the entries can be added to the batch Cancel Closes the window and the entries are not added to the batch 89 1 6 3 Creating and Running a Batch Setting up batch processing within ImaGene takes only a few minutes and requires only a few pieces of information to be defined The description below covers the basics for creating and running a batch as well as common tips and tricks for success l A AE aE a a Setup the initial parameters for use within ImaGene This needs to be done the first time prior to running a batch To do this perform the following a Load a sample image b Load a gene ID file c Create and place a grid on the image d Save the result as a template e Set the parameters on the ImaGene Parameter Settings Window f Save these configurations You will probably want to save this to the same location as the template previously created From the ImaGene Menu Bar select File then Batch Editor Click the Add Batch Entries Button Specify the images to be batched For Grid specify the template previously created Like
179. vigate to the data file within the file system 7 Open the quantified data file using either MS Excel or Microsoft WordPad 128 Begin Header version 6 0 0 Beta Date Tue Aug 24 10 08 25 PDT 2004 Image File C TEMP Agilent Rat_Agilent tif Page 1 Page Nam Green Inverted FALSE Begin Field Dimensions Field Metarows Metacols Rows Cals A 1 1 105 215 End Field Dimensions Begin Measurement parameters Segmental auto Signal Low Signal Higl Backgroun Backgroun Backgroun Backgroun 1 End Measurement parameters Begin Alerts Control Ty Minimum t If tested Percentagilffailed Maximum Iftested Percentagilf failed CV threshilftested If failed End Alerts Begin Quality Flags Begin Flagging Settings Empty Spi TRUE Threshold Poor Spots FALSE Negative S TRUE End Flagging Settings Begin Flagged spots of Empty Spots 500 of Poor Spots 0 of Negative Spots 0 of Manually Flagged Spots 0 End Flagged spots End Quality Flags End Header 39 Begin Raw Data Field Meta Row Meta Colui Row Column Gene lD Flag Signal Me Backgroun Signal Mec Backgroun Signal MocBackgroun A 1 1 1 1 Pro25G 0 22062 05 46 8561 24797 5 46 26089 59 45 0789 8 To save the normalized data or analysis results choose the Save Report options beneath those respective data tables Loading GAL GEML or MAGE templates The use of templates through ImaGene provides a convenient and simple way to perform grid placement and associate gene information with c
180. w control type is created by specifying several parameters 11 a Type parameters Type name kl Minimum expression 0 0 Maximum expression H000 0 Maximum C 1 0 Color iii J Highlight on image Pass Fail image for Percentage of Failed controls alloyed Minimum value Panpin pnpa a pagg a a OS 20 40 60 BO 100 Percentage of Failed controls allowed i Mazzimu value i SUE LLELLU CELAA ELLEI CLEES LU LLELLE LEELLE PEE TEELE fo Oo 20 40 60 BO 100 Inter spot cy OK Cancel Type name provide a meaningful name for the type Minimum expression provide a minimum value for signal mean Maximum expression provide a maximum value for signal mean Maximum CV provide a maximum coefficient of variation between signal means for the spots should be gt 0 Highlight on image specify if all spots of this type are to be highlighted on the image If this box is checked the selection of a color for presentation of this type will be enabled Spots of this type will be marked with squares the problematic spots corresponding to violation of above thresholds will be highlighted with diamond shapes inside the squares Pass Fail image for minimum value whenever the number of spots belonging to this type that violate the minimum expression criterion become higher than the specified percentage the warning box will pop up after the quantification procedure is completed or Update Vis
181. wer Yes on the dialog that appears to confirm these settings 7 Select the license services a Click on the Service License File tab b The following screen will appear LMTOOLS by Macrovision Corporation http www macrovision com 2 ioj x File Edit Mode Help Sermvices allow FLEsnet Servers to run in the background Server List Configuration using License File f Configuration using Services FLExIm Service 1 c Select the Configuration using Services radio button d Select the Service Name you used from step 7 8 Start the License Server a Click on the Start Stop Reread tab b The following screen will appear 177 LMTOOLS by Macrovision Corporation http waww macrovision com i iol x File Edit Mode Help FLEXnet license services installed on this computer Stop Server ReRead License File T Force Server Shutdown _ Advanced stings gt gt NOTE This bos must be checked to shut down a license server when licenses are borrowed Using License File C FlexsNetslicense lic c Double check that the Service Name selected is the same as the one you configured in step 6 Click on the Start Server button e The text at the bottom of the window should change from Using License File to Server Start Successful 9 Did the license server start successfully a If yes then you re almost done All you need to do now if configure each of the clients to run the software
182. wing i Time Settings System Tire corms Paciic Standeed Time GMT Time Tue Api 0317 34 47 2002 IF Addie pane ooo TIG Difference From UCT s0 Ethemet Addes ko MSDOS Time 10 34 47 Disk Volume Serial Maree Local Time DEEN FLEXID z Windows Directory CAWINNT d The computer s Ethernet Address is used as the computer ID e The Ethernet Address is a 12 digit hexadecimal number In this example the address is 00065b1bb4ee f The computer s Hostname is used to identify the computer on the network In this example the hostname is BIOINTERN g You can close LMTools by select Exit from the File menu or clicking the small X button in the top right corner 4 Request license file from BioDiscovery a Email support biodiscovery com for your customized license file b Please include 1 The Computer s Ethernet Address from step 4 2 The Computer s Hostname from step 4 3 Your name or the name of who purchased the software 4 Your company 5 Your BioDiscovery Product Serial Number if you ve purchased a copy You can find your serial number on the case of the CD shipped to 175 you or in the accompanying User Registration form If you haven t purchased a copy just list the products you wish to evaluate c BioDiscovery will reply with a small text file called license lic This file contains all of your licensing information 5 Copy license lic into your FlexNet directory In tru
183. wise for configuration specify the configuration previously saved Specify the output directory This is the directory which will store both the data and the snapshot file or sst file Click the Run Button 1 6 4 Saving a Batch Once entries have been added to the batch the batch can be saved and recalled later To save the batch to a file perform the following l 2 3 Click the Save Batch Button In the Save As Dialog that appears navigate to the target location for the file Specify the name of the file A bch file extension will automatically be added to the file name 90 1 6 5 Loading a Batch If a batch file was saved to the file system previously it can be easily recalled and used again The following steps outline the process for loading a batch file 1 Click the Load Batch Button 2 Browse to and select the desired batch file 3 Select Open to load the file 4 The batch entries are now visible within the Batch Editor 1 6 6 Editing a Batch Once entries are added to a batch they can be easily modified thus eliminating the need to create a new batch file To modify an existing entry within the Batch Editor follow these steps l 2 P Select or highlight the desired entry by left clicking on its row within the Batch Table Only one row at a time may be modified Click Edit Selected Entry Button to open the Create Batch Entry Window for editing Modify the desired parameters Click OK to co
184. y between the Original and Segmented images This image is key in determining if the best settings have been entered on the Measurements tab Use this view to modify the settings until the desired signal and background values are included while removing contaminants Histogram Represents the distribution of pixels along the intensity scale for the spot The y axis vertical represents the number of pixels and the x axis horizontal represents the range of intensities of pixels across the spot The histogram is intended for a simple qualitative preview and should not be used for any form of data analysis Within the histogram colored vertical bars indicate specific values The colors and their meaning are e Blue Signal Mean Value e Yellow Signal Median Value e Cyan Signal Mode Value 32 Under the Segmented Composite and Histogram Views colors are used to indicate pixels to be included within the signal and background measurements If a pixel is not colored then the pixel is being ignored and is not being used for calculation of either signal or background values The Preview Panel colors include e Green Indicated the pixel is used in the background calculation e Red Indicated the pixel is used in the signal calculation e Black Indicated the pixel is not used in either calculation of signal or background Quantification Table The Quantification Table displays the quantified numerical intensity value
185. y level between 0 5 and 1 A flag will be issued whenever a particular contamination confidence goes over that threshold Background mode is used as a background level estimate Background level can be tested against the information from all spots on the image or against information extracted only from those spots that fall within the same subgrid as the current spot The latter approach can be useful if subgrids are large enough gt 100 spots and some kind of acceptable background level change is observed from one subgrid to another 2 Flag for the signal being contaminated Signal Y C Combine signal test with background test 71 The next check box corresponds to the signal abnormality detection method This approach was designed to flag any spot with unusually high volatility in pixel wise intensities within the signal area For every individual spot the signal variance is compared to the distribution of signal variance measurements across the image However before a t test analogous to the one for background contamination is performed spots are grouped according to their signal mean measurements This is performed to compensate for a well known observation that intra spot intensity volatility tends to be different for different expression levels This method will ensure that each spot is tested only against spots similar in their expression The resulting confidence number is then compared to the threshold similar to the
186. yntax Header Anything other than the Gene ID information located at the beginning of the file For header information this line must start with a percentage sign The percentage sign is an indication to ImaGene not to read this line as gene information but rather continue until a line is encountered matching the structure found below Columns Metarow Number MR Metacolumn Number MC Subgrid Row Number SR Subgrid Column Number SC Gene ID Annotation 1 Annotation 2 Separator Tabs are utilized to delimit the fields in each row Order ImaGene does not care about the order in which the rows are sorted It s also unnecessary to specify Gene ID information for all the spots in the array Gene ID The Gene ID field can be any alpha numeric string including blank spaces following the Subgrid Column Number A database entry can be specified followed by the gene name as one option Comment Lines Use the percentage sign to indicate comment lines Creating the Gene ID File Any text processing program spreadsheet or database program may be used to generate the Gene ID file CloneTracker software can also be utilized to generate this file CloneTracker manages the information about cDNA plates and the mapping of plates onto the slide It can also generate the necessary gene ID file to be used by ImaGene Insert the accession number followed by a colon and then insert the Gene ID name For example all of
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