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Imagene User Guide

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1. BioDiscovery License Management Wizard A oj xj HodeLocked Floating Computer ID 00 06 5B 18 B4 EE User Namel Joe Green Company Name Green Corp Serial NumberlBon 2345 Ml Use as Serice NT2000 P only Save HodeLocked Cancel d 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 Ifyou do not have a serial number because you re only evaluating this product you can enter BDI DEMO h Ignore the Use as Service checkbox I Click the Save NodeLocked button to save this information and exit 12 Start the BioDiscovery software If the license server is running the software will start up normally 4 4 FLEXIm Server Installation Guide for Windows The default install directory for FLEXIm is C FLEXIm In the following examples it is assumed that this is the directory being used If FLEXIm is installed into another directory then make the appropriate modifications to the path 1 Extract FLEXIm Server files a Double click on the self extracting executable FLEXIm zip b You will see the following screen Winzip Self Extractor FLEXIm Server Installexe j xj To unzip all files in this self extractar
2. Quantification Click the Quantify Button under the Main Tab Once the quantification completes click the Save Button located under the Results Tab Selected Freeze Unfreeze Restore default column order 0 See oS eT Manel Specify a file name and location to save the data file 16 Using Windows Explorer or My Computer navigate to the data file within the file system 17 Open the file using either MS Excel or Microsoft Wordpad Notice the GeneID column contains gene names as defined by Research Genetics ee Eod ee ASIDE eal eae e ainia DFS any IRB EI Du MEL NE im faa 10 e s D P Seis Fda IEE 5 Dain Mon Age 062155 PET SO Ieee File Y Sample DataiknaGereSamplestResSen it Page U Page Hama Immrted TRUE Begn Field Dimensions Frald Metar ws Metecok Rowe Cala Main Fj oO a T2 End Field Dimensions Begn Moasuniment parameters segmentaliized circle Signal Live Signal Higl 1 Backgrown D Backgrawn 1 Backgroun Backgroun J End Moasurement paraenetors Begn Alaris Canteod Ty kleurum tH iasted Fercantagel talad Maximum Wished Parentah faded Cw thrashelt tested i tailed Erd Ales Begn Gualay aetterge Empty Spe TRUE Thresheld J Poor Spots TRUE Begin Poor Spots Parameters Batbgroun FALSE Threshold 155 Barkgroun TRUE gral com FALSE Threshold 1 a Signal con FALSE Ignored pe FALSE Threshold 2 Upan peer FALSE
3. Listed under the Segmentation Tab are the following parameters Background Buffer Parkground buffer 30 The distance in pixels between the signal and the background measurement regions Within this region all pixel values are ignored during quantification 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 Background Width SLUM UML RAY 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 Do not allow donut shapes Elbo ve elis When you select this option ImaGene will make sure that a donut 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 Note The background width will not extend beyond the snip or rectangular boundary around each spot Even though you can set the background to an extremely high value the background measurements will stop at t
4. INCLUDING BUT NOT LIMITED TO THE IMPLIED WARRANTIES OF MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE ARE DISCLAIMED IN NO EVENT SHALL L2FPROD COM OR ITS CONTRIBUTORS BE LIABLE FOR ANY DIRECT INDIRECT INCIDENTAL SPECIAL EXEMPLARY OR CONSEQUENTIAL DAMAGES INCLUDING BUT NOT LIMITED TO PROCUREMENT OF SUBSTITUTE GOODS OR SERVICES LOSS OF USE DATA OR PROFITS OR BUSINESS INTERRUPTION HOWEVER CAUSED AND ON ANY THEORY OF LIABILITY WHETHER IN CONTRACT STRICT LIABILITY OR TORT INCLUDING NEGLIGENCE OR OTHERWISE ARISING IN ANY WAY OUT OF THE USE OF THIS SOFTWARE EVEN IF ADVISED OF THE POSSIBILITY OF SUCH DAMAGE Packages cern colt cern jet Copyright O 1999 CERN 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 1s 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
5. OFFSET gt lt CONTROL TYPE IFUPALERT 0 gt false lt CONTROL TYPE IFUPALERT 0 gt lt SPOT AREA gt true lt SPOT AREA gt lt REGULARITY THRESH gt 0 65 lt REGULARITY THRESH gt lt LOG SCALE gt false lt LOG SCALE gt lt STANDARD CHECK gt false lt STANDARD CHECK gt lt HOT HIGH gt 100000 0 lt HOT HIGH gt lt REGULARITY FLAG gt true lt REGULARITY FLAG gt lt CONTROL TYPE N gt 1 lt CONTROL TYPE N gt lt MEASURE MEDIAN gt true lt MEASURE MEDIAN gt lt CONTROL TYPE IFCVALERT 0 gt false lt CONTROL TYPE IFCVALERT 0 gt lt CONTROL TYPE COLOR 0 gt 52429 lt CONTROL TYPE COLOR 0 gt lt IGNORED FLAG gt true lt IGNORED FLAG gt lt CONTROL TYPE LOWALERTTHRESH 0 gt 0 0 lt CONTROL TYPE LOWALERTTHR ESH 0 gt lt OPEN PERIMETER gt true lt OPEN PERIMETER gt lt CONTROL TYPE ID N 0 gt 1 lt CONTROL TYPE ID N 0 gt lt BLANK HIGH gt 1000 0 lt BLANK HIGH gt lt BACKGROUND HIGH gt 1 0 lt BACKGROUND HIGH gt lt HOT LOW gt 30000 0 lt HOT LOW gt lt IGNORED THRESH gt 25 0 lt IGNORED THRESH gt lt ImaGene Parameters gt 5 1 4 Batch File The ImaGene batch file is an XML file which contains links to all the configuration files required to batch process images The file is created saved and loaded from within the Batch Editor Window As with ImaGene configuration files the contents are usually only relevant to advanced users and those who need to create a batch file from scratch As ImaGene can also be launched from the command line cr
6. 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 BioDiscovery 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 FLEXIm directory you specified in step 2 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 FLEXIm directory b Click on the Config Services tab c You will see the following screen LMTOOLS by Globetrotter Software http vw globetrokter com Service License Fle Spitem Settings Utiiies Start Stop Retead Server Status Server Diage Config Services Banewing Configue Senica SewiceNeme FlEMinSencet Beneit Pathiothelngdeve fle E Miewniimodexe o Paihiothelcensefle Tviesimenee eee Pathtathe debug logtie CWksindebuges Browse Ve Lo F Stat Server at Power Lip FF Use Services d You can leave the Service Name as its de
7. checking at all f ImaGene 5 0 x Check for Sotware Updates O Daily i Weekly gt Monthly O When Prompted On Check for Updates How When ImaGene detects a new version available for download the following message will be shown x 2 Version 5 0 of ImaGene is available 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 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 Technical Support at support biodiscovery com before removing any version of ImaGene from your computer as un installation may affect your licensing ImaGene Un Installation 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 second ImaGene confirmation dialog box displays when the un installatio
8. right click and select Create Metagrid 18 In the window that appears enter the following information a Metarows 6 b Metacolumn 1 19 Click Place MetaGrid 20 In the top subgrid where we just placed the grid click on the top left most spot 21 Using the scroll bar located along the right of the main image panel scroll downward until the last subgrid is reached 22 Click the top left most spot within this subgrid as well ImaGene automatically places the remaining subgrids in the proper locations 23 Using the scroll bar along the right of the main image panel scroll up so that the second to last subgrid is visible 24 Click the Adjust Subgrid button on the toolbar E 25 Click on and drag this subgrid slightly to the left to provide a better fit 26 Or alternatively click the Automatically Place Grid button and the subgrids will be adjusted automatically 27 Click the Adjust MetaGrid Button from the toolbar and click on any subgrid Ea 28 Click the Auto Adjust Spots Button from the Toolbar to perform automatic spot finding l Quantifying Pre Analyzing and Saving the Data 29 Click the Quantify Button located along the Bottom of the Main Tab Main Preview Images E 1 7hrs2 gel E i7hrs1 gel Reverse Display Colors Display Control N7 Grid Main 6x1x163x11 Gene IDIz Not Selected gt Quantify 30 After a few moments the quantified da
9. selected criteria fails spot is highlighted with mark on image and receives flag value 3 in the results table Note You can use Multi Channel Flagging button to change logic for combining automated flags between multiple channels For all three types of automated flags Empty Poor and Negative you can choose to assign a flag to a spot if it is flagged in at least one channel or in all channels Manual flagging user can manually flag spots for the following reasons No particular reason code 1 X mark Empty spot code 5 X mark Poor spot code 6 mark Negative spot code 7 mark Manual flag can be placed using a Marking tool from the tool bar Pad Click on the Marking button right click on image to select a type of a flag Place flags by clicking on corresponding spots Manual flag marks will have red color 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 ImaGene Parameter Settings aime Measurements e Mean w Median w Mode i Total r Standard Deviation Area Ignored Area W Spot Area w Ignored Median Area to Perimiter Ratio Offset from Expected Position k Shape Regularity 7 Open Perimeter Select all Unselect all Load Save As OK Mean signa
10. 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 the warping of the medium or potentially bent pins during printing spots and sometimes even entire subgrids require spot 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 very dubious indeed p ImaGene Parameter Settings x Controls and Alerts Quality Flags Spot Finding Image Preferences Measurements Segmentation r Find Negative Spots l Enforce Grid Constraints Local Flexibility 4 0 pixels Global Flexibility Rigid Flexible The following parameters affect how ImaGene performs spot finding Find Negative Spot While not a problem for most arrays negative spots can potentially cause problems for ImaGene spot finding If you suspect that 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 If you suspect all the signal values within the image are negative due to specific scanner settings then you may need to invert value for the images Occasionally the scanner software does not save information about which values are hi
11. Browse to the Research Genetecs folder the Human folder and select the gf200 template Look in Ci Human v l cal H i gf200 final data 047998 ipl gf201 final data 052798 tpl gf202 final data 090498 tpl gf203 final data 123198 tpl gf204 final data 047699 tpl gf205 final data 072799 tpl ni gf211 final data 110498 tpl 9f221 final data 122998 p1 E B gf223 final data 052599 ipl ae Place in saved position File name 91200 final data 042898 tp Open Files of type Template Files tpl v Cancel feaLoad Template 10 Select Place Manually 11 Click the Open Button 12 Click the four corners of the entire array The grid will be placed exactly on the array taking into account differences in scale and rotation 13 14 15 ImaGene 5 0 E i l0 x File Grid Spot Tools Help esse He RRE es E RO eE Composite ResGenitif Images 5 IE ResGenttifinverted C Reverse Display Colors Display Control EEVA i j 5 i gz Grid Gene ID gf200_final_data_042898 tpl B ImaGene 5 0 _ File Grid Spot Tools Help else fee RRE 18 sjel sd vea main Preview m ResGen tiflinverted VE E Reverse Display Colors Display Control Grid ran 2x8x30x12 Gene IDlyf200 final data 042898 tpl Quantify data
12. Gene ID field can be any alpha numeric string including blank spaces following the Subgrid Column Number You can specify a database entry followed by the gene name as one option Comment Lines Use the percentage sign to indicate comment lines Creating the Gene ID File You can use any text processing program spreadsheet or database program to generate the Gene ID file You can also use the CloneTracker software tool 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 the following are valid formats for the Gene ID File Header information MR MC SR SC Gene ID MR MC SR SC Ascension MR MC SR SC Ascension Gene ID Note The actual accession numbers are not provided nor determined by ImaGene or BioDiscovery The information must either be located by you or provided by the microarray chip manufacturer 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 Header Information Field MR MC SR SC Gene ID Field MR MC SR SC Ascension Field MR MC SR SC Ascension Gene ID 5 1 2
13. ImaGene e Adding Entries to the Batch Entries are the images and the corresponding settings to be used when performing batch processing e Post Processing Start times stop times and any errors which occurred during the batch are recorded to a simple text file for later analysis Like wise results can be reviewed to determine processing quality 2 4 1 Preparing for batch Processing D Prior to performing batch processing a configuration file and either a grid or a template file must exist To generate these files perform the following Launch ImaGene 1f the software is not already started Load a sample image which is indicative of the group of image to be batched In this case load Studyl cy3 TIF from the ImaGene samples directory The next step is to setup the configuration 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 and Poor spots set empty spot threshold to 1 Click Change Parameters button and click Default on Quality Parameters dialog close dialog Finally click the Save As button to save the configuration file to the file system to a location you choose Remember this location as we will recall it shortly gt ImaGene P
14. 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 this Agreement shall not exceed that license fee paid to Licensor by Licensee for the use of the Software In no event shall Licensor and or Licensor s suppliers be liable for any indirect incidental consequential special or exemplary damages or lost profits even if Licensor and or Licensor s suppliers have been advised of the possibility of such damages 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 9 TRADEMARK ImaGeneQ is a trademark of Licensor No right license or interest to such trademarks 1s granted hereunder and Licensee agree that no such right license or interest shall be asserted by Licensee with respect to such trademarks 10 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 or SEVEN 7 calendar days following mailing by U S Registered Mail postage prepai
15. Mean amp BG Mean amp Median amp BG Median amp Total Existing Color Schemes 4 F crestepertion a sources 2 genes 96 DataSet DefaultDS gs Tran Note if you have only one file with measurements drag this file directly into the right most window of DataSet Builder and hit Done Select the data set and go to Tools menu of GeneSight Lite Select Data Preparation tool It will ask you if you want to use Preview Mode Answer No The window for Data Preparation tool will pop up Select one of preset data preparation sequences from Preset Preparation Sequences menu t Data Preparation DefaultDS gs File Sub Select Genes View Help E E A i E E amp i Log Scale Log Scale Replicates ataset Contents Row No Gene ID 1205_g pairMean ratio 1205 g pairBG Mean ratio 1205_g pairMedian ratio Row 1 Gene 0 8364 1 0611 1572 Row 2 Gene 1 1 0571 1 0729 Row 3 Gene 2 i 1 0274 6389 Apply Data Preparation Row 4 Gene 3 1 0261 7296 Row 5 Gene 4 1 8817 Row 6 Gene 5 Row 7 Gene 6 Row 8 Gene Or alternatively you can create your own sequence by dragging preparation icons into the area of under toolbar where the sequence will be displayed Click Apply Data Preparation button Close data preparation window when the preparation a YP 8 sequence is finished For more information on how to construct data preparation sequence please refer to Gen
16. 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 1s 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 5 0 Date Tue Mar 26 09 19 34 PST 2002 Image File C BioDiscovery ImaGene Samples 1205_g g TIF Page 0 Page Name Inverted false Begin Field Dimensions Field MetarowsMetacols Rows Cols A 1 1 8 12 End Field Dimensions Begin Measurement parameters Segmentation Method auto Signal Low 0 0 Signal High 0 0 Background Low 0 0 Background High 0 0 Background Buffer 3 0 Background Width 3 0 End Measurement parameters Begin Alerts Control Type Minimum threshol
17. 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 pick location in intensity distribution 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 signal area of a spot is inscribed into a circle Than number of non signal pixels that fall within this circle 1s computed and divided by circle s area This ratio is subtracted from 1 as is called shape regularity Ignored Area Area of ignored regions directly neighboring touching the signal area 1s computed Spot Area Signal Area plus Ignored Area Ignored Median Median pixel intensity computed over the local ignored region Area To Perimeter This quality measure defines spot s circular
18. Set 4 52 Enter Hot Genes for the name of this sub selection 53 Close the Scatter Plot Window 54 From the menu bar of GeneSight Lite select Utilities then Generate Report 55 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 Er select the look of the Report E BEE PefauttDS ys _ Show Only Selected Genes i Row Mo Gene ID Group 17hrst pair 17hrs1 pair 117hrs1 Row 1 Gene 29 8060 9024 Row2 senest 948 ooe 8183 Rowa Genets8 pozor aare 7668 a tenrstraean tah Row4 loene1e2 haron hor 1304 tthrstpairBGMean Row5 Gene1er eon 4947 eose Rows Genter asee 2600 7020 vi 17hrstpairmedianr Row7 Geneteg foor 8014 aon oomega Rows Gene193 12924 nansa 13269 t rste Med rowa Gene226 19773 19345 19491 Zi 17hrs1Mean Row10 Gene238 1 2875 8949 TOR Row11 Gene255 9855 ena BBS vit7hrstBGMean Row12 Gene 255 1 0972 8820 0 0538 Row13 Gene266 9886 5o55 e759 saves a Rowt4 Gene277 8097 8337881 J Rows Gene 20a 13925 6872 25800 Row16 Gene320 1 1373 7930 19475 Row17 Gene323 8501 13855 9341 Select AllCelmmns JRow18 Genea9t 1 0467 e328 aser Row19 Genes53 4 058 ma ame Deselect ANCohumns Rrow20 Genes84 42500 8
19. Start Stop Retead Server Status Server Diage Config Services Banewing Coniigue Seniesa Sevice Nara FLEXm Service 1 Brows Path to ther lege ara file E Aflesdrn Leng exe Boine Path to the kcense fle C iedmicense bc Bowe Path tothe debug log fle Clexinivdebug bd Bronse View Lea FF Start Server at Power Up R Use Services n You can leave the Service Name as its default FLEXIm Service 1 o Enter the Path to the Imgrd exe file or click the Browse button to the right In this example it is C flexlm Imegrd exe p Enter the Path to the license file or click the Browse button to the right In this example it is C flexlm license lic q Enter the Path to the debug log file or click the Browse button to the right In this example it is C flexlm debug txt r Make sure you place a check mark in Use Services and then Start Server at Power Up Note that the Use Services checkbox may be disabled under Windows 95 98 ME s Click on the Save Service button t Answer Yes on the dialog that appears to confirm these settings 7 Select the license services e Click on the Service License File tab f The following screen will appear LAITOOL 5 by Globetrotter Soft ware http ere globetrotter com Service License File System Settinge Unites Start Stop Fl ead Server Status Server Diags Config Services Botrowing Servicer allow FLEALM Server bo nun in the backgroun
20. TO IMAGENE 5 QUICK START GUIDE 6 PART 1 LAB USERS GUIDE 7 1 1 IMAGENE MAIN WINDOW OVERVIEW 7 1 2 CONTROL TABS 11 1 2 1 MAIN TAB 11 1 2 2 PREVIEW TAB 19 1 3 IMAGE DISPLAY PANEL 27 1 4 IMAGENE PARAMETER SETTINGS WINDOW 32 1 5 IMAGENE WIZARD 43 1 6 BATCH EDITOR AND BATCH PROCESSING 44 1 6 1 THE MAIN BATCH EDITOR WINDOW 44 1 6 2 CREATE BATCH ENTRY WINDOW 46 1 6 3 CREATING AND RUNNING A BATCH 47 1 6 4 SAVING A BATCH 48 1 6 5 LOADING A BATCH 49 1 6 6 EDITING A BATCH 49 1 7 REVIEWING RESULTS 50 1 7 1 THE SNAPSHOT FILE SST FILE 50 1 7 2 LOADING RESULTS TO REVIEW 50 1 8 IMAGENE TOOLS 52 1 8 1 AUTO ALIGNMENT TOOL 52 1 8 2 AUTO GRID PLACEMENT TOOL 52 1 8 3 SAVE DISPLAY IMAGE TOOL 53 1 8 4 ZOOM TOOL 53 1 8 5 SCROLL TOOL 53 1 8 6 RULER TOOL 54 1 8 7 INTENSITY TOOL 54 1 8 8 FLAGGING TOOL 54 1 8 9 UNDO AND REDO TOOLS 55 1 8 10 GRID TOOLS 55 PART 2 IMAGENE TUTORIALS 58 2 1 BASIC ANALYSIS 58 2 2 PREMANUFACTURED ARRAY ANALYSIS 68 2 4 BATCH PROCESSING T2 PART 3 GENESIGHT LITE 78 3 1 WHAT IS GENESIGHT LITE 78 PART 4 INSTALLATION AND LICENSING 84 WINDOWS 84 IMAGENE UPDATE 85 UN INSTALLATION INSTRUCTIONS 86 MACINTOSH 86 UNIX 86 4 2 LICENSE MANAGEMENT 87 PART 5 APPENDICES 104 5 1 FILE SPECIFICATIONS 104 5 1 1 GENE ID FILE 104 GENE ID FORMAT 2 IMAGENE FORMAT 105 5 1 2 OUTPUT FILE 105 5 1 3 CONFIGURATION FILE 107 5 1 4 BATCH FILE 109 lt BATCH gt 5 2 TECHNICAL SUPPORT 111 5 2 TECHNICAL SUPPORT 111 5 3 WARRANTY INFORMATION 112 L
21. Trrashald Ar Shape reg FALSE Tr ashold Ur Anba To Py FALSE Threshold oF Offset fag FALSE Threshold bu End Pow Spots Parameter Hegaine amp TRUE a E arj 5 5 ci a 4 Un Ti 12 13 taj 15 16 17 ve t 20 Fil Fr 23 M d m x 2 rod m a1 a2 a M J5 EJ 4 a Al Eng Baader d2 Hagin Raw Daia 43 Field Mata Rive Mota Gola Raw Cohen Gen ID Flag Signal hi Backgrun Signal Me BackgrounSsgnal Mo Backgrew Signal Sid Backgrem N oord Yasid Oo d d Mari 1 1 AAN SBS O 4856061 13 65 515 14 B25 13 1705602 13518681 11708 S0 30257 a5 Mar 1 l 1 AASE TE D Sharla 16 37143 5 IES BTS 1E Se VISEN SO 13 EE EI R 45 Maia 1 1 I AMP 196 D 225 120005 12 225842 905712 TOMI SAS 015265 ST eae Mai len di 1 AAAS OM RADA ang 11 SO NARRA OR RARER 5 47 H Aknee Pa ja Ready End ually Settings The use of templates through ImaGene provides a convenient and simple way to perform grid placement and associate gene information with clone locations 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 e Preparing for batch processing Prior to performing batch processing several files need to be made available to
22. area failed Offset failed Empty spot Negative spot A 1 1 1 1 0 109 89393939393939 4 926565874730022 129 5 5 0 132 9090909090909 4 738562091503268 66 0 463 0 7293 0 2281 0 55 13623039946595 2 9050872336804874 0 584070796460177 0 0 66 0 NaN 1 0 0 0 33 427956043956044 21 692307692307704 9 0 0 3435990182607777 0 3298958928437372 0 09606760762402544 33 09806015111231 21 78837529993173 33 984848484848484 21 151515151515152 1 0917803796972039 0 8867883337361775 0 6368601484165772 8 319720247072127 10 339058029748454 0 0 0 0 0 1 0 0 0 0 A 1 1 1 2 0 163 88571428571427 4 821350762527233 228 5 5 0 248 61111111111111 4 571428571428571 70 0 459 0 11472 0 2213 0 102 5569587883649 1 2165800511090508 0 6481481481481481 0 0 70 0 NaN 0 9773843811168245 0 0 55 77061338661339 21 72727272727274 9 0 0 2811541783968894 0 269943687738035 0 07860074732736422 55 500669698875356 21 805873474600105 56 0 21 514285714285716 0 5782336652136307 0 49933030112464394 0 29158776031438904 8 236983309361506 11 051234910005755 0 0 0 0 0 1 0 0 0 0 A 1 1 1 3 0 105 26470588235294 4 836776859504132 138 0 5 0 173 3 4 7482993197278915 68 0 484 0 7158 0 2341 0 68 92421619325587 1 4288247004403603 5 1 3 Configuration File The configuration file stores information about virtually all the parameters within ImaGene This file is a simple XML file format which is easily saved and loaded from the ImaGene Parameter Settings Window While the contents of this file are not important to most us
23. file to the specified folder press the Unzip button Run Wirdp Browse Close h Overwrite files without prompting Unzip to folder About Help PEPE Select Unzip to folder C FLEXIm 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 c Ifyes then skip to step 5 d Ifno then proceed to step 3 3 Determine the server s Ethernet Address and HostName h Start the LMTools program by double clicking on Imtools exe in the FLEXIm directory I Click on the System Settings tab j You will see the following screen LMTOOLS by Globetrotter Software http srww globetrotter com ml 1 Fie Edt Mode Help Service License File System Seitings Utter Start Stop Retead Server Status Server Diags Contig Services Banowing Hoste Settings Tine Stings Computer Hosinan BIDINTEAN J System Tine Zone Pacis Slandacd Time Username f rer o Fenn a GMT Tine Tue Apr 09 17 34 47 2002 CPU ID i Difference From UCT 480 MSDOS Time 3 47 Loca Time 1016373697 Windows Directory CAWINNT k The computer s Ethernet Address is used as the computer ID l The Ethernet Address is a 12 digit hexadecimal number In this example the address is 00065blbb4ee m The c
24. name 4 The image is now saved and 1s available to be viewed 1n virtually any graphics program such as Microsoft Imaging or Adobe Photoshop 1 8 4 Zoom Tool The Zoom Tool EN as its name implies allows you to zoom a specific region of the image The several methods which ImaGene currently supports to zoom 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 Similarly to above 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 key board left click with the mouse and drag a rectangle around the section of interest from within the either of the areas mentioned previously e Drag the Zoom slider bar located directly below the Main Image Panel Note To return to the original image size double right click with the mouse 1 8 5 Scroll Tool The Scroll Tool Ri provides a convenient way to move about the image without requiring zooming out from the current 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 View The yellow box indicates the region currently being view
25. 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 Matthew 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 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 YO
26. the remaining portions or provisions hereof shall not be affected thereby 14 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 15 ENTIRE AGREEMENT The Parties hereto acknowledge that each has read this Agreement understands it and agrees to be bound by its terms The Parties further agree that this Agreement and any modifications made pursuant to it constitutes the complete and exclusive written expression of all terms of the Agreement between the Parties and supersedes all prior or contemporaneous proposals understandings representations conditions warranties covenants and all other communications between the Parties relating to the subject matter of this Agreement whether oral or written The Parties further agree that this Agreement may not in any way be explained or supplemented by a prior or existing course of dealing between the Parties by any usage of trade or custom or by any prior performance between the Parties pursuant to this Agreement or otherwise 16 AMENDMENTS No amendments or other modifications to this Agreement may be made except by a writing signed by both parties 17 ACKNOWLEDGMENT By Licensee s installation or use of this Software Licensee acknowledges th
27. time remaining This particular image demonstrates the fact that not all images contain the proper information regarding intensity In this image the default is that the background contains high intensity values and the signal regions possess low intensity Since this is not how the image is in the real world we must Invert the image with ImaGene ImaGene 5 0 Full version w Batch Processing 10 x File Grid Spot Tools Help eske HEE aRe KRSER KP Al r al Main Preview Images E ResGen tif Composite ResGenitif _ Reverse Display Colors Display Control Grid Gene IDICOVERYDesktopiDesktopiStudy1 cy3 sst Right click on the Images panel and select Invert Values Load Images amp Remove Selected Images Select Color 0 Invert Values 1 Hide CO Flip Horizontal Rotate Slide the top triangle of the Display Control Panel to the left 3 4 of the panel s length The image should now be clearly visible 3 ImaGene 5 0 Full version w Batch Processing este eee aaa ey 1 sjel sk Jed Images I Reverse Display Colors Display Control E Grid Gene ID COVER DesktopiDesktopiStudy1 cy3 sst Quantify Template Selection A From the menu bar select Grid followed by Load Template 9
28. web site you can access latest technical documentation request a new feature obtain sample images or templates etc e About ImaGene Displays the About ImaGene dialog box This interface contains information license number mode etc about your copy of 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 Olle e Fre ERERKET 8 So Ak ee Context Menu Context Menus are created by right clicking on the various elements within the ImaGene graphical user interface GUI Not all elements support context menus Elements which do support this feature are listed below Images Panel Grid Panel Image Display Panel Gene ID Selector Flagging Tool Load Images amp Remove Selected Images Select Color E 1 Hide C Flip Horizontal Rotate Control Tabs Located to the left of the window The control tabs provide information immediately relevant to the images being quantified Please see section 1 2 for additional information on the control tabs Image Display Panel Located to the right of the window This panel displays loaded images and is where grid placement takes place Reverse Display 1 play Control Compneiia Sample Sampler Status Bar Located at the bottom of the window The status bar provides feedback to the user regarding loading and handling of images Also ot
29. 133 148 Row21 Geneso2 43388 88 1278 Update Table View Row22 Genes43 1 6208 5120 14649 Row23 Gene552 43574 3382 1 3907 Row24 Genesee 03582 5720 1 152 Row25 Gene 585 1 1158 4552 7609 5 2 2 Premanufactured 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 genelist or Gene IDs 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 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 premade templates uo he 6 i Quantification Describes the quantification and subsequent output of data Image Selection Launch ImaGene from the desktop icon of from the ImaGene folder within the Windows Start Menu From the menu bar select File followed by Load Images Browse to the Samples folder within the ImaGene folder The location is typically C ImaGene Samples Select ResGen tif Click the Open Button to complete the image loading A progress bar displays
30. Editor follow these steps Select or highlight the desired entry by left clicking on its row within the Batch Table with the left mouse button 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 commit the changes or cancel to return to the Batch Editor without any changes 1 7 Reviewing Results One of the most powerful features of ImaGene 1s the ability to review results of previously processed data ImaGene is designed to load and display data exactly as it was processed days or even months earlier 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 a result of quantification within ImaGene Upon saving the quantified data to the file system ImaGene also saved another file which contains a snapshot of that data as it existed after quantification It is this snapshot file ending with an sst file extension which ImaGene loads and allows for results to be reviewed The snapshot file itself is simply a proprietary binary file format which is not useful outside of ImaGene Due to the large amount of information contained within the file its size can gro
31. Gene Parameter Settings Dolz Controls and Alerts Control type list Minimum expression allowed Maximum expression allowed Maximum CV allowed Gene ID list Add gene ID Remove gene ID Load gene IDs from file Update Visual Load Save As OK Using Add button on the panel user can create a new control type by specifying several parameters Type parameters Type name Minimum expression Maximum expression Maximum CV Highlight on image Pass Fail image for Percentage of failed controls allowed g Minimum value 0 20 40 60 80 100 Percentage of failed controls allowed a I Maximum value 0 20 40 60 80 100 Inter spot CV Type name provide arbitrary name for the type Minimum expression provide minimum value for signal mean for the type Maximum expression provide maximum value for signal mean for the type Maximum CV provide maximum coefficient of variance between signal means for the spots within this type should be gt 0 Highlight on image specify if you want all spots of this type to be highlighted on image If you select this check box you will be able to select a color for presentation of this type 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 s
32. Grid ImaGene allows the grid to be hidden enabling a better view of the bare spots Hiding the grid hides any lines circles and flagging that may exist within the Main Image Panel E 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 you have spots with irregular signal values and you set grid flexibility 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 button Note If you use this button several times spot searching will start from the position where 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 redoing 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 Ea A sample application would be to perform spot finding for grid geometry on an array image with a local flexibility set to a large numb
33. ICENSE AGREEMENT AND LIMITED WARRANTY 113 SKIN LOOK AND FEEL 0 3 1 LICENSE 119 PACKAGES CERN COLT CERN JET 120 PACKAGE COM IMSL MATH 120 PACKAGES JAL 120 JAVA 3D 1 2 1 01 BINARY CODE LICENSE AGREEMENT 121 JAVA 3D TM SOFTWARE VERSION 1 2 1 01 SUPPLEMENTAL LICENSE TERMS 124 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 or minutes while at the same time generating extensive quality control information ImaGene performs microarray image analysis for users Microarray image analysis 1s used to quantify the relative expression levels as exist 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 a data analysis package such as BioDiscovery s GeneSight for further analysis Quick Start Guide ImaGene is designed to be the fasted and easiest software solution for quantification of microarray images The description below outlines the essential steps to required to setup ImaGene For additional information please view the remaining sections of this user manual To setup ImaGene Perform grid placement Quantify the Image 1 Install ImaGene 2 Submit registration information and
34. ImaGene User Manual Version 5 5 March 2003 BioDiscovery Inc Copyright Notice 1997 2003 BioDiscovery Inc All Rights Reserved The ImaGene Users Manual was written at BioDiscovery Inc 4640 Admiralty Way Suite 710 Marina Del Rey CA 90292 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 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 IMAGENE 1 COPYRIGHT NOTICE 2 TRADEMARKS 2 INTRODUCTION
35. License License File File File local lic local lic local lic 4 3 FLEXIm 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 FLEXIm is C FLEXIm In the following examples it is assumed that this is the directory being used If FLEXIm is installed into another directory then make the appropriate modifications to the path 1 Install the BioDiscovery software with the FLEXIm license manager service a Make sure that the account you are using has administrative privileges on this computer b Make sure that you select the option to install the FLEXIm Service component during the software install CloneTracker 2 Setup 5 xj Select Optional Components d To instal component chok the check box next to If the check box is clear that Component wall mot be installed Inaalam 2 Remember which directory you ve selected for FLEXIm in during the install a Note that C flexlm is the default I select FLEXIm Folder xj Please choose the installation folder Path CI Flexdm Directories Desktop H E My Documents age My Computer Heg 3t Floppy 4 3 aS Local Disk C HO BACKUP ee bd H build TJ CloneTracker 1 4 h can
36. ND 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 RETURN TO LICENSOR THE 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 SUBJET OF THIS LICENSE ARE PROVIDED ON AN AS IS BASIS WITHOUT ANY WARRANTIES OF ANY KIND INCLUDING ANY AND ALL IMPLIED WARRANTIES OR CONDITIONS OF TITLE NONINFRIGEMENT MERCHANTABILITY OR FITNESS OR SUITABILITY FOR 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 AND DISTRIBUTOR EXPRESSLY DISCLAIM ANY WARRANTY OR REPRESENTATION TO ANY PERSON OTHER THAN LICENSEE WITH RESPECT TO THE SOFTWARE OR ANY PART THEREOF 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 8 LIMITATION OF LIABILITY Notwithstanding any other provision of this Agreement the cumulative liability of Licensor and or
37. OT SOLD l LICENSE GRANT For consideration promised and or received Licensor hereby grants to Licensee one non exclusive nontransferable internal end user license the License to use the basic software product entitled ImaGene version 5 0 and those software modules expressly authorized in writing by Licensor if any the Software and the accompanying documentation in the form delivered to Licensee 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 CPU 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 this 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 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 use on two or more computers nor use in a local area network or other network is permitted Upon having purch
38. RO PRSI RD gt Also located within the data output directory is a file called Log txt This file contains start and stop times for all the batches It also contains any errors that may have occurred Begin Batch Mon January 01 08 02 50 PST 2002 Begin Run Info current image Y Sample Data ImaGene Samples Study1_cy3 TIF Info channel name End Run Begin Run Info current image Y Sample Data ImaGene Samples Study1_cy5 TIF Info channel name End Run Begin Run Info current image Y Sample Data ImaGene Samples Study2_cy3 TIF Info channel name End Run Begin Run Info current image Y Sample Data ImaGene Samples Study2_cy5 TIF Info channel name End Run End Batch Mon Apr 01 08 02 58 PST 2002 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 at 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 Part 3 GeneSight Lite 3 1 What is GeneSight Lite After intensity measurements are extracted from an image there is a need for further data processing ImaGene incorporates a powerful tool that provides creating simple types of experimental design d
39. SSNNNN NNN EE Generating a Report Via GeneSight Lite 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 p DataSet Builder 10 x Lin Tools gt pen Heg hd a LEN All ME RSA GE kp DE pan Select files on the left to add to the DataSet on the right Ca v El Experiment E Control xperiment Info File a Browse 3 CloneTracker2 DE cr5ap 5 2 No handling Files are consistent v O amp CI cygwin a 9 C Data i E 17hrs1 bt E 17hrs2 t eer Ratio Sp Perform Ratio amp Add as Replicate JE Repeated Experimental Conditions tat Add to Data Set Add as Experiment Add as Control rjj Add as Replicate as 42 Select and drag 17hrs1 txt to the Experiment Panel 43 Select and Drag 17hrs2 txt to the Control Panel 44 Click the Pair Data Perform Ratio Button 45 Click Done from the Dataset Builder Toolbar Done 46 Select User Defaults for all Experiments and click the OK Button 17hrsl txt Enter Experiment Information Enter Experimental Information for DataSource 17hrs1txt Enter information for this data file Y
40. Segmentation Preview Use the Segmentation Preview to view the effects of current parameter settings on the segmentation both before and after quantifying the data You select the spot to view and the corresponding information about the spot displays in the segmentation panel At the same time selected spot can be seen on the image and two of the plots Scatter plot and Box plot 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 in the dialog box Gene ID Genelia Field Right Metarow 1 Metacolumn 1 Row Column 2 Diameter 29 0 original Segment CompositdHistogra The following textual spot information is available via the Segmentation Preview Gene ID Lists the corresponding information typically name or accession from the Gene ID file af you imported a Gene ID file into ImaGene Field Lists the field that the selected spot belongs to This name is specified when the grid was first constructed Metarow Identifies the row within the metagrid where the selected spot 1s located Metacolumn Identifies the column within the metagrid where the selected spot is located 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
41. TDEV gt true lt MEASURE STDEV gt lt CONTROL TYPE VARTHRESH 0 gt 1 0 lt CONTROL TYPE VARTHRESH 0 gt lt MEASURE_AREA gt true lt MEASURE_AREA gt lt PERIMETER THRESH gt 25 0 lt PERIMETER THRESH gt lt STANDARD CV gt 5 0 lt STANDARD CV gt lt AUTO SEG gt true lt AUTO SEG gt lt CONTROL TYPE UPALERTTHRESH 0 gt 0 0 lt CONTROL TYPE UPALERTTHRESH 0 gt lt CONTROL_TYPE_IFLOWALERT_0 gt false lt CONTROL_TYPE_IFLOWALERT_0 gt lt BLANK_LOW gt 0 0 lt BLANK_LOW gt lt BACKGROUND_BUFFER gt 3 0 lt BACKGROUND_BUFFER gt lt CONTROL TYPE NAME 0 gt hot lt CONTROL TYPE NAME 0 gt lt EMPTY FLAG gt true lt EMPTY FLAG gt lt MEASURE MODE gt true lt MEASURE MODE gt lt ENFORCE GRID gt true lt ENFORCE GRID gt lt OVERALL FLAG gt false lt OVERALL FLAG gt lt MEASURE TOTAL gt true lt MEASURE TOTAL gt lt SIGNAL HIGH gt 1 0 lt SIGNAL HIGH gt lt PERIM TO AREA gt true lt PERIM TO AREA gt lt SIGNAL FLAG gt false lt SIGNAL FLAG gt lt BACKGROUND WIDTH gt 3 0 lt BACKGROUND WIDTH gt lt BLANK CHECK gt false lt BLANK CHECK gt lt ON TOP gt false lt ON TOP gt lt CONTROL TYPE UPTHRESH 0 gt 65000 0 lt CONTROL TYPE UPTHRESH 0 gt lt SIGNAL THRESH gt 0 9995 lt SIGNAL THRESH gt lt FLEXIBILITY gt 0 0 lt FLEXIBILITY gt lt FIND NEGATIV ES gt false lt FIND NEGATIVES gt lt SIGNAL LOW gt 0 0 lt SIGNAL LOW gt lt STANDARD HIGH gt 10000 0 lt STANDARD HIGH gt lt BACKGROUND LOW gt 0 0 lt BACKGROUND LOW gt lt OFFSET gt true lt
42. U 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 e SELECT THE DO NOT ACCEPT BUTTON AT THE BOTTOM OF THIS PAGE AND THE INSTALLATION PROCESS WILL NOT CONTINUE e RETURN THE UNOPENED SOFTWARE TO THE PLACE OF PURCHASE FOR A REFUND OR e DO NOT DOWNLOAD THE SOFTWARE l Licensed Software Licensed Software means the JAVA 3D 1 2 1 01 software in binary 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 2 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 3 License Restrictions The following restrictions apply to your license e The Licensed Software is conf
43. a files as well as the snapshot or sst files 8 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 are to be batched this process of adding entries can be repeated as many times as needed i ImaGene Batch Editor E D x Configura Output Dir Channel Studyl c Template Template Config xml Desktop Studyt c Template Template Config xml Desktop Study c Template Template Config xml Desktop Ste ER LE study oc Template Template Config xml Desktop Remove Selected Entries Available Unlimited Edit Selected Entry Show Full Path i Channel 1 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 Finally click the Run Button to begin the batch A small Batch Processing Window appears displaying both the progress through the current image and the progress through the entire batch Upon completion of the batch this small window simply closes Loan Baten Save raten ram 2 Processing Batch J OD x Wample DatailmaGenetSampl Sample DatalmaGenetSampi 18ample DatalmaGenetSampi Batch Completed 3 of 4 Des O Sample DatallmaGene Sample Merke O 2 4 3 Post Batch Processing Ulti
44. ad Template Displays the Load Template dialog box Use this interface to select and open a template tpl file A template is a erid file which contains gene ID information Save Template Displays the Save Template dialog box Use this interface to save a template Adjust Metagrid Allows you to select and move an entire metagrid Adjust Subgrid Allows you to select and move an individual portion of the metagrid subgrid Adjust Spot Allows you to select and move one spot in a metagrid Lasso Adjust Allows you to select and move a specific free form area of the metagrid Rectangle Adjust Allows you to select and move a specific rectangular area of the metagrid Auto Adjust Spots Tells ImaGene to automatically adjust each spot to better align them with the corresponding image Wrangle Enforces new local spot flexibility parameters Essentially reduces the distance used in spot finding without requiring spot finding to be reapplied e Tools Menu Zoom Turns the cursor into a magnifying glass Use this tool to adjust the on screen display size of images Scroll Turns the cursor into a hand Use this tool to scroll all images and grids at the same time Undo Cancels the last executed command For example 1f you moved a spot you could select Tools gt Undo to cancel this action and in effect deselect the spot Redo Restores the last canceled command For example if you used the Un
45. 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 6 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 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 F 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 8 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 documen
46. amp Remove Selected Images hale Save Display Image Click the Add Batch Entries Button on the Batch Editor Window 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 four image files that begin with Study There should be four files Holding the lt ctr gt key while selecting the images allows several to be selected at once O x Entries Available Unlimited Gene ID Configuration Output Dire ChannelNa Add Batch Entries Remove Selected E Create Batch Eni ait Selected Entry Images _ Show Full Path L Ilx Grid None Selected gt Gene ID None Selected gt Configuration None Selected PEE Output Directory lt None Selected Cancel Load Batch Save Batch Run 4 Next click the Gird Button Either a grid or a template may be specified here Browse to and select the template we created previously 5 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 6 Click the Configuration button and specify the configuration previously created 7 The last parameter to specify is the output directory ImaGene needs to know where to store the quantified dat
47. 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 Blue Signal Mean Value Yellow Signal Median Value Cyan Signal Mode Value d Under the Segmented Composite and Histogram Views color is used to indicate pixels to be included within the signal and background measurements If a pixel is not color then the pixel is being ignored and is not being used for calculation of either signal or background values dal The Preview Panel colors include Green Indicated the pixel is used in the background calculation Red Indicated the pixel is used in the signal calculation No color or black on histogram Indicated the pixel 1s used in neither calculation of signal nor background Quantification Table The Quantification Table displays the quantified numerical intensity values 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 is 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 in
48. arameter Settings E x Controls and Alerts Spot Finding Segmentation Quality Flags Find Negative Spots r Enforce Grid Constraints Local Flexibilityl5 0 pixels Global Flexibility Rigid Flexible Save As Close 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 8 for the Rows and 12 for the Columns The Min and Max Diameters should be set to 9 Click the Place Grid Button and click on the four corners of the array Specify a gene ID to be used with this array To do so from the menu bar select Grid followed by Load Gene IDs Locate and select the study Gene IDs 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 which 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 Spot Tools H Load Grid Save Grid Clear Grid Load Gene IDs Clear Gene IDs Load Template 2 4 2 Adding Entries to a Batch U Once the configuration file and the template files exist these can be combined with 1 or more images to create a batch To add entries to a batch From the menu bar select File followed by Batch Editor Grid Spot Tools Help Load Images
49. ased 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 The terms and conditions of this Agreement shall apply to all additional subsequent or multiple Licenses obtained by Licensee for the Software Licensee agrees that it will not assign sublicense transfer pledge lease rent or share its rights under this License Agreement nor will Licensee 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 attempt to reverse engineer decompile disassemble modify reproduce reverse assemble reverse compile or otherwise translate the Software or any part thereof 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 Software Diskettes are damaged or destroyed Licensee may make one copy of the Users Manual for backup purposes only Any such copies of the Software or the Users Manual shall include Licensor s copyright and other proprietary notices Except as authorized under this paragraph no copies of the Software or any portions thereof
50. at Licensee has read and understands the foregoing and agrees to be bound there Skin Look and Feel 0 3 1 License Copyright 2000 2001 L2FProd com All rights reserved Redistribution and use in source and binary forms with or without modification are permitted provided that the following conditions are met 1 Redistributions of source code must retain the above copyright notice this list of conditions and the following disclaimer 2 Redistributions in binary form must reproduce the above copyright notice this list of conditions and the following disclaimer in the documentation and or other materials provided with the distribution 3 The end user documentation included with the redistribution if any must include the following acknowledgement 4 This product includes software developed by L2FProd com http www L2FProd com 5 Alternately this acknowledgement may appear in the software itself if and wherever such third party acknowledgements normally appear 6 The names Skin Look and Feel SkinLF and L2FProd com must not be used to endorse or promote products derived from this software without prior written permission For written permission contact info L2FProd com 7 Products derived from this software may not be called SkinLF nor may SkinLF appear in their names without prior written permission of L2FProd com THIS SOFTWARE IS PROVIDED AS IS AND ANY EXPRESSED OR IMPLIED WARRANTIES
51. ata cleansing transformation normalization visualization etc This tool is called GeneSight Lite and can be launched at any time using 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 refer to Data Preparation tool for data cleansing transformation and normalization After all necessary preprocessing is applied to the data you can start using various visualization and analysis tools of GeneSight Lite Aforementioned tools include Histogram GenePie tool for easy comparison of gene expressions across different experiments Scatter Plot 2 and 3 dimensional Partition Manager Hierarchical Clustering 2 linkage methods and 5 distance measures Interface with web based public genetic data Time series visualization 3 2 How to use GeneSight Lite For details on GeneSight Lite usage 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 Scatter plot To build a scatter plot on a data you just qua
52. ation quantification and analysis of array 1mages 1 8 1 Auto Alignment Tool The Auto Alignment tool 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 cam handle up to 10 degree s of rotation as well The Auto Alignment tool is intended to provide quick and highly accurate means 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 used 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 ImaGene s Auto Grid Placement Tool E automatically repositions a grid structure to the proper location within the image A common application of this tool is with the use of grid and template files Of the geometry of an array will stay the same through and between experiments If this is the case then the use of a common template for all experiments would be beneficial However due to differences in arraying and scanning the location of the array within the image may vary across experiments and time The solu
53. ations are required BioDiscovery recommends GeneSight for full data analysis and visualization However there is an access to ratio data directly from ImaGene through Histogram plot under the Plots tab on Image Display Panel Export to XML Before saving the data 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 mode for export as signal background value For more details go to http www rosettabio com products conductor geml default htm MAGE 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 Note once you export the data the results table will not be closed you can continue analyzing the data or save the results in usual format Cancel Cancel clears the quantified data values without saving them Once the quantification is cancelled the image will need to be reprocessed before the data may be saved again Upon selecting Cancel you will be prompted to verify your action Clicking yes will then clear the data The Quantification table will no longer be visible Selection Starting with 5 5 version of ImaGene multiple spot selection is allowed You can s
54. ave through either technical support or by sending you a replacement copy of ImaGene For warranty information review the license agreement or contact us via E mail info biodiscovery com Phone 310 306 9310 United States Mail 4640 Admiralty Way Suite 710 Marina Del Rey CA 90292 USA 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 SOFWARE 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 THAT IT CONTAINS TO YOUR PLACE OF PURCHASE NO LATER THAN TEN 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 THAT IS THE SUBJECT OF THIS AGREEMENT IS LICENSED UNDER THIS AGREEMENT N
55. basic structure is the same between 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 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 syntax Header Anything other than the Gene ID information located at the beginning of the file If you want to include header information you must start each line 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 Separator You must use tabs to separate the fields in each row Order ImaGene does not care about the order in which the rows are sorted It s also not necessary to specify Gene ID information for all the spots in the array Gene ID The
56. 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 need be Load Batch Load Batch loads from the file systems a batch file that has 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 via the Load Batch Button 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 Batch ImaGeneGridFile grd Load Baten Save Baten ram 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 Entries Available Unlimited Note When saving the batch only basic information about the location of configuration files and image files is included The actual files remain unchanged in their original location Do not confuse saving the batch file with saving the configuration files used to ru
57. be changed but the results will not be visible until spot finding has been performed again O Max Diameter The maximum expected diameter of the spots to be used during spot finding This value may be changed but the results will not be visible until spot finding has been performed again Grid Create Grid Create MetaGrid Delete selected Field Clear Grid Convert to single Subgrid Properties Create Grid g x Field Name Right Rows Columns Min Diameter 0 Max Diameter 9 0 Rectangular Grid Staggered Left Grid O Staggered Right Grid Place Grid Cancel How to create Grid Perform the following steps to create a subgrid within ImaGene Load the desired images into ImaGene Oo WN Re Right click with the mouse on the Grid Panel and select Create Grid Specify the parameters within the create grid window Ifa 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 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 within ImaGene Click on and select the desired subgrid to use as a basis for the metagrid This can be done by left clicking on the grid within the Grid Panel Right click and select C
58. browse to and select the desired file Multiple files may be selected by holding the lt shift gt or lt ctr gt keys and left clicking on the image name Note ImaGene supports the following image file formats e 6 Tiff The file extension is tif e MD Gel The file extension is gel e Fuji Bas This is a two file format with one file ending in inf and the other ending in either img or bas Reverse Display Colors Mark this check box to reverse the displayed colors of all open images Activating this feature has no affect on the Composite tab Changing the appearance of an image does not affect the pixel intensity of your original data This tool just makes it easier to see dim spots in an image Reverse Display Colors Display Control The Display Control allows adjustment of how the images are displayed By moving the triangle located at the top and bottom the display control the image can be lighted and darkened 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 the Display Control these are applied only to the Image s selected within the Images Panel Display Control mms The specific elements of the Display Control are and perform the following e Top Triangle This sets the minimum intensity to be displayed e Middle Triangle Allows the rate of change of pixel intensi
59. button evel 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 Note all GeneSight Lite tools can be used simultaneously and any gene sub selection in one tool will propagate into all other tools Part 4 Installation and Licensing Windows 10 11 12 Introduction ImaGene uses a standard InstallShield installation under Windows Most users are familiar with this easy to use format 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 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 Review the list of selected components to be installed Note the default location for the ImaGene program folder If you do not want to use this location click the Browse button and select another
60. can use Load gene IDs from file button to load a list of gene IDs from a text file Such file can contain a column of multiple gene IDs one gene ID per line Once loaded such gene IDs will be assigned to the current control type This feature is useful if you want to bring a list of selected genes from some alien program GeneSight for instance and analyze corresponding spots on microarray images To see all spots that belong to the specified types highlighted on image click Update Visual This action will also show all control violations If current image failed the controls test corresponding warning box will pop up Control type for every spot will also be shown in the results table in one of the columns 1 5 ImaGene Wizard ImaGene wizard is designed to help user understand ImaGene tools better and to assist in going through necessary steps Wizard can be switched on and off at any moment through Help menu Even when Wizard window is switched on it does not limit user s access to any of ImaGene tools ImaGene wizard E E X You are using Change settings tool Here is how to use It Change advanced settings for orid spot finding segmentation quality flagging controls To launch Wizard select Wizard On option from Help menu Wizard window will be shown in the bottom right part of your screen ImaGene Wizard works in two modes First mode switches on whenever Wizard detects that user per
61. ck that the Service Name selected is the same as the one you configured in step 7 Click on the Start Server button 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 d b If yes then you re almost done Skip to step 11 If no then you need to do some troubleshooting Proceed to step 10 10 Troubleshooting the FLEXIm license server a b gg rho ao Double check that you entered the correct file paths in step 7 View the license file by right clicking on the license lic in the FLEXIm directory and selecting Open With Select Notepad from the list Place a checkmark next to Always use this program to open these files Click the OK button 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 4 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 FLEXIm debug txt 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 or by double clicking runwiz bat in your product directory Click on the NodeLocked tab c You will see the following screen
62. cted spots in full scale Scatter Plot Scatter plot offers visualization of one measurement plotted against any other Plots Choose the plot type Bas a Scatter plot Signal Madian 0 1e4 2e4 3e4 ded Se4 Ged Signal Mean Signal Mean m Signal od If multiple images are loaded 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 can give a rough idea about regulation of the genes Selection of points on the scatter plot will result in selection of corresponding rows in the results table and highlighting of selected spots on the image Note you can add to current selection by simply continuing to select other groups of spots To start a new selection right click on the plot first to clear the previous selection Box Plot This type of plot allows you to visualize measurement s distribution between different categories of spots belonging to different subgrids different rows having different flags etc You choose between different categories for X axis and between different measurements for Y axis The box on such graph 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 Such plot can be usef
63. d g Select the Configuration using Services radio button h Select the Service Name you used from step 6 8 Start the License Server f Click on the Start Stop Reread tab g The following screen will appear LMTOOLS by Globetrotter Software http weww globetratter com Fie Edt Mode Help Service License File System Settings Litiities Start Stop F h 1 j Double check that the Service Name selected is the same as the one you configured in step 6 Click on the Start Server button 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 C d If yes then you re almost done All you need to do now if configure each of the clients to run the software Please see the FLEXIm Client Installation Guide for details If no then you need to do some troubleshooting Proceed to step 10 10 Troubleshooting the FLEXIm license server J k mepop gr NM Double check that you entered the correct file paths in step 6 View the license file by right clicking on the license lic in the FLEXIm directory and selecting Open With Select Notepad from the list Place a checkmark next to Always use this program to open these files Click the OK button The text file will contain a line that looks similar to this SERVER HOSTNAME 00065b1bb4ee Make sure that HOSTNAME i
64. d 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 Agreement unless change of such address and or facsimile number has been provided previously in writing 11 GOVERNING LAW AND VENUE This License Agreement shall be construed and governed in accordance with the laws of the State of California The parties 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 The parties 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 12 ATTORNEYS FEES If any action is brought by either party to this License 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 granted reasonable attorney fees and costs 13 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
65. dIf tested Percentage allowed If failed Maximum threshold If tested Percentage allowed If failed CV threshold If tested If failed hot 45000 0 false 0 0 false 65000 0 false 0 0 false 1 0 false false End Alerts Begin Quality settings Empty Spots true Threshold 0 3 Poor Spots true Begin Poor Spots Parameters Background contamination flag true Threshold 0 9995 Background tested against subgrid data only true Signal contamination flag false Threshold 0 9995 Signal contamination test connected to background contamination threshold false Ignored percentage flag true Threshold 25 0 Open perimeter flagtrue Threshold 25 0 Shape regularity flag true Threshold 0 65 Area To Perimeter Ratio flag false Threshold 0 65 Offset flag true Threshold 60 0 End Poor Spots Parameters Negative Spots true End Quality settings End Header Begin Raw Data Field Meta Row Meta Column Row Column GeneID 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 Area Ignored 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
66. do command to cancel a spot movement you could select Tools gt Redo to move the spot again Translate Images Turns the cursor into a cross with arrows Use this tool to move selected images Rotate Images Turns the cursor into a circle Use this tool 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 Use this tool to measure the size of spots or distances between them Image Intensities Turns the cursor into a small light bulb Use this tool to measure the intensity of spots Tag Spots Turns the cursor into the letter X Use this tool to manually flag spots Right click with this tool to see a list of available flagging options ImaGene Help Displays the ImaGene Online Help documentation Wizard On Off Turns on and off the ImaGene Wizard The wizard provides guidance on the proper steps to be performed within ImaGene Change Skins launches a dialog for choosing between different skins for ImaGene interface using skinlf When a specific skin 1s selected the change take effect next time you launch ImaGene Auto Update This option allows you to automatically update ImaGene package with the newest version available for download from BioDiscovery website You can also set a frequency for automatic update check at ImaGene start up Support Center launches Web Browser with customer support page for ImaGene Through that
67. e 3 Do you already have a license file called license lic a If yes then skip to step 6 b Ifno 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 b Click on the System Settings tab c You will see the following screen LMTOOLS by Globetrotter Software http es globetrotter com i bel 2 Fie Edt Mode Help Service License File System Seitings Utter Start Stop Retead Server Status Server Diags Contig Services Banowing Hoste Settings Tine Stings Computer Hosinan BIDINTEAN J System Tine Zone Pacis Slandacd Time Username f rer o Fenn a GMT Tine Tue Apr 09 17 34 47 2002 CPU ID i Difference From UCT 480 MSDOS Time 3 47 Loca Time 1016373697 Windows Directory CAWINNT 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 00065blbb4ee 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
68. e Composite Tab The default color 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 o 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 you determine the expected values for the signal and the background measurement to be opposite to what is expected selecting Invert Values will solve the problem o Hide The selected image will no longer be visible under the Composite Tab While not visible 1f quantified data will be generated for the image o Rotate Rotates the image 90 180 or 270 degrees around the top left of the image F Load Images amp Remove Selected Images select Color I Invert Values Hide I Flip Horizontal Rotate k How to Load an Image s The following steps describe the process of loading Images within ImaGene 1 From the menu bar select File followed by Load Images Alternatively click the first icon on the toolbar li or right click on the Images Panel and select Load Images 2 From the Load Images dialog that appears
69. e can be threshold at any level from 0 5 to 1 and the flag will be issued whenever particular contamination confidence goes over that threshold Background mode 1s used as a background level estimate Background level can be tested against 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 Flag for signal being contaminated I Signal Y O Combine signal test with background test 0 5 0 6 0 7 0 8 0 9 1 0 Next check box corresponds to signal abnormality detection method This approach is design to flag any spot with unusually high volatility in pixel wise intensities within the signal area For every individual spot 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 done spots are grouped according to their signal mean measurements This is done to compensate for a well known observation that within spot intensity volatility tends to be different for different expression levels This way we make sure that each spot is tested only against spots close to it in their expression The resulting confidence number is compared to the threshold analogously to previous pr
70. e menu bar select Tools followed by Zoom 8 Drag a rectangle around the top subgrid of the array to zoom into this region of the image ON 9 Select Tools then Ruler from the menu bar to measure the approximate sizes of the spots within this array This information will be used slightly later 10 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 in pixels 11 Next count the number of rows and columns of spots within this subgrid 12 Right click on the Grid Panel and select Create Grid from the menu that appears Create Grid 13 Enter the following information into the Create Grid Window that appears Field Name Main Rows 16 Columns 11 Min Diameter 18 Max Diameter 26 Rectangular Grid is selected monoge Create Grid E x Field Hamel hain Rows Columns 11 Min Diameter 15 Max Diameter 6 Rectangular Grid gt Staggered Left Grid Tit 0 Staggered Right Grid Place Grid Cancel 14 Click the Place Grid Button 15 Use the mouse cursor and click on the four corner spots of the subgrid Upon placement of the fourth spot the grid becomes visible on the subgrid 16 Click on the name Main in the Grid Panel Create Grid Create MetaGrid N 17 With Main selected
71. eSight manual Chapter 7 Select any pair of measurements in GeneSight Lite main window Click Scatter Plot button on the toolbar posta 2 D scatter plot window will pop up GeneSight Lite 3 1 5 des View Tools Plots Utilities NE om son ratio EF Scatterplot rr E E BG Total ratio E 12059 ken Edit Sub Select Genes Color Scheme Choose URL Help Reference amp BG Median E Total amp BG Total 1205_g control amp Mean amp BG Mean amp Median amp BG Median 100 150 200 250 Existing Color Schemes Find Gene A IT m righ Reset Schemes 1205_qMean Create Partition 7 Show Gene IDs sources 2 genes 96 DataSet DefaultDS gs Transformations done 1205_g controfMean 50 Note If you select three measurements 3 D scatter plot will be shown For manipulations with within Scatter Plot window refer to GeneSight manual Chapter 9 Histogram Follow steps 1 4 from the previous section Select the measurement of interest in GeneSight Lite main window Click Histogram button on the toolbar stoam Histogram window will pop up O NDE GeneSight Lite 3 1 5 View Tools Plots Utilities den A o oon LETTET Help gT Total ratio aa EUA Z a Mr rd pe Be ge Gene Got E BG Total ratio amp BG Mean 1205 gMean amp BG Median E Total amp 8G Total 1205_g control amp Mean amp BG Mean amp Median amp BG Median Ex
72. eating a batch manually and passing this information in as a command line argument provides a viable alternative than simply running the software from the Batch Editor Listed below is a sample batch file Note that the information contained here matched the information required within the Batch Editor lt xml version 1 0 encoding UTF 8 gt lt Batch gt lt Entry gt lt Image gt C BioDiscovery ImaGene Samples jk88 amp 2 TIF lt Image gt lt Template gt C Documents and Settings data Desktop Desktop tpl lt Template gt lt Configuration gt X data configuration xml lt Configuration gt lt Destination gt C Documents and Settings data Desktop lt Destination gt lt Channel gt 0 lt Channel gt lt ChannelName gt lt ChannelName gt lt Imageld gt 0 lt Imageld gt lt Entry gt lt Entry gt lt Image gt C BioDiscovery ImaGene Samples 1267 rTIF lt Image gt lt Template gt C Documents and Settings data Desktop Desktop tpl lt Template gt lt Configuration gt X data configuration xml lt Configuration gt lt Destination gt C Documents and Settings data Desktop lt Destination gt lt Channel gt 0 lt Channel gt lt ChannelName gt lt ChannelName gt lt Imageld gt 1 lt Imageld gt lt Entry gt lt Entry gt lt Image gt C BioDiscovery ImaGene Samples 18998 TIF lt Image gt lt Template gt C Documents and Settings data Desktop Desktop tpl lt Template gt lt Configuratio
73. ed within the Main Image Panel 1 8 6 Ruler Tool The Ruler Tool u allows objects to be measured within the Main Image Panel The most common use of this tool is to determine the minimum and the maximum diameter for spot sizes and distance between the spots The tool can also be used 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 provided 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 measured in pixels 1 8 7 Intensity Tool The Intensity Tool EI 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 is displayed Note The values provided here are raw intensities meaning they are not mean or median values but rather a single pixel value 1 8 8 Flagging Tool The Flagging Tool Pad provides a simple means 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 defect within the spot or array Once a spot has been flagged a numerical value indicating the type of flag 1s then outputted with the quantified data T
74. elect several spots at a time pressing Ctrl or Shift keys when making selection in the results table Indicator in the bottom of the table will show you how many spots are selected You can also freeze the selection using corresponding button When frozen table rows will be highlighted in dark blue color and they will have flag Selected in the right hand end of the table turned to 1 When you freeze the spots you can analyze them one by one by selecting only one row at a time You can unfreeze the selection at any time and the spots will become highlighted with a usual selection color This feature 1s useful if you selected the spots using one of the plots selected regulated genes using Histogram for instance and want to analyze their images segmentations and quantifications results You also can Save the frozen rows into txt file Selectect Freeze Unfreeze Restore default column order Save frozen rows Savel Export To XML Cancel 1 3 Image Display Panel This panel displays loaded images A tab appears along the top of the panel for all currently 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 which ImaGene can load is limited based upon the computer hardware specifications C
75. entioned previously Histogram allows to choose only one measurement Plots Choose the plot type Err Histogram in number 100 L v Tails Lower bound Upper bound 709 78271 Total genes 1250 eft selected Genes 0 Middle selected Genes Right selected genes 0 Total selected genes Signal Mean hud The histogram demonstrates distribution of the measurement values over their domain One can make interval selection on the histogram and see how many genes fall into that selection By changing selection of Tails check box you can select genes either inside the interval or outside You can switch between untransformed measurement and log transformed measurement using log check box You can also change bin density print histogram and save its image this feature is especially useful for publications Histogram provides not only ability to plot any of the measurements available in quantification table but also gives you an access to such useful values as inter channel signal ratios including background corrected Using these values in combination with logarithmic option you can obtain a histogram of a log ratio natural logarithm will be used Selection of the tails of such distribution provides you a quick way to analyze up and down regulated genes Note when selection is made on the histogram same spots will be selected in measurements table and on the image allowing you to analyze sele
76. er of pixels After spot finding if the resulting circle placement has high variability the Local Flexibly can be reduced After reducing Local Flexibility click the Wrangle Button to apply the new setting without waiting for spot finding to be performed again Part 2 ImaGene Tutorials 2 1 Basic Analysis This tutorial demonstrates performing basic quantification through ImaGene The tutorial covers the essential steps from loading the initial array images until a report 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 grid properly e Quantifying and Saving the Data Describes the quantification and subsequent output of 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 icon or from the ImaGene folder within the Windows Start Menu 2 From the menu bar select File followed by Load Images File Grid Spot Tools Help k Load Images 3 Browse to the Samples folder within the ImaGene folder The location is typically C ImaGene Samples 4 Select 17hrs1 ti
77. ere between 0 and 4 The user can either use a slider on Quality Flags panel of Settings dialog box or type the threshold value directly in the text field Negative spots this tool will automatically flag any spot with signal mean lower than background mean A green mark will be issued to this spot on image and corresponding flag in the results table will have value 4 Poor spots 00 ImaGene has a complex tool for detecting low quality spots It is designed to accommodate various possible interpretations of spot quality This tool includes 7 different criteria for spot Negative Spots 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 Test against whole image vi Background 0 5 0 6 OF 08 09 10 Test against the subgrid only Flag for background being contaminated 9905 The top most check box on quality parameters dialog corresponds to flagging method based on background abnormality detection Background level of each individual spot is compared to local background level distribution over the image through a t test Resulting p value is subtracted from 1 and is called confidence in contamination presence This confidenc
78. ered trademarks of Sun Microsystems Inc in the U S and other countries 4 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 CONDITIONS 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 De 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
79. ers you should 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 IGNORED_MEDIAN gt true lt IGNORED_MEDIAN gt lt PERIMETERTOAREA FLAG gt false lt PERIMETERTOAREA FLAG gt lt BACKGROUND FLAG gt true lt BACKGROUND FLAG gt lt POOR FLAG gt true lt POOR FLAG gt lt PERIMETER FLAG gt true lt PERIMETER FLAG gt lt PERIMETERTOAREA THRESH gt 0 65 lt PERIMETERTOAREA THRESH gt lt TOLERANCE gt 5 0 lt TOLERANCE gt lt STANDARD LOW gt 5000 0 lt STANDARD LOW gt lt OFFSET FLAG gt true lt OFFSET FLAG gt lt SHAPE REGULARITY gt true lt SHAPE REGULARITY gt lt CONTROL TYPE LOWTHRESH 0 gt 45000 0 lt CONTROL TYPE LOWTHRESH 0 gt lt NEGATIVE FLAG gt true lt NEGATIVE FLAG gt lt BLANK_CV gt 5 0 lt BLANK_CV gt lt IGNORED AREA gt true lt IGNORED AREA gt lt POOR THRESH gt 0 995 lt POOR THRESH gt lt CONTROL TYPE ID 0 0 gt Gene17 lt CONTROL TYPE ID 0 0 gt lt OFFSET THRESH gt 60 0 lt OFFSET THRESH gt lt HOT CV gt 3 0 lt HOT CV gt lt CONNECT FLAG gt false lt CONNECT FLAG gt lt EMPTY THRESH gt 0 3 lt EMPTY THRESH gt lt HOT CHECK gt false lt HOT CHECK gt lt MEASURE MEAN gt true lt MEASURE MEAN gt lt BACKGROUND THRESH gt 0 9995 lt BACKGROUND THRESH gt lt MEASURE S
80. f and 17hts2 tif Multiple selections can be performed by holding the lt ctl gt key on the keyboard and left clicking x Look in sampes cal El DO ee Load images e r Samples 17hrst gel 17hrs2 gel Samples tif SampleH tif Study _cy3 TIF Study cys TIF Study2 eva TIF Study2 cvs TIF File name h 7hrs1 gel Open Files of type Image files tif gel inf Cancel 5 Click the Open Button to complete the image loading A progress bar displays time remaining 6 Once the images are loaded and are visible within the Main image display located along the right of the primary ImaGene window the 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 click the Align Images button from the toolbar In additional to auto alignment ImaGene offers manual tools for rotation and translation of images B ImaGene 5 0 Full version wiBatch Processing l E mfa x File Grid Spot Tools Help ejst ua REDRESS Main Preview Composite 17hrs2 gel 17hrs1 gel Images E 17hrs2 gel E i7hrs1 gel Reverse Display Colors Display Control Gene ID lt Not Selected gt LE R Grid dx 121 333 dy 4 33333 distance 121 410 Grid Placement and Manipulation 7 From th
81. fault FLEXIm 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 flexlm lmegrd exe f Enter the Path to the license file or click the Browse button to the right In this example it is C flexlm 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 flexlm debug txt 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 I Click on the Save Service button 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 LATDOLS by Globetrotter Software https saw globetrotter com Serice License File System Settings Litter Stat Stop Reread Server Status Server Diags Config Services Borrowing Servicer allow FLEXLM Server bo nun in the background 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 LMTOOLS by Globetrotter Software http weww globetratter com Fie Edt Mode Help Service License File System Settings Litiities Start Stop F C Double che
82. formation Yellow Quality criteria failures and controls warnings Gene ID FlaglXCoordControl Background Model Gene34O0 282 0 343 33333333 Gened2W0 3245 7 1 8 GeneSQ00 369 5 47 2265157094 penegi 415 2 55 561 5763546 GenebbO0 447 0 66 2060301507 Gene 40 500 7 41 796875 Genes20 3544 5 47 3009350315 Gene900 565 2 149 97 0 632 0 T2 4493392070 0 675 7 680995215311 UES r 15 5 51 5000000000 eee 63 2 24 5256G66866 Genes 0107 2 116 180602006 Gene 0 1151 0 103 349593495 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 Column Number of column in the subgrid where the spot is located GeneID Gene ID information 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
83. formed some action that should be logically followed by some other action Then Wizard will prompt what action was performed and what should be done next To follow this advice user can either use a tool 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 recommended action 1s not necessary user will have an option to go to next advice Sequence of recommended actions will start immediately after the 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 If user tries to apply some tool that does not fall into a pre defined sequence of actions Wizard will switch 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 user has an opportunity to learn how to use the software in an interactive fashion 1 6 Batch Editor and Batch Processing The ability to batch process images 1s an optional component that can be added to the base ImaGene A batch is simply a series of similar images to be processed with the same settings to be processed autonomously Through the use of this module ImaGene can be used in high throughput environments where large numbers of arrays are processed everyday The same data is exported from ImaGene whether run in batch mode or not During ba
84. gh and low within the tiff image The result is that ImaGene does not know whether white or black pixels are the high values If all signal values are negative which can be deduced through quantification or use of the intensity tool then select Invert Values and re quantify The Find Negative Spots option is not intended for use in these situations 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 you will want to enforce grid constraints as this will limit the movement of circles during spot finding and help prevent erroneous spot finding due to dust and other contaminations Local Flexibility Local Flexibility defines the radius measured in pixels that ImaGene is allowed to search for spots The origin for the search 1s 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 Grid Flexibility Grid flexibility 1s an indication of the extent to which ImaGene should deform the grid to match a given set of spots The measurement is a qualitative notion based on a large part due to the unique properties of the image Most users should set this to the midd
85. hat you enter your actual Serial Number since this information is used to determine eligibility for upgrades using the BioDiscovery Auto Update feature The serial number can be found on the case of the CD shipped to you or in the accompanying User Registration form If you do not have a serial number because you re only evaluating this product you can enter BDI DEMO Click the Save Floating button to save this information and exit Start the BioDiscovery software If the license server is running the software will start up normally 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 The Gene ID file allows you 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 For example you will be able to click on a GenePie from within GeneSight Lite and see the specific Gene ID associated with the selected spot in addition to all the other detailed quantification information 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
86. he 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 e Does not distribute additional software intended to replace any component s of the Licensed Software e Agrees to incorporate the most current version of the Licensed Software that was available 180 days prior to each production release of the Program e Does not remove or alter any proprietary legends or notices contained in the Licensed Software e Includes the provisions of Sections 2 3 4 5 6 7 8 9 of the Binary Code License and Sections and 2 of the Supplemental terms in Licensee s license agreement for the Program e 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 e 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 e Only distributes the Licensed Software pursuant to a license agreement that protects Sun s interests consistent with the terms contained in t
87. he Agreement 2 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 oy 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 Java related trademarks logos and icons including the Coffee Cup and Duke Java Marks and agrees to e Comply with the Java Trademark Guidelines at http java sun com trademarks html e 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
88. he flag value can then be used 1n later 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 shape is also added to make the flag visually unique Each shape corresponds to a different flag and is explained below ImaGene supports a variety of automatic and manual flagging types To change the type of flag right click with 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 Spots O Empty Spots O Poor Spots O Negative Spots Flagging codes Manual flags color red I Flag spots 5 Empty spots cross 6 Poor spots plus 7 Negative spots dash Automatic flags color green 2 Empty spots cross 3 Poor spots plus 4 Negative spots dash Note Manual flags override auto flags 1 8 9 Undo and Redo Tools ImaGene supports an unlimited number of undo and redo operations Anytime you wish to go back and not do an action this tool will provide means to do so Note The action to be undone or redone is displayed next to the text within the menu bar For example if I move the grid then wish to undo this action from within the Tools Menu the text will read Undo Move Grid This is a convenient means to see what action will be reversed 1 8 10 Grid Tools Adjust Metagrid This tool allows ma
89. he 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 7 1205_g txt Enter Experiment Information Enter Experimental Information for DataSource 1205_g txt Enter information for this data file You may also specify a file containing this information by entering the filename in the area above Dataset name in DataSetBuilder Experiment Descriptor 1 205 g Experiment User User Name Experiment Date 3 29 02 OK _ Use defaults for all Experiments Specify the information and click Ok Additional dialog will pop up asking you to select measurement that you want to import into GeneSight Lite EG 1205 g txt Select Experiment Columns elect the fields to load vi v MetaRow MetaCol Row Col vi X coord Y coord Diameter v Flag vi Median vi Area Select measurements and click Ok New data set will be created and corresponding measurements will be shown on the left hand side of GeneSight Lite window For more information on how to build a new data set please refer to GeneSight manual Chapter 6 E GeneSight Lite 3 1 5 File View Tools Plots Utilities Help GE E u E New Open Data Prep Chromosome Histogram 21 DefaultDS gs 1205 gpair amp Mean ratio amp BG Mean ratio amp Median ratio amp BG Median ratio amp Total ratio amp BG Total ratio 11205 g amp
90. her tools such as the Ruler and Image Intensity display their information here dxi l21l 333 dy 4 33333 distance lel 410 1 2 Control Tabs Located along the left of the main ImaGene window are two tabs containing essential information about the analysis 1 2 1 Main Tab The Main Tab is used to perform the essential steps of microarray image analysis loading the image s to be processed and placing a grid on the image and its corresponding structure The following Panels are contained within the Main Tab Main Preview Images E Samplea tif E Samples tif Reverse Display Colors Display Control NZ S Grid Gene ID lt Not Selected Images Panel The images panel displays the names of images currently loaded within ImaGene Each loaded image 1s 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 Images E Samples tif GO Sample tit The Images Panel fully supports context menus and as a result provides the following menu choices o Load Images Allows browsing to and selection of images from the file system o Remove Selected Images The images which are selected or highlighted from within the images panel are removed or unloaded from analysis o Select Color Select the color to be used for the select image when seen under th
91. his boundary and not include signal values from surrounding spots 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 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 with a dust particle assuming they will be fluorescing at a high intensity level will be filtered out 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 AutoSegmentation Selecting this option will turn on BioDiscovery s patent pending automatic segmentation Under most circumstances this should be selected Under AutoSegmentation the only parameter which ImaGene will use is the Background Buffer The typical value should be slightly less than the radius Under AutoSegmentation the slider bars for the signal and background percentages become disabled Note An important difference between automatic and manual segmentation is the fact that under manual the parameters and thei
92. idential 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 modify or create derivative works of the Licensed Software decompile disassemble or 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 No right 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 regist
93. isting Color Schemes v Tails Lower bound Upper bound rotai genesjog Find Gene Left selected Genes Midale selected Genes Right selected genes Total selected g Eg create partiton sources 2 genes 96 DataSet DefaultDS gs Transformations done For manipulations with within Histogram window refer to GeneSight manual Chapter 9 Gene Pie Follow steps 1 4 from the Scatter Plot section Select any number of measurements of interest in GeneSight Lite main window Click Gene Pie button on the toolbar Senere Gene Pie window will pop up Gene pie schema shows relative value of every selected measurement within each spot GeneSight Lite 3 1 5 File View Tools Plo ayers Aa Po Edit Sub Select Genes Color Scheme Choose URL dn New Save D Gan E e amp Total ratio amp BG Total ratio 1205 g amp Mean amp BG Mean amp Median amp BG Median amp Total amp BG Total 1205 g control amp Mean amp BG Mean E Median amp BG Median Existing Color Scheme Find Gene Reset Schemes create Patton sources 2 genes 96 DataSet DefaultDS gs 1205_gMean 1205 g control Mean _ Diameter encoding max intensity vi legend 4 For manipulations with 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 and vy click on the Go To Web
94. ity Area of a spot is divided by a square of spot perimeter and multiplied by 4z As a result this measure ranges from 0 highly non circular shape to I 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 Y Coord 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 Expected Y Y 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 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 positio
95. l and background mean intensity Median signal and background median intensity Mode signal and background mode intensity mode corresponds to the pick of corresponding histogram Total signal and background total intensity all pixel intensity summed up 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 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 User can check and uncheck these measurements at any time results table will be updated immediately Controls and Alerts ImaGene incorporates a powerful tool for testing expressions for control spots located on image This tools allows to group several gene IDs into a unique control type and to set up multiple test parameters for the type Ima
96. le values Note You can press 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 aspects of array image analysis Proper segmentation yields more robust data free from contamination and other adverse affects Bi ImaGene Parameter Settings Ioj x Controls and Alerts Image Preferences Quality Flags Measurements Spot Finding Segmentation K Background buffer fo v Auto Segmentation ale Lt so 7 Do not allow donut shapes Signal percentages Background percentages Low High Low High 10 90 10 90 100 100 100 100 60 a0 80 a0 60 60 60 60 40 40 40 40 ImaGene supports two primary type of segmentation manual and automatic With Manual Segmentation you must specify the appropriate values for ImaGene to use when analyzing the image Often these proper values can be arrived at through experimentation or through experience Alternatively through a robust patent pending statistical approach ImaGene can calculate the appropriate segmentation parameters for you Under normal circumstances BioDiscovery recommends use of the Automatic Segmentation due to its superior contamination removal Regardless of the segmentation in use both methods assist is in providing the highest quality data available
97. license granted under this License Agreement are non refundable and shall not be returned to Licensee under any circumstance including but not limited to any request for a pro rata refund by Licensee gt TERM This License 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 uninstall 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 uninstalled 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 Subject only to SECTION SIX 6 of this Agreement under no circumstances 1s Licensee entitled to a refund or credit of any licensee fees paid in consideration of the license g
98. location Then click the Next button 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 Note that ImaGene requires that Java Runtime Environment JRE 1 3 be installed on the computer During installation ImaGene determines if the Java 1 3 Runtime Environment JRE 1 3 needs to be installed If ImaGene cannot locate the proper runtime version you will be prompted to install Java before continuing the installation The ImaGene installation will automatically proceed after Java is installed When the installation is complete a message box appears Click the Finish button to complete the installation and close the install program A webpage now appears with the latest release notes for this particular version of ImaGene Close the web browser when done reviewing these release notes Prior to use please review the next section on licensing ImaGene ImaGene Update 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 Five different frequencies are available from checking at every start up to not
99. lows selection of a snapshot or sst file which will allow for the results of previous analysis to be reviewed or Grid reexamined Simply browse to and select the desired sst file and ImaGene will load previously created settings data segmentation and flagging information 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 generation of publication 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 Load Grid Displays the Load Grid dialog box Use this interface to select and open a previously saved grid grd file Save Grid Displays the Save Grid dialog box Use this interface to save a grid Clear Grid Removes all displayed grids from the Image Display Panel Load Gene IDs Displays the Load Gene ID File dialog box Use this interface to select and open a gene ID txt file Clear Gene Ids Displays the Confirm Deletion dialog box Use this interface to verify that you want to remove the displayed gene ID information Lo
100. mately the utility and convenience batch processing provides means very little without accurate quantification To view the results of Batch Processing ImaGene includes two tools the results reviewer and the ImaGene log file Launch the ImaGene software From the File Menu select Review Results Grid Spot Tools Help Load Images 6 Remove Selected Images 1 Batch Editor hl Save Display Image Browse to and select any one of the four snapshot files or files ending with an sst extension which were produced during the previous batch processing The file will be located within the same directory as the data output Click the Open Button 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 5 ImaGene 5 0 Full version w Batch Processing Ek m xd File Grid Spot Tools Help gle Hue SRS E ROAA a Main Results Composite Study1_cy3 TIF Study1_cy3 TIF seg Gene ID Field Main Metarow 1 Metacolumn 1 Row 2 Column 2 Diameter 9 0 Study1 cy3 TIF Field MetarowMetacolumnRow Column Main 2 gt Main Main Main Main Main Main Main Main Main Main Main Main Main Main Main ae oe EN oe ee oe oe EN e eo oe ee me oe an KTR
101. may be made by Licensee or any person under Licensee s control or authority 2 LICENSOR S RIGHTS Licensee agrees and acknowledges that the Software and the Users Manual 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 including associated intellectual property rights are and shall remain with Licensor and or Licensor s suppliers This License Agreement does not convey to Licensee an interest in or to the Software but only a limited right of use revocable in accordance with the terms of this License Agreement 3 RESTRICTED RIGHTS Licensee hereby covenants that neither the Software product nor any information or know how embodied in such Software will be authorized to be directly or indirectly transported or removed to any source for use in any country or countries 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 4 LICENSE FEES _ The license fees paid by Licensee in consideration of the
102. n 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 text output file The file itself is a simple XML file that can be saved and loaded to the ImaGene Parameter Settings Window e Output Directory ImaGene requires the location where to deposit the subsequent text files after analysis Click browse to and select the output directory to use The location can be either be local or on a network drive e OK Once all the required parameters have been specified the OK Button becomes enables and the entries can be added to the batch e Cancel The window is closed and the entries are not added to the batch 1 6 3 Creating and Running a Batch OAO TP SS See Setting up batch processing within ImaGene takes only a matter or minutes and requires only a few pieces of information The description below covers the basics of creating and running a batch as well as common tips and tricks for SUCCESS Setup the initial parameters for use within ImaGene This needs to be done the first time prior to running a batch To this perform the following Load a sample image Load a gene ID Create and Place a grid on the image Save the result as a template to a location of your choosing Set the parameters on the ImaGene Parameter Settings Wi
103. n completes Click the OK button to acknowledge that ImaGene has been removed your computer Macintosh Please see the release notes provided with your ImaGene software distribution Unix Please see the release notes provided with your ImaGene software distribution 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 recently switched over to GlobeTrotter s FLEXIm license manager This popular license manager provides an excellent balance of features flexibility 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 FLEXIm based products the addition of BioDiscovery licenses will be very easy Many specific FLE XIm questions are answered in this online FAQ http www globetrotter com flexlm Imfaq shtml It is not necessary to review this FLEXIm FAQ since the basic setup of FLEXIm is covered in the accompanying text The FLEXIm license manager can be configured in tw
104. n gt X data configuration xml lt Configuration gt lt Destination gt C Documents and Settings data Desktop lt Destination gt lt Channel gt 0 lt Channel gt lt ChannelName gt lt ChannelName gt lt Imageld gt 0 lt Imageld gt lt Entry gt lt Entry gt lt Image gt C BioDiscovery ImaGene Samples 198fdddty65 TIF lt Image gt lt Template gt C Documents and Settings data Desktop Desktop tpl lt Template gt lt Configuration gt X data configuration xml lt Configuration gt lt Destination gt C Documents and Settings data Desktop lt Destination gt lt Channel gt 0 lt Channel gt lt ChannelName gt lt ChannelName gt lt Imageld gt 1 lt Imageld gt lt Entry gt 5 2 Technical Support BioDiscovery is available to answer any questions that you have 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 310 306 9310 United States Fax 310 306 9101 Mail 4640 Admiralty Way Suite 710 Marina Del Rey CA 90292 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 h
105. n 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 the spot s signal region can be inscribed in Control Name of a control type for current spot no name means the spot is not a control spot Failed Control 0 if the control passed all tests 1 1f at least one of the tests failed Background Contamination Present 0 if the spot passed background contamination test I 1f it did not see section 1 4 Signal Contamination Present 0 if the spot passed signal contamination test I if it did not see section 1 4 Ignored failed 0 if the spot passed ignored percentage test I 1f it did not see section 1 4 Open Perimeter Failed 0 if the spot open perimeter test 1 1f it did not see section 1 4 Shape Regularity Failed 0 if the spot passed shape regularity test I if it did not see section 1 4 Perim To Area 0 if the spot passed perimeter to area test 1 1f it did not see section 1 4 Offset failed 0 if the spot passed offset test 1 1f it did not see section 1 4 Empty spot 1 if the spot was qualified as empty 0 if it was not see section 1 4 Negative spot I if the spot was qualified as negative 0 if it
106. n the batch 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 bottom right of the ImaGene Batch Editor Window is the Channel Selector The channel selector is designed for use with multichannel images also known as composite images Essentially a composite image is a single file which contains several images within it This makes it possible to load a single file within ImaGene yet have 2 4 or more images then visible Several scanner manufacturers on the market today produce such file formats While most of the scanner software can also export the images in a single one image per file format ImaGene is flexible enough to support both formats Channel 1 r Channel 2 There may be times when only a single channel of a multichannel scan is of interest to you If this is the case then only processing the desired channel may save time 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 The window requires four parameters to be entered in order to batch a set of Images All images which belong to a common set can be created here and added to the batch as a group For example if I have 5 images generated with Molecular D
107. ndow Save this configuration to disk 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 Gird specify the template previously created Likewise 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 Click the Save Batch Button In the Save As Dialog that appears navigate to the location where you wish to save the file Specify the name of the file A bch file extension will automatically be added to the name 1 6 5 Loading a Batch sae ge ea If a batch file has been saved to the file system it can be easily recalled and used again The following steps outline loading a batch file Click the Load Batch Button Browse to and select the desired batch file Select Open to load the file The batch entries are now visible within the Batch Editor 1 6 6 Editing a Batch OI Once entries have been added to a batch they can be easily modified eliminating the need to start anew creating the batch file To modify an existing entry within the Batch
108. ned within the table Image The name of the image Grid The name of the grid Gene ID The name of the Gene ID file Configuration The name of the configuration file Output Directory The name of the output directory Channel Name The Channel to be processed when using composite images Image Grid Gene ID Coantigur Output Di Channel Sample Templat Templat Config xml Desktop Sample Templat Templat Contig xml Desktop Sample Templat Templat Conrfigxml Desktop Buttons From within the Batch Editor Window buttons exist to do the following Add Batch Entries Adding batch entries allows several images to be associated with a settings and grid file for processing For example if I want to add 10 images to be processed then I would only need to click this once specify the 10 images some other parameters and the 10 images would be added to the batch If I then had 13 more images of a different type I would repeat this process of adding batch entries until all images were added As I add batch entries the 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 use of the lt shift gt and lt ctrl gt keys Add Batch Entries Remove Selected Edit Selected Entry Edit Selected Entry This opens a window that allows the
109. ng on the Grid Panel provides the following options Create Grid The option launched the Grid Creation Window where the essential information about the design of the grid must be entered Remember that within ImaGene the grid is the most elemental structure followed by the metered and finally field The following parameters must be specified O 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 Ifa gene ID file is being used in conjunction 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 1s specified here and that is contained within the Gene ID file For example 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 If the name does not match then ImaGene with NOT use the gene name within the data output file O Rows The number of rows of spots contained within the grid Typically this is simply counted visually O Columns The number of columns of spots contained within the grid Typically this is simply counted visually O Min Diameter This specifies the minimum expected diameter of the spot and is measured in pixels The size of the spot can best be determined by use of the R
110. nipulation of entire metagrids The metagrid can be moved stretched or resized ET Adjust Subgrid This tool allows manipulation of entire subgrids The subgrids can be moved stretched or resized E Adjust Spot This tool allows for individual spots to be moved and resized EJ Lasso Adjust This tool allows you to select irregularly shaped regions of circles for adjustment Instead of shifting entire subgrids you can now specify unusually shaped regions of circles for fine tuning For example you would want to use this tool with membrane arrays because edges could become warped quite often zi Rectangle Adjust This tool which is similar to the lasso adjust tool allows you to make a sub selection of 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 a definite rectangular anomaly exists within the array making it ideally suited for the Rectangle Adjust tool R Duplicate MetaGrid Duplicating an existing metagrid can save time and produce better results rather than manually recreating the metagrid several times To use it click on the button and select a metagrid to duplicate by left clicking on it After that you can multiply this metagrid in any place of image by left clicking there To stop copying make a right button click Show Hide
111. ntified you need to follow these steps 1 Make sure that you saved quantification results from current image clicked Save button at the bottom of quantification table 2 Click GeneSight Lite button on the toolbar E And wait until GeneSight 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 result files saved by ImaGene in the file browse window on the left hand side Drag the file considered to be control data into Control window and the file considered to be experiment data into Experiment window Pa Dataset Builder P i JE XI Cancel Done Save Sort All Rema Selected Prat ins Selec fies on the kert to aki tothe DataSet on the tight y ie joa CEspaimen 7 f EEn E 1305 g E 120509 E La S EF 1505 g bath an SP 1505 gda iwa p data oa O 1205 gbi D iih a i EG 1205 gala pro S 1505 9 dat pre S EF 1505 g dala ba P 1505 g data stan 4 9 1205 data star Pai Data Perform Ratio O 1205 0 EE imeanregl 75noig ap Poriorm Ratio amp Add as Replicat0 DJ iit B JE Rencated Experimental Conditoris Addin Mma Sai Adil s Experiment Add as Control HE P Add 33 Raplicaia Click on Pair Data Perform Ratio button the new data set will show up in the window on t
112. o 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 1s 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 FLEXIm 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 The FLEXIm 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 manager service every time Below is an example of a node locked computer being both a license server and a client This diagram 1s also a good reference since it shows the file names for each part of the license FLEXIm Node Locked Dataflow Server 2 Vendor vie Daemon File biod exe Daemon license lic Sand Rev License Manager Daemon Imgrd exe SendiRey Licensed Client Application ImaGene Read Only Local License File local lic 4 2 3 Floating Licenses A floating license allows BioDiscovery products to run on a set number of different c
113. ocedure 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 background contamination threshold Flag for high ignored percentage Wnoredpercentage As it was mentioned before some pixels may be assigned neither to signal nor to background in the presence of local contamination Such contamination will be excluded from the measurements and called an ignored region Ignored percentage flagging works as follows It computes the area of ignored regions directly neighboring touching the signal area Let us denote it by Z Than the following ratio is computed R I x100 I S S is the signal area in the above equation AWN re 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 Oen perimeter percentage This tool is designed to catch a specific type of spot deformation It computes the percentage of 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 abnormal form Whenever such percentage crosses the pre set threshold spot gets flagged as of low quality Flag for abnormal shape regularity Shane regularity This algorithm computes a shape regularity measure that characterizes clo
114. omposite sampleA tif SampleB tit 4 All image manipulation tools such as zoom and rotation 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 The Zoom Slide bar is one of four tools available for zooming 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 Some of these new tabs display the segmentation as it has been performed for the given image From this view you have the ability to see the macro view of the image and notice any large defects and the corresponding segmentation Typical use of the segmentation tabs is for detailed post processing quality assurance analysis Composite 1205 r TIF 1205 g TIF 1205 r Tlf seg 1205 o T11f seg 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 Red All pixels within the red lines are signal
115. omputer s Hostname is used to identify the computer on the network In this example the hostname is BIOINTERN n 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 ne d Email support biodiscovery com for your customized license file e Please include The Computer s Ethernet Address from step 3 The Computer s Hostname from step 3 Your name or the name of who purchased the software Your company 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 f 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 FLEXIm directory In truth the license lic file can be anywhere on your computer but we put it in the C FLEXI m directory to make it easier to keep track of 6 Configure the license services k Start the LMTools program by double clicking on Imtools exe in the FLEXIm directory Il Click on the Config Services tab m You will see the following screen MTOOL 5 by Globetrotter Sotware http i eee globetrotter com ale Fie Edt Mode Help Service License File Spitem Settings Utiiies
116. omputers 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 FLEXIm 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 1s 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 that 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 FLEXIm Floating Client Server Dataflow server Daemon Vendor meee KE Daemon ape File biod exe Log report log license lic Send Rev License Manager Daemon Imgrd exe SandiRev Licensed Licensed Licensed Client Client Client Application Application Application CloneTracker ImaGene GeneSight Read Only Local Local Local License
117. ou may also specify file containing this information by entering the filename in the area above Dataset name in DataSetBuilder Experiment Descriptor 17hrsi Experiment User User Mame Experiment Date 402 OK Use defaults for all Experiments 47 Click OK on the next window to use all experimental conditions available within the data files ey 17hrsl txt Select Experiment Columns elect the fields to load ra vi MetaRow MetaCol Row Col X coord Y coord Diameter e Flag 1 Mean Wi Median Total Area Standard Deviation 48 Click on while holding the lt ctl gt key Mean listed under 17hrs1 and 17hrs control in the primary GeneSight Lite User Interface Be certain both Mean labels are highlighted El DV MELUPPNINI ali 17hrst ET Mean ET BG Mean ET Median ET BG Median 17hrst control E Mean EF BG fan Existing Color Schemes Laas 49 Click the Scatter Plot Button from the toolbar Scatterplot 50 Drag with the mouse to select all the genes located along the top right of the plot or those genes with high intensity values Scatterplot D x File Edit Sub SelectGenes Color Scheme Choose URL Help Reference 17 hrs1 controfMean 17hrs1 Mean _ Show Gene IDs 51 From the menu bar select Sub Select Genes followed by Sub select Chosen Genes Color Sche Create Gene Subset Use Full Gene
118. pots that belong to this type and violate the minimum expression criterion becomes higher than specified percentage the warning box will pop up after the quantification procedure is completed or Update Visual button is pressed Pass fail image for maximum value whenever the number of spots that belong to this type and violate the maximum expression criterion becomes higher than specified percentage the warning box will pop up after the quantification procedure is completed or Update Visual button is pressed 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 Select a type from the list click on Add gene ID button A new dialog box will pop up If gene ID file 1s loaded you can select any gene IDs from the list on the right hand side of the dialog and click Add You alternatively may just type an ID in the text field and click Add In this field you can also type a key for assigning gene IDs The key is created using a symbol For example by adding a key BLANK you assign all gene IDs that start with BLANK to the current control type Whenever you are done adding gene IDs click Close Alternatively you
119. r corresponding values are applied uniformly across all spots of the image Under automatic segmentation each spot is calculated independently usually generating more accurate segmentation Quality Flags Once the segmentation is done and the spot expressions are extracted the next question would be how much we can trust 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 ImaGene Parameter Settings P cualtyrns Spot Quality Labeling Threshold signal strength Empty Spots r e Tie POL ee Lo Ce A Tyee cL a 0 0 1 0 i JO 40 2 0 Poor Spots r Change Parameters Negative Spots r Multi Channel Flagging Load Save As OK Threshold signal strength Empty Spots j iaa are Tat TA i Empty spots i this option allows you to automatically flag low expressed spots or missing spots This tool needs to have a sensitivity threshold set up 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 value can be chosen to be anywh
120. ranted hereunder regardless of the reason for termination of this Agreement 6 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 disclose the Software to any third party Licensee further acknowledges and agrees that any written information or documentation provided by Licensor to Licensee which contains a legend upon such documentation whether or not such legend be a single legend affixed upon a multiple page document which legend identifies such documents 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 such information contained within such documents 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 written informa
121. reate MetaGrid from the menu Inthe Metagrid Parameters Window specify the number of rows and columns of subgrids contained within the metagrid Click Place Metagrid and click on the top left spot in each of the corner subgrids Typically this process will require four clicks but never more How to Save a Grid Once a grid has been created the following steps will save the grid for later use From the menu bar select Grid followed by Save Grid In the Save as Dialog browse to the location where you wish to save the grid file Specify a file name ImaGene will automatically add the grd file extension to the end of the name How to Load a Grid To load a previously created grid perform the following steps From the menu bar select Grid followed by Load Grid From the Load Grid Dialog that appears browse to and select the desired grid file Click on one of the following radio buttons to select a grid placement method Place Manually Select this option to set the location and size of the saved grid yourself 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 positioned based on this information Place in Saved Position Select this option to place the grid in the identical position of the original grid Use this option 1f the size and resol
122. request a license from BioDiscovery 3 Start ImaGene 4 Load an image 5 Establish Settings 6 ie 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 You use this window 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 area fits into the array analysis and data extraction process z4 ImaGene 5 0 File Grid Spot Tools Help eske Bee UES J uR main nen composite GE GE Images E SampleB tif E Samplea tit I Reverse Display Colors Display Control VZ Eoo Grid A 2x2x20x16 Gene ID lt Not Selected gt Quantify Menu Bar Located along the top of the window Click on one of the menus File Grid Spot Tools or Help to view the program commands available on that menu The following options are available from the Menu Bar File Grid Spot Tools Help e File e Load Images Allows selection of images to be loaded from the file system e 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 Al
123. s of subgrids Delete Selected Fields The currently highlighted field will be removed Clear Grid All fields that have been created will be removed l 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 meta grid with 15x10 subgrids after selecting this conversion the resulting subgrid size will be 30x10 During this conversion nothing changes except for the how the individual spot location are represented This feature is option and may used if required to construct a multi level metagrids for example Properties This option opens a window which displays the parameters for the currently selected field Only certain options can be changed while the remainder required the grid to be deleted then recreated a new The following parameters are displayed within the properties window O Field The name of the field This value may be changed O Metarows The number of metarows previously specified O Metacolumns The number of metacolumns previously specified O Row s 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 Min Diameter The minimum expected diameter of the spots to be used during spot finding This value may
124. s the visual pixel intensities into numerical values to be used later in expression analysis The Quantify Button starts this computation within ImaGene and should be performed after all other parameters have been set and the grid has been placed The amount of time quantification takes is directly proportional to the speed of the computer hardware Once quantification 1s completed the Preview Tab becomes highlighted and the numerical values become visible Additionally after quantification complete an additional tab for each image becomes available over the main image panel This tab is the segmentation tab and displays the segmentation which has been performed across the entire image 1 2 2 Preview Tab The Preview Tab displays information about the image both prior to and after quantification Numerical values and segmentation can easily be viewed and reviewed to determine that optimum settings are established The Preview Tab is divided into two primary parts Segmentation Preview Quantification Preview Main Results Gene ID Gene101 k 13 a11 Field A Metarow 1 Metacolumn 1 Row 16 Column 6 Diameter 30 0 n de ERIE Row _ Column 15 1 p p it f fi fi 1 1 an ami mb fom fo fot mb mb mb mb Po Po mb 1 4 r 4 Selectec Freeze Unfreeze _ Resto feat column order Setene Su ent To XML Case
125. s 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 FLEXI m debug txt file to support biodiscovery com 4 5 FLEXIm 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 l D SF WA NodeLocked Save Floating Start the License Wizard by clicking on Start gt Program Files gt Product gt License Wizard or by double clicking runwiz bat in your product directory Click on the Floating tab You will see the following screen BioDiscovery License Management Wizar i ol x Floating Server Name BIOGREEN User Namel Joe Green Company Name Green Corp Serial NumberlBon 2345 Fen 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 It is important t
126. seness of spot s border to circular shape First step of this algorithm is to inscribe a signal area of a spot into a circle Than number of non signal pixels that fall within this circle is computed and divided by circle s area This ratio is subtracted from as is called shape regularity Obviously such a variable will range from 0 highly non circular shape to I a perfect circle Whenever this ratio falls below the pre set threshold spot gets flagged as of low quality Flag for low Area to Perimeter ratio H e4to Perimeter ratio This measure of spot s quality plays a role similar to the one of shape regularity just mentioned several lines above Area of a spot is divided by a square of spot perimeter and multiplied by 4x As a result this measure ranges from 0 highly non circular shape to 1 a perfect circle Whenever this ratio falls below the pre set threshold spot gets flagged as of low quality Flag for significant offset from expected position set Expected position of a spot in current grid is computed by fitting least square lines to grid s columns and rows Whenever the offset becomes larger than pre set percentage of the distance between grid nodes spot gets flagged for low quality User can use any combination of the above quality measures with arbitrary thresholds However the quality parameters dialog has a Default button that allows to set a default configuration for the thresholds Whenever at least one of
127. 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 versus background The following visual spot information 1s 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 pixel determination 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 Composite Displays an overlay between the Original and Segmented images This image is key to determining 1f 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
128. ta is visible under the Results Tab 31 Select Plots tab on the Image Display panel 32 Select Histogram on the Plots panel 33 Select Signal Mean Ratio chl ch2 from the list of measurements 34 Make sure that log option is not selected for the Histogram 35 Select 2 fold regulated genes by specifying 2 in the Upper bound text box 36 Freeze the selection using corresponding button in the bottom of the table 37 Sort the data by column Selected clicking on its header All selected genes will be in the bottom of the table 38 Go through the spots one by one analyzing spot images 39 40 41 Gene ID Gene101 9 2 a7 Field sprik gt Metacolumn F le 7 GE Name e e SampleA tif SampleB tif Samplea it SampleB tif Field Row Colarm Gene ID Flag Sig D 20 2 20 i a 2 oe 2 165 TE TJ oe 386 Choose the plot type Histogram FE TOTKRIN To ae 13 Gene10 11 Gene10 11 Gene10 2 Gene10 2 Gene10 1 Gene10 2 Gene10 16 Gene10 8 Gene10 16 Gene10 16 Gene10 14 Gene10 16 Gene10 112 Gene10 14 Gene10 Selectec Freeze Uniinez Te ae A aaa FBP 7 ST 1 Save frozen rows Save Export To XML Cancel Signal Mean ratio ch1 ch2 Me Click the Save Button to save the Quantified data and snapshot file to an output directory of your choice Harr Pr PPP PP PP PPP ee NNNNNNNNNN a a NNNNNNNN
129. tation 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 9 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 Court for the Northern District of California 10 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 11 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 au
130. tch 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 creates saved and loaded Batched can be created and saved for later use or as a template to be modified at a later date Even details such as which channels of mutichannel image composite to process can be selected loj x Entries Available Unlimited Add Batch Entries Remove Selected Edit Selected Entry l Show Full Path ge ImaGene Batch Editor Gene ID ConfigurationQutput Direc ChannelMa Dw vorkKish Docume CADocume CADocume ClDocume Dworki Docume C1Docume CIDocume CIDocume DAAorkSh Docume CIVDocume ClADocume CIDOCume DAWarkSh C1Docume CVDocume CADocume ClDocume Dev orkKiSh Docume Clbocume Clbocume CIDocume DvvorkiGh Docume CADbocume CADocume ClDocume Dw vorkKish C1Docume CAbocume CIDocume CIDOCume DewvorkKiSh Docume Clbocume Clbocume CIDocume DavvorkiGh Docume CADocume CAbocume ClDocume Dw vorkKish Docume Clbocume CIDocume GlDocume Channel 1 Loan Baten Save Baten run Batch Table Once a batch has been created the information about the batch is displayed The following information is contai
131. thorized representative of each party 12 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 13 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 Sun may assign this Agreement to an affiliated company 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 l 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 e Distributes the Licensed Software complete and unmodified except for t
132. ties to be specified by changing the curvature of display curve e Bottom Triangle This sets the maximum intensity to be displayed Grid Panel The Grid Panel lists all fields that have been created and 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 Grid Main 8 x 12 Control 12 x 20 Fight 1 x 2 Before explaining how and why to created 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 ina 2 x 4 configuration the region of the slide containing the resulting printing 1s 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 are printed by a single pin 12x12 for example The accompanying diagram demonstrates the relationship between the three levels of structure The bounding box represents the field The MetaGrid is 2 x 4 NR CO se eeeaeaeaeeaeee The Grid Panel like other components of ImaGene fully supports context sensitive menus Right clicki
133. tion 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 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 SIX 6 shall not extend to any information which 1s lawfully known to Licensee prior to receipt from Licensor or 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 The provisions of this SECTION SIX 6 shall survive termination of this Agreement ve LIMITED WARRANTY Licensor warrants for a period of THIRTY 30 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 Users Manual LICENSOR FURTHER WARRANTS THAT DURING THE WARRANTY PERIOD THE MEDIA THAT CONTAINS THE SOFTWARE SHALL BE FREE FROM DEFECTS IN MATERIAL AND WORKMANSHIP LICENSEE S SOLE A
134. tion is to use the Auto Grid placement tool once the image and the grid have been loaded The tool will automatically move the grid to the proper location 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 Grids placement on the image Ifthe Auto Grid Placement cannot locate the proper array geometry then the mage will probably need to be processed manually 1 8 3 Save Display Image Tool ImaGene S save Display Image Tool Fas 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 of use in publications 1 8 3 1 Saving the Display Image Perform the following step to save the current composite overlay to a tiff image file 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 the location you wish to save the image 3 Specify a file name ImaGene will automatically add the tif file extension at the end of the
135. u 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 GEML v 1 0 XML standard representing a pattern that can not fully describe a grid structure but rather provides location and gene ID info for every spot 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 Map View Panel The Map View provides a comprehensive and unabridged view of the image while indicating exactly where within the image the primary image display panel is zoomed to 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 for proper gridding and segmentation 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 Quantify The act of quantification covert
136. ul 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 Plots Choose the plot type Em y Q S Zoom Print Image Save Image 75 Box plot pk a i 8 8 EE ED ad 000 8 SubGrid Number Y Signal Mean ch1 naa Note any selection that you make using one of the plots will be common for all other plots results table and image tab There is an exception though if you select a group of spots using Scatter plot Box plot or Results table the selection will not be visible on Histogram plot because of its specifics But if you make selection using Histogram it can be viewed by all other tools 1 4 ImaGene 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 Grid Spot Tools Help Loalsmages Remove Selected Images z Review Results Batch Editor lal Save Display Image The window is organized into several tabs which group related parameters
137. uler 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 the 10 of the smallest spots of the array Depending on the type of array 1f the spots are highly uniform then the minimum diameter specified here will be close or equal to the maximum diameter specified next O Max Diameter Similar to the Min Diameter specified above this parameter reflects the maximum anticipated spot size measured in pixels Ts with the minimum diameter this value can be approximated by using the top 10 of large spots and measuring the sizes with the Ruler Tool O 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 is the most common all the possible selected are m Rectangular Staggered Left Grid m Staggered Right rid 6 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 To create a metagrid first highlight the desired subgrid then select Create Metagrid The Create MetaGrid Window appears and requires the following parameters to be specified O Metarows The number of rows of subgrids O Metacolumns The number of column
138. ution of the images has not changed Finally click the Open Button to place the grid Gene ID The Gene ID file allows you 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 If a Gene ID has been selected the name of the corresponding file is displayed here The Gene ID also supports right click context menus with the following options Gene ID12046_genelDs ted Load Gene ID s Selecting this option will open a window allowing for the selection of the gene ID file to be used Likewise you may also select Grid from the File Menu followed by Load Gene IDs to accomplish the same task 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 Clear Gene ID s If a gene ID has already been selected this option will remove the selected file from use Likewise you may also select Grid from the File Menu followed by Clear Gene IDs to accomplish the same task Load Gene IDs Clear Gene IDs Template You can load and save a template file that will contain both grid structure and corresponding gene IDs For this purpose you can use BioDiscovery s serialized data format tpl or yo
139. values Yellow All pixels within the yellow lines are ignored pixels Note The description and use of colors between the Preview Panel and the segmentation tab does vary slightly The segmentation tab includes the use of yellow to indicate ignored pixels where as the Preview Panel uses no coloring 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 is accounted by the fact that due to customer requests the segmentation tab is designed to prevent eyestrain during extensive visual inspection Plots Tab This tab appears also only when the image is quantified It contains a set of useful data visualization tools that can help speed up the analysis process Choose the plot type Histogram You can take virtually any measurement available from the results table and plot it in a manner corresponding to one of three tools Histogram Scatter Plot Box Plot Once you choose desirable type of visualization choices of the measurement for X and Y axes become available If Histogram is selected only the measurement choice for X axis will be available Any plot can be printed or saved as an image file A axis T axis Signal Mean T signal Median T Change in measurement selection will be followed by update of the current plot Histogram As we m
140. w quite large often surpassing the image size itself The snapshot file contains the following components within it Grid Gene ID Segmentation Quantified Data Quality Measures Flag Values While the sst file contains almost all the required information to reload and review 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 ask you 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 From the ImaGene menu bar select File followed by Review Results Likewise you may click on the Review Results icon located along the toolbar With the Open dialog browse to and select the snapshot file you wish to open Depending on the file ImaGene may prompt you to specify the location of the original images as well The information and data should now be visible within the main ImaGene user interface If you wish to reprocess the images click the Cancel button along the bottom of the Results tab and use Quantify button of the main panel 1 8 ImaGene Tools ImaGene includes many handy tools designed to help facilitate array analysis These tolls are designed to allow manipul
141. was not see section 1 4 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 you are satisfied with the results the final step 1s to Save the data ImaGene will save the data to common tab delimited test files which can easily be opened in other programs such as Microsoft Excel or Notepad ImaGene will save each image s data to a separate file and will automatically name the file based upon the name of the image Note By default ImaGene does not compute ratio values between images and save this information for a text file To compute ratio information please use GeneSight Lite Due to the large number of post image analysis options and to be as flexible as possible BioDiscovery as separated all post image analysis processing out of its ImaGene Analysis Basic mathematics such as generating ratio value can be performed with GeneSight Lite If additional more advanced comput
142. ynamics hardware then I may wish to add all five images here since they contain the same settings and grid within ImaGene This allows me to specify a single settings file and template file for several similar images Create Batch Entry 3 Images gilentGreenJX_STD_2 tif gilentysreent STD 4tif Grid EC Documents and S ta Gene ID C Documents and Setti Configuration lt Documents and S ta Output Directory Documents and sett The required parameters include e Images The images to be included with the batch and to be associated with the setting mentioned next Typically while the hybridization information about the images may be unique the geometry of the arrays is the same If this is the case then multiple images can be loaded here e Grid The grid file that matched 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 I grid file does not contain gene IDs e Gene ID The Gene ID file allows you 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 Please see section 5 1 for the proper file specification for a ImaGene gene ID file e Configuratio

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