AGScan USERS MANUAL
Contents
1. Unit a s n non SE E Rotate Unit 4 NW aime a et ea Tam AO01 401 7 40 0 6923 0 2 A01 B01 6093 157 6090 004 1 2 1 1 d 3 ADI CO1 8456 314 6874 726 1 3 1 1 3 Example the grid has been moved and rotated AGScan User Manual page 20 46 Sigenae February 2006 Edition You can choose the unit of your rotation degrees radian the unit of your translation or resizing pixels um or spots Thanks to this tool you can manually correctly place the grid on the image In order to verify the position of each spot you can apply a spot detection presented below Note that it is possible to perform an automatic local alignment on a manually aligned grid It is interesting to know how manually place a grid but in the most cases you can perform automatic global and local alignments In the next pages we will see how to perform these automatic alignments Let us first introduce the notions of global and local alignments The global alignment places the grid as good as possible on the entire image The local alignment searches to correctly align each block separately These two algorithms have been initially developed for the BZScan software So they have a lot of parameters that can be modified and memorized IL Ac Global alignment algorithm iim Quantification Batch Tools About MovelRotateiResize Spots detection Global aligrimenie Local alignment Ed2. User interface Loading an image file Creating a grid Aligning a grid on an image The table Quantifying an alignment Analyzing quantification results Processing a batch First it will start by presenting the user interface layout III 1 User interface 2A AGScan January 27 2005 Version E E File Edition Display Image Grid Alignment Quantification Batch Tools About AGScan Title Toolbar Menu bar Display window Status bar The user interface of the software wants to be simple readable and as ergonomic as possible It is composed of five elements The title window presenting the used AGScan version in the selected language The menu bar containing the different menus of the application The toolbar icons wich are shortcuts to some sub menu actions The display window the main window containing experiment view Several elements may be displayed in the same time images grids alignments They are open in different tabs The status bar displays current information like status and progress bar AGScan User Manual page 11 46 Sigenae February 2006 Edition As we want the software to be simple to use we tried to place the menus in a logical order from left to right We hope that menus are explicit enough to be easily memorized Shortcuts to common actions have been added below the menus in order to open align and quanti
3. ZS D marrage Q Derni res nouvelles Summary i i Surveys 4GScan is a micro array image processing software It is an evolution of B2Scanz2 It is based on Image and i purposes fluo micro array images support multi language menus It is also a plugin based application user can Project Admins develop functionalities Remi See Developers Christophe Klopp Development Status 4 Beta Intended Audience Developers End Users Desktop Other Audience License GNU General Public License GPL View Members Request to join Natural Language English French German Operating System Windows Linux Programming Language Java Topic Bio Informatics Registered 2005 11 16 09 21 Activity Percentile 100 View project activity statistics Just click on the Files subsection Date GEESS SEENEN EER EE NE EN EN EN N N EE IE MEN AGScan Full v270106 2006 01 27 14 35 AGScanFull270106 zip 19 5 MB 01386 Zip v081205 2005 12 08 00 00 4GScanFull081205 zip 19 48 MB 01386 zip v061205 2005 12 06 00 00 4GScanFull061205 zip 19 48 MB 01386 zip v211105 2005 11 21 00 00 AGScanFull211105 zip 19 49 MB 01386 Zip AGScan Light v270106 2006 01 27 14 38 AGScanLight270106 zip 3 3 MB 01386 Zip v081205 2005 12 08 00 00 AGScanLight081205 zip 3 28 MB 11386 zip v061205 2005 12 06 00 00 A4GScanLight061205 zip 3 28 MB 01386 zip v211105 2005 11 21 00 00 4GScanLight211105 zip 3 29 MB 01386 zip Data ex
4. February 2006 Edition AGScan USERS MANUAL Version 0 1 February 2006 Edition Remi Cathelin Copyright 2006 SIGENAE INRA Permission is granted to copy distribute and or modify this document under the terms of the GNU Free Documentation License Version 1 2 or any later version published by the Free Software Foundation with the Invariant Sections being just Background no Front Cover Texts and no Back Cover Texts A copy of the license is included in the section entitled GNU Free Documentation License at the end of this manual AGScan v0 1 User Manual page 1 February 2006 Edition Revision table Version 0 1 Date February 15in 2006 Acknowledgment wrote this guide in English having the aim to give access to AGScan to the largest possible scientific community But English is not my mother language so any remark about the orthography or the grammar is welcome Major modifications First release Author s R Cathelin AGScan v0 1 User Manual page 2 February 2006 Edition INDEAX TIN RG SR L WEE 4 KEREN 4 1 2 MAIN FEATURES OF AGSCAN ee ee ee ee ee ee ee ee ee ee Ge ee ee ee ee ee ee ee ee ee ee ee ee ee ee ee Ge ee ee ee ee ee ee ee 4 e e ies EE 4 II GENERAL OVER VIE ee 6 OAK ey ies ME OE EE N OE EE N EO EE OO RE Ee NG 6 E SE Oie ie E OE EE EE EE RE N AE OE OR OE 7 IL 3 MAGE QUALITY E 7 ILA INSTALLING AGSCAN ON YOUR e NEE 8 MES TIO EE S EE
5. Image Constant diameter Quantification for each spot the quantification value is computed thanks to the image pixels intensities and the constant diameter given by the grid structure Image Variable diameter Quantification for each spot the quantification value is computed thanks to the image pixels intensities and a variable computed diameter Fit Constant diameter Quantification the quantification value is computed thanks to the estimated intensity of the spot with a fit function and the constant diameter given by the grid structure Fit Variable diameter Quantification the quantification is computed thanks to the estimated intensity of the spot with a fit function and its variable computed diameter The QM value is an indication of quality of the spots Each spot has a value of QM ranging between 0 and 1 the closer the value is to 1 the more it is reliable The fit correction and the overshining correction are two corrections values that can be used with quantification Be careful to apply them you need a specific formula I1 6 b analyze your results Thanks to the table columns options you can sort the result values Example of a sorted column click right on the column header and choose Sort gt Descending Order 10636 ABBA 96840 GER An useful option is also the color tool You can choose to give a color to some spots of the alignment in order to locate them Example we wa
6. e Introduction e General overview e Processing steps e Plugins presentation e Frequently asked questions 1 1 A bit of history AGScan is a micro array image processing software developed by the SIGENAE team http www sigenae org It is an evolution of BZScan BZScan is a radioactive DNA micro array image processing software based on the JAI library It was developed by the TAGC hitp tagc univ mrs fr from 2002 to 2005 In 2005 the SIGENAE team decided that it was worthwhile to go on with the development of BZScan and mainly by going through the new Sigenae version named AGScan This software is freely available using the following address http mulcyber toulouse inra fr projects agscan You can also download the source code report bugs and propose enhancements at the same address I 2 Main features of AGScan The main features of AGScan are 1 Grid conception 2 Able to use single or multiple up to 3 channel images as input 3 Image manipulation and processing based on ImageJ 4 Manual and automatic global alignment 5 Automatic local alignment 6 Automatic quantification 7 Batch processing 8 Data export 9 Plugin support to add new file formats alignment or quantification methods 10 Multi language support These different features will be presented in the rest of this document 1 3 Vocabulary Image is a file produced by a micro array scanner and sometimes modified cut Grid is first
7. 10 IL4 c Launching Athen 10 II PROCESSING STEPS rosies ese de N ee Se Ge N ee Ee Ge de ee ee ee ee Ge ee ed ee oe Eie og ee EEN 11 MI N GER EE 11 III RS Ee Ee EEN 12 HEL EA ius de N EG Ed OE N ON N N OE N N ER EE Corey 16 ULA tee ON IG EE 18 IE SE OR OR RE EA RE EE HE EE IS PET FED A OR EE 19 E Global alignment alone ER AE eN OE de ed n ia eo pe es EE EE 21 TTA doraka Tne aUl SOT a EErEE EE E been a E EE EE DE EG 23 EENEG 26 NIESE oi ER vet EE EE EE EEA sca eaten os pen needs ees 26 UU 6 EE ING AN EE 27 ER AIA eo eN EE EE ete RE TE EE OT N ER IT N 27 TET O aa EE VOUT KEES eege 29 TO oe oe N OE EE EE N or 3 IT PROCESSING A BATCH ee ee ee ee ee ee ee ee ee ee ee ee ee ee ee ee ee ee ed 32 Eed TOUT EE 32 DTT Te TPO Eege 33 EE EENEG 36 RS PE GINS oasis seis EE GE ORE OE N N OO EE 37 6 RE PRESENTATION AA RE OO Oe OE OE OOR ON 37 IV EE EE MEMORY ts EE 37 V FREQUENTLY ASKED QUESTIONS c cccccsssssccsssescssscsssosccssnsccesssecsensescssscscsnsecssnsecsssscsesssccssnsccosseasesssessens 39 YEINDE A E 40 KA fa ai EE 40 MI AT Ee EE 42 VIIL GNU GENERAL PUBLIC LICENSE esss sod ses sosso sseiees iese ode dee oek oie oi ed oe ede see ies de ese ede Gee ede se se Ge N Ge ed ee ee Ge 44 AGScan v0 1 User Manual page 3 Sigenae February 2006 Edition lI Introduction This manual is aimed to provide the necessary information to biologists using AGScan image processing software The document is split in four parts
8. DE 32 s5 lie W I e EE ER OE N S 32 Bale Wiles COMO UNA E 33 Batch experiment miormaton ee ee EE ER EE RR ER EE Ee RE EE ER EE Re ee EE ee EE ee ee EE ee ee ee ee ER ee ee ee Ee ER ee EE ee Ee ede 33 Balen ui te e e DE 34 Following a Batch iN slee RE EE EE EE 35 Batch progress TMM SING Cs EK N EE EER EE EE OE EN 35 AGScan User Manual page 42 46 February 2006 Edition Elements of the output directory File gt Open gt Batch menu Plugins download page Memory plugin window AGScan User Manual page 43 46 Sigenae February 2006 Edition VII GNU GENERAL PUBLIC LICENSE Version 2 June 1991 Copyright C 1989 1991 Free Software Foundation Inc 51 Franklin St Fifth Floor Boston MA 02110 1301 USA Everyone is permitted to copy and distribute verbatim copies of this license document but changing it is not allowed Preamble The licenses for most software are designed to take away your freedom to share and change it By contrast the GNU General Public License is intended to guarantee your freedom to share and change free software to make sure the software is free for all its users This General Public License applies to most of the Free Software Foundation s software and to any other program whose authors commit to using it Some other Free Software Foundation software is covered by the GNU Library General Public License instead You can apply it to your programs too When we speak of free soft
9. Finally any free program is threatened constantly by software patents We wish to avoid the danger that redistributors of a free program will individually obtain patent licenses in effect making the program proprietary To prevent this we have made it clear that any patent must be licensed for everyone s free use or not licensed at all The precise terms and conditions for copying distribution and modification follow TERMS AND CONDITIONS FOR COPYING DISTRIBUTION AND MODIFICATION 0 This License applies to any program or other work which contains a notice placed by the copyright holder saying it may be distributed under the terms of this General Public License The Program below refers to any such program or work and a work based on the Program means either the Program or any derivative work under copyright law that is to say a work containing the Program or a portion of it either verbatim or with modifications and or translated into another language Hereinafter translation is included without limitation in the term modification Each licensee is addressed as you Activities other than copying distribution and modification are not covered by this License they are outside its scope The act of running the Program is not restricted and the output from the Program is covered only if its contents constitute a work based on the Program independent of having been made by running the Program Whether that is true depends on what the P
10. OS Z Zoom 10 4 6 37 38 4 11 14 24 28 10 12 13 15 29 4 7 11 27 29 31 37 38 11 12 1 4 21 22 5 6 17 20 24 26 27 29 30 11 15 16 26 16 18 15 16 18 32 33 35 2 7 11 16 18 20 24 26 29 31 11 12 19 21 23 26 28 6 9 46 10 14 16 AGScan User Manual page 41 46 Sigenae February 2006 Edition V1 2 Illustration index EE ad le les ME N N EE N 1 Mulcyber AGScan page 8 Be velle sek oi soo AE OR N N EE EE N EE 9 IDEN TON LR AE N OE EE EE EI EE EE EE Or 12 Opel SION CU tee 12 Re ET en ele 12 ODEN imade SMOMCU sd EG ER De ER GER ke EG EO RE ee Ee EE Ee GE Ee GE EE ER SEE 12 Open Images ale OW EE OE EE EE N N EE oe 13 mage parameters WINAOW ee ee ee EE RE EA Re ER EA EE ee AE ee ee ee ee ede Ee ee RR Ee ee Ee ede Ee ee RR Ee ee EE Ee ee Ee ee Re ee 13 Radioactive experiment open 13 V OVT OX SS IMIS ODE e T N EE ee EE Ge 14 lr Leg MAGI e A E E T E E E EAN EDE 14 ZOO SHON GUL IE DEE 14 idee ele E et 15 Be as ER EE OE RE E EE ER EEE TE EN 15 Windows display mode amp ZOOM WINdOWT ee see ee EE EA EE EE ee AA EE ee RR Ee ee AE ee ee Re ee ee EE ee RE ee ee Ee ee ee ee 16 Grid Structure Editor window ee ese see see see ee EE ee ee EE ee EE Ee ee Ge ee EE ee ee EE ee RE ee ee RE ee ee ee RE Ee ee ee eek Ee eke ee ee 17 Grid Eet 18 Eod et ICE Skiet d Le DEE 18 AGAS TIER sd N SE E De DE DE ie De eo ERA Ge SE EN AE EDE 19 Grid elements
11. The grid the table and the editor are linked So be aware that you need to move on an other box or hit ENTER carriage return after having entered a value in each text box of the grid structure editor in order to synchronize the view of the grid You have now define your grid and you can save it The default proposed name of the saved file has no sense It is just created randomly in order to be unique The resulting file has a grd extension and is in XML format Notice that you cannot create a grid directly on a loaded image You need before to load the image and after the grid above TIT 4 Aligning a grid on an image IL A a Loading a grid This is where all really begins After having opened an image you can load a grid on it To do this you can use the Grid gt Load menu or the following icon The selected grid will be loaded on the image placed on the top left and the tab is divided into two parts in order to add the table of spots characteristics AGScan User Manual page 18 46 Sigenae February 2006 Edition 01 401 401 601 401 01 AO1 DO1 401 E01 The next step consists in correctly aligning the grid on the image You have several possibilities to do it This step can be done manually or automatically by launching alignments algorithms All the alignment functionalities are available in the Alignment menu Alignment Quantification Be MovelRotatelResize Spots detect
12. To modify them you need to choose the 4 profiles menu The 2 profiles and the 4 profiles spot detection algorithms are similar The difference is that profiles of spots used to search the center of the spot since the center of the research zone are diagonal lines for the 2 profiles detection and diagonals vertical horizontal lines for the 4 profiles detection 2 profiles 4 profiles These two methods progress rapidly and give you the percentage of detected spot in the status bar Detected Spots TI1 5 The table When a grid is open the main window is split into two parts giving you access to a table view of the spot informations This table is synchronized with the alignment So coordinates have been updated by the move of the grid However no new columns are added AGScan User Manual page 26 46 Sigenae February 2006 Edition op ADI DOS 4400 071 6391 421 8 4 1 G 4 300 0 A selected spot and its table description Base columns are the first column is the number of the spot determined by the spotting plan Name ID of each spot defined by AGScan the first part is the block ID the second part is the spot ID The letter corresponds to the line position A B and the number is the column position 1 2 3 X X coordinate of the center of the spot in the image in micrometer Y Y coordinate of the center of the sp
13. WITH YOU SHOULD THE PROGRAM PROVE DEFECTIVE YOU ASSUME THE COST OF ALL NECESSARY SERVICING REPAIR OR CORRECTION 12 INNO EVENT UNLESS REQUIRED BY APPLICABLE LAW OR AGREED TO IN WRITING WILL ANY COPYRIGHT HOLDER OR ANY OTHER PARTY WHO MAY MODIFY AND OR REDISTRIBUTE THE PROGRAM AS PERMITTED ABOVE BE LIABLE TO YOU FOR DAMAGES INCLUDING ANY GENERAL SPECIAL INCIDENTAL OR CONSEQUENTIAL DAMAGES ARISING OUT OF THE USE OR INABILITY TO USE THE PROGRAM INCLUDING BUT NOT LIMITED TO LOSS OF DATA OR DATA BEING RENDERED INACCURATE OR LOSSES SUSTAINED BY YOU OR THIRD PARTIES OR A FAILURE OF THE PROGRAM TO OPERATE WITH ANY OTHER PROGRAMS EVEN IF SUCH HOLDER OR OTHER PARTY HAS BEEN ADVISED OF THE POSSIBILITY OF SUCH DAMAGES AGScan User Manual page 46 46
14. batch just click on Launch AGScan User Manual page 34 46 Sigenae February 2006 Edition File Edition Display Image Grid Alignment Quantification Batch Tools About Ba Fao PALTA i 1 1 1 et ee g EE microarray example bh microarray examplez tif Following a Batch in progress When you choose to display the process you can follow it into the same tab indeed a new part appears in the top of the current tab As for the left window you have to stretch it with the mouse The status of each image is updated In the statusbar of AGScan the total percentage of the batch is given When the process is successfully finished the image status is DONE microarray example microarray example Batch progress finished When the batch is finished you can see results in the output directory For each experiment you will have the archive zaf file the snapshot tif file rapid snapshot of the alignment the export files optional In our example we have analyze two images and we asked for the text result file So we have three files by image AGScan User Manual page 35 46 Sigenae February 2006 Edition WI ui microarray examples microarray examples microarray examples ay microarray example microarray example microarray example ze Elements of the output directory IIl 7 c Save Open a batch You can save a batch
15. image use the File gt Open gt Image menu or the following short icon of the toolbar wi AGScan User Manual page 12 46 Sigenae February 2006 Edition Alte magg al u5 Browse Pixel width um He Pixel height ym 25 Unit Pie Latitude 8 AGScan January 27 2005 Version A selection window appears Choose your type of experiment one channel radio or multi channels fluo and load the image s thanks to the Browse button Notice that only the images files extensions that AGScan supports are visible in the file chooser By default tif and inf The Properties button allows you to give some initial parameters of the image produced by the scanner These parameters are stored into the general parameters of the application Pixel width width of a pixel micrometers Pixel height height of a pixel micrometers Unit image unit QL or PSL IP type Image Plate size centimeters Latitude dynamic range of above number power of 10 If you don t know where to find these parameters ignore them default values will be apply Click OK to load the experiment File Edition Display Image Grid Alignment Quantification Batch Tools About alol Tela AGScan User Manual page 13 46 Sigenae February 2006 Edition The image is loaded into the Display window In the case of a multi channels experiment the RGB image
16. is displayed Z AGScan January 27 2005 Version File Edition Display Image Grid Alignment Quantification Batch Tools About chicken635_1_2DYES wm Once opened you can now modify it using the different widgets disposed in the Image menu Grid Alignment Invert Adjust ki EE Rotation Flip ROT Selection Crop TEE They allow to invert the LUT to modify the contrast and the brightness to rotate the image to flip it horizontally or vertically Some of these options are very interesting when you have scanned your array the wrong way You can also select a rectangular region and crop it to create a new sub image Sometimes due to low contrast or quality the spots can be quiet difficult to locate All these image tools can help you to visualize them However you have to know that the modification of the contrast and the brightness of the image doesn t affect pixel values of the image and isn t save Other general functionalities can be used in the Display menu An example the zoom Working with images we sometimes need a general view and sometimes a very precise one This can be obtained by using the zoom Clicking on the left of the progress bar will zoom out of the image clicking in the right will Zoom in the image AGScan User Manual page 14 46 Sigenae February 2006 Edition When the zoom is active the icon background becomes gray
17. or not the fact to save image into the archive created for each image Notice that if the image isn t in the archive you need to give its localization when you will open the experiment 4 Select the number of channels 1 to 3 5 Click on Add Images to select images Note you can add images one by one or several in the same time in a multi selection only in 1 channel mode After choosing several images the left part is filled microarray example tif microarray examples tif Batch experiment information You can now choose all the actions you want to process for each image thanks to the job right part AGScan User Manual page 33 46 Sigenae February 2006 Edition Job Images process wi Compute Rotation 90 90 3 180 wl Image Constant Diameter Quantification Image Computed Diameter Quantification Fit Constant Diameter Quantification wi Global Alignment wi Fit Computed Diameter Quantification 9 Edges Detection wei OM GE Parameters Sniffer Flip horizontal C vertical Fit Correction Oyershining Correction Local Alignment wl Export results C Entire Grid w l Text aramebers Block per block Launch Display the current alignment Batch parameters configuration You just need to check or un check process you want You can choose to display or not the current alignment processing by checking the last checkbox To launch the
18. By clicking a second time on the icon the zoom is disable Example image with zoom action enabled Seen Image Grid The display tools are Eat tabs or windows the display mode Each item image grid Windows alignment is on a tab or on a distinct window Tabs are the default EE display mode j b Shon zoom disable enable the zoom zoom window Allows to display a little window containing the SE entire image the zoom factor applied and that show the part of the w Display the state bar image zoomed Properties Display the ruler disable enable a ruler Disabled by default Display the State bar disable enable the status bar Enabled by default Properties allows to display the properties name size of the current element displayed into the current tab or window By default the display mode is tabs the zoom isn t active the ruler is disabled and the status bar is displayed see the image above AGScan User Manual page 15 46 Sigenae February 2006 Edition 7 AGScan January 27 2005 Version File Edition Ta r Grid Batch Tools About EE On this example the window mode is active the status bar is hidden the ruler is displayed and the zoom window is open the current zoom factor is 3 The next part will show you how to create a grid III 3 Creating a grid The grid is a very important element for the image anal
19. a file generated by AGScan describing the way the array has been spotted From this it can be aligned on an image and be a part of an alignment You will see later the importance of the grid structure Alignment is composed of an image and a grid It can have several states like not aligned globally aligned and locally aligned AGScan User Manual page 4 46 Sigenae February 2006 Edition Quantification once the alignment is finished the quantification is the process of extracting one or more figures for each spot giving an intensity value for the spot Batch is a group of alignments processed by AGScan from global alignment to quantification through local alignment AGScan User Manual page 5 46 Sigenae February 2006 Edition II General Overview This chapter presents an overview of the whole process some information about image file types and quality and gives the software install procedure II 1 The process To start the process you need at least e amicro array image different formats can be used e a grid file generated in AGScan see next chapter section e AGScan installed see install procedure below Notice that AGScan works with 16bits unsigned grayscale images By default AGScan input image format is the TIFF format but ISAC format INF IMG is also recognized by a plugin automatically provided into all AGScan versions The grid file can be generated with AGScan or provid
20. amples Radio grids 2005 11 25 16 15 GRID_AGScan_example_8x4 grd 6 KB 25 1386 Other GRID_AGScan_generic_16x4 grd 6 KB 23 1386 Other Mulcyber AGScan page AGScan User Manual page 8 46 Sigenae February 2006 Edition All versions of the software are available They are named using the date of the release The software releases are available in two different packages light and full versions With the light version Java Runtime Environment needs to be installed previously on your operating system The full version does not need any other software just to click on the icon Downloading AGScan is very easy First choose a release of the package For each release notes are available by clicking on the release name Next you can download the corresponding AGScanxxxxxx zip file Release Name v270106 Hotes AGScan z Full qa This zip file is an AGScan full version Includes Sun s JRE 1 Release notes To use it unzip the file and click on the exe file Windows OS command line using the jar file v2 70100 e SE 27th January 2006 Version AGScanFull270106 zip the grid structure editor supports Interblocks values in Addition of a ROI system Region Of Interest in order to VUn 1205 The CROP function is also modified to work with the ROI Fixed bug that displays always 0 for interblocks values AGScanFull081205 zip opening a saved grid hug 166 Fixed bug of wrong rotation in
21. can User Manual page 38 46 Sigenae February 2006 Edition V Frequently asked questions to be completed AGScan User Manual page 39 46 Sigenae February 2006 Edition VI Index VI 1 Word Index Xmx bah grd jar txt zaf A AGScan Alignment B Batch Block BZScan C ChangeLog txt D Display window Download E Export F File formats Fit correction Fuji BAS5000 scanner Full G Global alignment Grid Grid structure Image ImageJ INF IMG Intensity Interface ISAC J Java Runtime Environment L Languages Launcher configuration file Levell Level2 Light Linux OS Local alignment M Menu bar Mulcyber O Overshining correction P 10 7 36 7 18 10 7 131 35 1 2 4 13 17 24 27 29 35 37 4 7 11 15 19 21 24 26 27 29 31 34 35 37 38 4 5 7 11 31 36 17 19 24 27 4213 10 11 14 4 31 4 7 31 35 4 7 37 29 7 9 10 45 4 6 21 23 4 6 7 11 15 27 29 31 33 4 4 6 7 11 16 18 19 21 22 27 29 31 33 35 42 4 10 6 5 29 11 45 6 7 9 10 10 12 10 17 17 9 10 4 6 21 23 24 11 4 8 37 29 AGScan User Manual page 40 46 Sigenae February 2006 Edition Packages contents Plugin Plugins Progress bar Properties Q QM Quantification S Shortcuts SIGENAE Sniffer Spot Status bar Structure editor T Tab Table TIEF Toolbar V Versions W Windows
22. configuration in order to launch it several times or to re open and modify it The extension of a batch file is a bzb This file can only be used with AGScan The format is kind of XML compliant Note In the current version it is not possible to record a batch in multichannel mode To open a batch use the File gt Open gt Batch menu Edition Display Image Grid Create kb ER Grid Gr Alignment File gt Open gt Batch menu AGScan User Manual page 36 46 Sigenae February 2006 Edition IV Plugins IV 1 Presentation One of the most interesting feature of AGScan is the plugins system It allows to easily add functionalities to AGScan For example to add new file formats alignment or quantification methods The only thing to do is to add plugins files in the agscan directory named plugins Some plugins are already available on the mulcyber following page Plugins SPlotMatrixPlugin 06 12 2005 14 28 SPlotMatrixPlugin zip 18 KB 141386 zip SPlotPlugin 08 12 2005 14 21 SPlotPlugin zip 6 KB 121386 zip SAlignmentSnapshotPlugin 06 12 2005 15 12 SAlignmentSnapshotPlugin zip 3 KB 19 386 zip SSurfacePlot_3DPlugin 26 11 2005 16 04 SSurfacePlot 3DPlugin zip 49 KB 17 1386 zip SSurfacePlot 3DPlugin v3 zip 49 KB 01386 zip SQuantifiPlugin 26 11 2005 15 13 SQuantiflPlugin zip 27 KB 17 1386 zip SFilesMergePlugin 24 11 2005 14 45 SFilesMergePlugin zip 4 KB 27 386 zip SMemor
23. d to apply and the section as a whole is intended to apply in other circumstances It is not the purpose of this section to induce you to infringe any patents or other property right claims or to contest validity of any such claims this section has the sole purpose of protecting the integrity of the free software distribution system which is implemented by public license practices Many people have made generous contributions to the wide range of software distributed through that system in reliance on consistent application of that system it is up to the author donor to decide if he or she is willing to distribute software through any other system and a licensee cannot impose that choice This section is intended to make thoroughly clear what is believed to be a consequence of the rest of this License 8 If the distribution and or use of the Program is restricted in certain countries either by patents or by copyrighted interfaces the original copyright holder who places the Program under this License may add an explicit geographical distribution limitation excluding those countries so that distribution is permitted only in or among countries not thus excluded In such case this License incorporates the limitation as if written in the body of this License 9 The Free Software Foundation may publish revised and or new versions of the General Public License from time to time Such new versions will be similar in spirit to the present version but may d
24. de distributed need not include anything that is normally distributed in either source or binary form with the major components compiler kernel and so on of the operating system on which the executable runs unless that component itself accompanies the executable lf distribution of executable or object code is made by offering access to copy from a designated place then offering equivalent access to copy the source code from the same place counts as distribution of the source code even though third parties are not compelled to copy the source along with the object code 4 You may not copy modify sublicense or distribute the Program except as expressly provided under this License Any attempt otherwise to copy modify sublicense or distribute the Program is void and will automatically terminate your rights under this License However parties who have received copies or rights from you under this License will not have their licenses terminated so long as such parties remain in full compliance 5 You are not required to accept this License since you have not signed it However nothing else grants you permission to modify or distribute the Program or its derivative works These actions are prohibited by law if you do not accept this License Therefore by modifying or distributing the Program or any work based on the Program you indicate your acceptance of this License to do so and all its terms and conditions for copying distributing o
25. der the scope of this License 3 You may copy and distribute the Program or a work based on it under Section 2 in object code or executable form under the terms of Sections 1 and 2 above provided that you also do one of the following a Accompany it with the complete corresponding machine readable source code which must be distributed under the terms of Sections 1 and 2 above on a medium customarily used for software interchange or b Accompany it with a written offer valid for at least three years to give any third party for a charge no more than your cost of physically performing source distribution a complete machine readable copy of the corresponding source code to be distributed under the terms of Sections 1 and 2 above on a medium customarily used for software interchange or c Accompany it with the information you received as to the offer to distribute corresponding source code This alternative is allowed only for noncommercial distribution and only if you received the program in object code or executable form with such an offer in accord with Subsection b above The source code for a work means the preferred form of the work for making modifications to it For an executable work complete source code means all the source code for all modules it contains plus any associated interface definition files plus the scripts used to control compilation and installation of the executable However as a special exception the source co
26. ed by someone having generated it To share a grid you have to be sure that the micro array and the definition used to scan the image are the same The process can be split in five steps experiment image s loading grid loading global alignment This step can be done by AGScan or by the user It corresponds to the general alignment of the designed grid on the image s local alignment This step is done by AGScan Starting from the global alignment AGScan tries to figure out what the small local changes are to fit the sub grids as well as possible to the image quantification This step is done by AGScan Starting from the local alignment coordinates AGScan calculates for each spot several quantification values These values will be presented later on As presented before AGScan is able to process an image and a grid without any human help This is a very important and user asked AGScan feature AGScan User Manual page 6 46 Sigenae February 2006 Edition II 2 File formats AGScan can load and save several file formats depending on the element See table hereunder Element file extension comments image DI Standard format of compression of image without loss of information inf This file is the information file of the corresponding img file Both files have to be in the same directory to be used by AGScan This file type is named ISAC and is for example produced b
27. eral of them with the Selection sub menu or the following toolbar icon Sr WW a a Selection Triage Fixed diameter Quantification Image Computed diameter Quantification Fit Fixed diameter Quantification RES EET NE Fiti Computed diameter Quantification Diameter Correction of overshining Correction by the Fit The selection menu opens the next window TEL AE Quantifications selection Make vour selection wl Image Constant diameter Quantification wi Image variable diameter Quantification Fit Constant diameter Quantification Fit Variable diametre Quantification GM Fit Correction Oyershining correction Select al Deselect all After selecting the actions you want to process click on OK Each calculation is done one after the other You can follow advance thanks to the progress bar Results are available into the table AGScan User Manual page 28 46 Sigenae February 2006 Edition Computed Diameter JH kt onst OH Image Calr QM 344 91245 16 ae a 47 06 Overshining Correction OC Fit Correction 0 0 ath alata Zl ae je 46 0 6 0 0 O C Example of columns added results of quantifications Note the Computed diameter column is filled during quantifications that need this value By selecting the data column title you can change its position in the table Available Quantifications Four quantification are available in AGScan
28. fy an image in three clicks Shortcut icons are also duplicated next to each menu title to ease memorization Notice that if you point your mouse on a shortcut you will obtain a little description of the associated action Edition Display Image Grid A File Edition Display sie E Open an image Create be pa Grid ul Ga Alignment a Batch S e Example the Open Image action menu and related toolbar item The first menu you can use is the Edition Language one It allows to change the language of the application menus Notice that you need to reload the application to apply a new language Note in the current version three languages are available English French and German There can be spelling mistakes However it is easy to modify these faults Indeed all the words are stored in text files named messages en properties messages fr properties and messages de properties We can even easily generate new ones in other languages For that it is necessary to add the new messages_xx properties file into the subdirectory of AGScan named languages It will be automatically recognized by the software Now Let us present the different steps needed to quantify an image III 2 Loading an image file Preliminary remark an experiment can be composed from 1 to 3 images but in the following chapters we will refer to an experiment including only one image To open an
29. ge files available in AGScan the lib directory containing AGScan libraries and dependences used by the software ij jar for example Notice that the subdirectory called plugins of this directory contains imaged plugins that can be used by AGScan the plugins directory containing AGScan case plugins files the ChangeLog txt file containing the history of release notes the parameters properties file containing the current user preferences of the software Ac Launching AGScan On Linux OS use the command line java Xss10m Xmx420m jar AGScanxxxxxx jar On Windows OS If you choose a full version just click on the AGScan exe icon You can also run the command line or write it in a bat file java exe Xss10m Xmx420m jar AGScanxxxxxx jar The jar file name is the release name of AGScan Note the Xss and the Xmx parameters correspond to the memory allocation given to the Java Virtual Machine JVM in order to support the application If you encounter java OutOfMemory errors then increase the Xmx value By default AGScan full version is configured with 420Mo of memory allocated for the JVM You can modify this value in the launcher configuration file AGScan cfg file AGScan User Manual page 10 46 Sigenae February 2006 Edition Ill Processing steps This guided tour will walk us through the different parts listed hereunder
30. ges detection Sniffer Se aT eT Th EE The Global alignment sub menu contains two different algorithms the sniffer and the edges detection global algorithms The default algorithm is the sniffer one in bold in the menu You can also call it thanks to the following toolbar icon gt If you click on Default algorithm it is launched without proposing you to change the parameters To modify them you need to choose the Sniffer menu So the parameters windows appears AGScan User Manual page 21 46 Sigenae February 2006 Edition Global alignment parameters bloc alignment algorithm parameters spot detection Algorithm Detection by 4 profiles Research area of the center Computed area of the center Quality threshold local alignment algorithm parameters Bootstrap sensitivity Iterations number ist run sensitivity Iterations number 2nd ran sensitivity Iterations number Sra run sensitivity Iterations number Threshold of change of sensitivity _ vi Verifier la cohi Verence de la grille d Incrementation for the research of the angle degrees Contrast For the research of the angle Size of the sniffer spots Notice that all these parameters can be changed and saved It is also always possible to restore the default parameters So don t hesitate to change them For more information about
31. iffer in detail to address new problems or concerns Each version is given a distinguishing version number If the Program specifies a version number of this License which applies to it and any later version you have the option of following the terms and conditions either of that version or of any later version published by the Free Software Foundation If the Program does not specify a version number of this License you may choose any version ever published by the Free Software Foundation 10 If you wish to incorporate parts of the Program into other free programs whose distribution conditions are different write to the author to ask for permission For software which is copyrighted by the Free Software Foundation write to the Free Software Foundation we sometimes make exceptions for this Our decision will be guided by the two goals of preserving the free status of all derivatives of our free software and of promoting the sharing and reuse of software generally NO WARRANTY 11 BECAUSE THE PROGRAM IS LICENSED FREE OF CHARGE THERE IS NO WARRANTY FOR THE PROGRAM TO THE EXTENT PERMITTED BY APPLICABLE LAW EXCEPT WHEN OTHERWISE STATED IN WRITING THE COPYRIGHT HOLDERS AND OR OTHER PARTIES PROVIDE THE PROGRAM AS IS WITHOUT WARRANTY OF ANY KIND EITHER EXPRESSED OR IMPLIED INCLUDING BUT NOT LIMITED TO THE IMPLIED WARRANTIES OF MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE THE ENTIRE RISK AS TO THE QUALITY AND PERFORMANCE OF THE PROGRAM IS
32. ion Global alignment b Local alignment IL Ab aligning manually a grid How to manually place the grid You can either use the mouse and the Move Rotate Resize menu Let us present to you the color code of a grid a loaded grid on an image appears in blue if it is not selected and orange if you select it To select in the grid just click on it Note that you can easily only move a sub grid by changing the selection mode These three icons of the toolbar allow to change the grid selection mode By default the first is enabled It corresponds to the entire grid If you select the second icon you turn into the block mode AGScan User Manual page 19 46 Sigenae February 2006 Edition that allows you to only select a block The third icon corresponds to the spot mode that allows to only select one spot A selected item spot block or the grid can be moved using the mouse For this move it by maintaining it selected left mouse button down Notice that the table view corresponding selected spots lines are yellow Block selection mode Spot selection mode DO OO You can also deform the grid by placing your mouse at the corners and drag them A specific and useful tool allow to easily manually align a grid the Move Rotate Resize menu Click on it a window appears offering you the possibility to rotate move and resize the grid of given values i MovelRotate Resize Ed
33. ity In the actual version two different local alignment algorithms are available else Quantification Batch Tools About o sjel Move Robate Resize Spots detection Global alignment Alignment of the entire grid ock by block PE aad ment bl The Local alignment sub menu contains two different algorithms the entire grid and the block by block local alignment algorithms The default algorithm is the block by block one in bold in the menu You can also call it thanks to the following toolbar icon If you click on Default algorithm it is launched without proposing you to change the parameters To modify them you need to choose the Alignment block by block menu Alignment block by block algorithm quick presentation Each block is processed individually The position of each spot is verified and adjusted The block may be moved from the initial global alignment place AGScan User Manual page 23 46 Sigenae February 2006 Edition Progression of the local algorithm In this process AGScan takes each block and tries to fit it as precisely as possible to the spots it finds underneath Each block is processed separately and can therefore move from the previously calculated global position to a new one It is quiet amazing to see how a block is progressing towards its end position Once the block position is optimized AGScan goes on with the next b
34. l but not always the case The level defines a block of spots Information needed is the dimension of a block number and size of columns and lines and the diameter of a spot level2 the grid level A grid can often be compared to a set of blocks So if you have this second level you just need to inform how many blocks you have vertically and horizontally So you can also notify inter blocks space size To sum up a grid is seen by AGScan as a set of elements The smallest element is the spot When you have two levels a set of spots forms a block and a set of block a grid AGScan User Manual page 17 46 Sigenae February 2006 Edition In the next table the first line gives you number of levels used the second gives you the structure of the first level and the third line gives you the structure of the second level In the last line you can see the graphical view of the type of spotting pattern you have used 1 level 2 levels 2 levels 20 x 12 spots 12 x 12 spots 1 x 1 spots 4x 2 blocks 10 x 10 blocks ODOOOOOOOOOOOOOOOOOOO DOOOOOOOOOODOOOOOOOO ODOOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOODOOOOOO DOOOOOOOOOOODOOOOOOOO DOOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOODOOOOOO DOOOOOOOOOOOOOOOOOOO DOOOOOOOOOOOOOOOOOOO DOOOOOOOOOODOOOOOOOO DOOOOOOOOOOOOOOOOOOO O0000000000000000000 HOO ooo oe Oppppppoppp HHO RRO THOR ooo Hoo oo ee N El Fi Fi Fi Fi Hi Ed Ed E ra Ed Ed Ed FA EA Fi EA Ed EA DH EE ER El Ed EF Examples of grids
35. lock The red circles indicate that the program identify a spot on the opposite the green ones indicate that the spot is deduced due to the grid structure and the red spots locations Note that the coordinate values of the spots have changed during the local alignment process You can see values in the alignment table The progress of the alignment is given by a progress bar At the end of the process your display windows will look like this AGScan User Manual page 24 46 Sigenae February 2006 Edition File Edition Display Image Grid Alignment Quantification Batch Tools About AGScan User Manual page 25 46 Sigenae February 2006 Edition IL Ae Spot detection The spot detection is the method used to define if a spot is found red or estimate green A spot is recognize as one if its form fit the model You can call it on a selected a grid Indeed a selected grid becomes orange so you don t see the spot nature Quantification Batch Tools About Move Rotate Resize Spots detection Global alignment 2 profiles Local alignment 4 profiles The Spots detection sub menu contains two different algorithms the 2 profiles and the 4 profiles algorithms The default algorithm is the 4 profiles one in bold in the menu You can also call it thanks to the toolbar icon 88 If you click on Default algorithm it is launch without propose you to change parameters
36. ncluding an appropriate copyright notice and a notice that there is no warranty or else saying that you provide a warranty and that users may redistribute the program under these conditions and telling the user how to view a copy of this License Exception if the Program itself is interactive but does not normally print such an announcement your work based on the Program is not required to print an announcement These requirements apply to the modified work as a whole If identifiable sections of that work are not derived from the Program and can be reasonably considered independent and separate works in themselves then this License and its terms do not apply to those sections when you distribute them as separate works But when you distribute the same sections as part of a whole which is a work based on the Program the distribution of the whole must be on the terms of this License whose permissions for other licensees extend to the entire whole and thus to each and every part regardless of who wrote it Thus it is not the intent of this section to claim rights or contest your rights to work written entirely by you rather the intent is to exercise the right to control the distribution of derivative or collective works based on the Program In addition mere aggregation of another work not based on the Program with the Program or with a work based on the Program on a volume of a storage or distribution medium does not bring the other work un
37. nt to color in orange spots that have an image constant quantification value higher than 10000 To do that 1 select the Modify menu of the Ou Image Const column 2 Check the superior case fill the value 10000 and choose a color orange AGScan User Manual page 29 46 Sigenae February 2006 Edition Modify a column Name Qtr Image Const Type Default value 1 O values Colors vl iF superior to C If between and J iF inferior to Modify a column window 3 Click on Apply and Close 4 select the Color spots menu of the Qtf Image Const column The text header of the column becomes red Spots concerned by this condition will appear in orange on the aligned image In our example only 4 spots are colored as the sorted column shows us before Spot coloration example You can also color spots between two values or lower than an other one AGScan User Manual page 30 46 Sigenae February 2006 Edition IIl 6 c Export results You can save the experiment in every process step An alignment and a quantification are considered as the same thing In fact there are two aligned grid with a table Only tables columns differ So use the save menu to save it The extension of an experiment file is zaf You can also export only the table values in order to use them into a spreadsheet To do that selec
38. ot in the image in micrometer R row number in the entire grid C column number in the entire grid Block number of the current block Row row number in the current block Column column number in the current block Diameter constant diameter of the spot defined in the grid structure Note the Computed diameter column is present but not used values are 1 until the quantification IS processed The table and the alignment are interactive You can select a spot in the table and see its location at the same time on the alignment the spot or the set of spots are highlighted into the alignment see the figure above So it s also possible to sort columns values copy it color some values hee Deel Ki cl Ke EE E EE Sort Ascending order D Color spots Descending order gt Hide gt Modify ES Copy prey la The columns options can be active by clicking on the right click of the mouse The interest of these options will be describe to you after the quantification part ITII 6 Quantifying an alignment Now you have an aligned grid So the next step is the quantification IL Ga quantifications AGScan User Manual page 27 46 Sigenae February 2006 Edition several computations are available To launch a quantification use the Quantification gt Compute menu and select your action You can choose the type of quantification you want to call or select sev
39. r modifying the Program or works based on it 6 Each time you redistribute the Program or any work based on the Program the recipient automatically receives a license from the original licensor to copy distribute or modify the Program subject to these terms and AGScan User Manual page 45 46 Sigenae February 2006 Edition conditions You may not impose any further restrictions on the recipients exercise of the rights granted herein You are not responsible for enforcing compliance by third parties to this License 7 If as a Consequence of a court judgment or allegation of patent infringement or for any other reason not limited to patent issues conditions are imposed on you whether by court order agreement or otherwise that contradict the conditions of this License they do not excuse you from the conditions of this License If you cannot distribute so as to satisfy simultaneously your obligations under this License and any other pertinent obligations then as a consequence you may not distribute the Program at all For example if a patent license would not permit royalty free redistribution of the Program by all those who receive copies directly or indirectly through you then the only way you could satisfy both it and this License would be to refrain entirely from distribution of the Program If any portion of this section is held invalid or unenforceable under any particular circumstance the balance of the section is intende
40. radian unit hug 180 06O12705 CROP suffix is added for each channel image when a s i that is freezed after a save action hug 177 AGScanFulld61205 zip creation of plugins categories associated to the differen 211105 AGScanFull211105 zip Download link E Ouverture de AGScanFull270106 zip Vous avez choisi d ouvrir AGScanFull270106 zip qui est up Fichier de type Fichier Winzip partir de hEtp f fmulcvber aulause inra fr Que doit Faire Firefox avec ce Fichier C 3 Ouvrir avec WinRAR d faut Ki 3 Enregistrer sur le disque L Toujours effectuer cette action pour ce type de fichier Annuler Download AGScan AGScan User Manual page 9 46 Sigenae February 2006 Edition Il 4 b packages contents The AGScanFullxxxxxx zip archive file contains the following directory ET ag Cl AGScanFull270106 The AGScanLightxxxxxx zip archive file contains the following directory Package contents are similar except 3 elements added to the Full version the install directory contains a Java Runtime Environment the AGScan cfg file the launcher configuration file the AGScan exe file the icon to launch AGScan on Windows OS Note the Full version launcher is only usable for Windows OS The file structure of files is composed of the AGScanxxxxxx jar file is the java executable archive file the languages directory containing langua
41. rogram does 1 You may copy and distribute verbatim copies of the Program s source code as you receive it in any medium provided that you conspicuously and appropriately publish on each copy an appropriate copyright notice and disclaimer of warranty keep intact all the notices that refer to this License and to the absence of any warranty and give any other recipients of the Program a copy of this License along with the Program You may charge a fee for the physical act of transferring a copy and you may at your option offer warranty protection in exchange for a fee 2 You may modify your copy or copies of the Program or any portion of it thus forming a work based on the Program and copy and distribute such modifications or work under the terms of Section 1 above provided that you also meet all of these conditions a You must cause the modified files to carry prominent notices stating that you changed the files and the date of any change AGScan User Manual page 44 46 Sigenae February 2006 Edition b You must cause any work that you distribute or publish that in whole or in part contains or is derived from the Program or any part thereof to be licensed as a whole at no charge to all third parties under the terms of this License c If the modified program normally reads commands interactively when run you must cause it when started running for such interactive use in the most ordinary way to print or display an announcement i
42. selection shortcut ons 19 kle seile kie CO I ads seed EE EE N 20 Move Rotate Resize od 20 Global alignment men 21 Glopa alignment Sone de E 21 Global alignment parameters window eie ee se ee ee EE Re EE Ee AR EE ee AE Ee ee ee ee ee ee ee ee EE ee ee ee ee ee ee ee 22 Sniffer local alignment algorithM ee EE EE RR EER RR EEN EE RR RR SEE NENNEN EE EER Re EE EER ER GEE EER RR Re NEEN EEN ER Re EEN 22 Local alignment HE DEE 23 L bal alignment SNOMCUL ICOM sesse ER EE ER EE Ee GE ER Re EE se ee GE sig ee ie RE ER GE RRN 23 Progression of the local algomthbm ee ee ee EE AR EE ee ER EE AA EE ee ER Ee ee AE ee ee ee ee AE ee ee RE ee de ee ee ee ee ede 24 An aligned od 25 Spot detectiont men 26 beelde ore aie aie dd IC ON EE OE EE E EE N EE EE EE id 26 Percentage of detected Ke 26 A selected spot and its table descrpion ee ee ee ER EE EA EE ee RE RE AE Ee ee Ee ee EE ee Ee ER ee AE Ee ee ee ee ede Ee 27 Gie Erin io eege 27 Quantification gt Compute gt Selection shortcut con 28 Ouantification Compute Selection men 28 Quantifications selection window ee ee ee ee ee ee ee ee Re ee ee Re Re Re ee ee ee 28 Example of columns added results Of ouantfcatons ee ee ee EE RR EE RE Ee RA ee ER ee ede ee ee ee ee ede 29 Esaias CO MN Ee AE AE EE ER EE EN EE EE 29 Modify a column di ele 30 SEENEN 30 Quantification gt Export gt Text File men 31 Example of output text file content 31 Batch shortcut ICOM
43. t the menu Quantification Quantification gt Export gt Text File The file saved content looks likes Compute Batch Tools b P ee IBI MI KI hd KI KI hd EI zech ch ch ch 1300 0 2300 0 3300 0 4300 0 6 300 0 6 300 0 7 300 0 6300 0 9 300 0 10300 0 11300 0 12 300 0 1300 0 2300 0 3300 0 4300 0 5 300 0 6 300 0 7 300 0 8300 0 m WEE 344 91243 344 91243 344 91243 344 91243 344 91243 346 0 271 35695 344 91243 344 91243 346 0 344 91243 344 91243 344 91243 344 91243 256 43436 344 91243 121 14123 295 16754 344 91243 344 91243 d d d d d de Block Row Column Diameter Computed Diameter lat Fit i onst 16 On FitfCalc ER Now you have done a complete analyze of your image Do you know that this process can also be repeat automatically using the batch mode Text file output example AGScan User Manual page 31 46 Sigenae February 2006 Edition III 7 Processing a batch II da Batch tab A batch allows to analyze automatically several experiments Important note all these expermiments are analyzed with the same grid To create a batch you can use the File gt Create gt New Batch menu or the Batch gt New Batch The associated icon of the toolbar is A3 The batch tab contains two parts Pe AGScan January 27 2005 Version File Edition Display Image Grid Alignment Quantifica
44. their meaning please contact us Sniffer algorithm quick presentation At first the angle of the image is calculated Then about 10 of the process the algorithm try to parse all the image with a sniffer block to find all spots To do this it already searches to place the sniffer block a defined block of spots at 40 in width and in height of the image the condition to place it is that the center spot must be a detected one When the algorithm detects this spot it considers this position as the first position of its route pict1 The sniffer block moves after in all the direction of the image and flags every spot it finds Each time it will find something considered a spot it will put a little colored cross on its center pict2 After having recovered the entire image the algorithm computes how to place the grid in order to include the most of detected spots At the end of the process you will have a yellow selected grid aligned on the image picts The Sniffer block Spots are found Grid is placed aA Le vd gek Va Some visual steps of the sniffer global alignment AGScan User Manual page 22 46 Sigenae February 2006 Edition Our grid is now globally aligned The next step of the process is the local alignment II A d Local alignment algorithm A local alignment is an alignment that you must launch on a already aligned grid It allows to improve the alignment qual
45. tion Batch Tools About elme mus ae ao ea BATCH_11393924241609 b2b 3 E Grid Output directory A Save images with alignments Channels Number Add images Left part Images process Compute Rotation 90 Co 30 180 Image f Constant Diameter Quantification Image Computed Diameter Quantification Fit Constant Diameter Quantification Global Alignment wl Fit Computed Diameter Quantification O Edges Detection E Sniffer Overshining Correction Flip 7 horizontal 7 vertical Local Alignment Export results Entire Grid C Text Parameters Block per block 1 Display the current alignment Batch tab The tab is split into two parts the left part that displays experiments status and images open and the right part that contains the configuration of the batch Note that the left part is by default hidden You have to increase it with the mouse to see it better AGScan User Manual page 32 46 Sigenae February 2006 Edition IL D Preparing the batch Grid GRID AGScan example 6x4 grd EC Output directory Documents and Settings rcathelinitest ECH i Save images with alignments Channels Number 7 Add images Batch files configuration In the top of the right part of the tab 1 Select the grid of the batch 2 Choose the output directory where results will be saved 3 Check
46. ware we are referring to freedom not price Our General Public Licenses are designed to make sure that you have the freedom to distribute copies of free software and charge for this service if you wish that you receive source code or can get it if you want it that you can change the software or use pieces of it in new free programs and that you know you can do these things To protect your rights we need to make restrictions that forbid anyone to deny you these rights or to ask you to surrender the rights These restrictions translate to certain responsibilities for you if you distribute copies of the software or if you modify it For example if you distribute copies of such a program whether gratis or for a fee you must give the recipients all the rights that you have You must make sure that they too receive or can get the source code And you must show them these terms so they know their rights We protect your rights with two steps 1 copyright the software and 2 offer you this license which gives you legal permission to copy distribute and or modify the software Also for each author s protection and ours we want to make certain that everyone understands that there is no warranty for this free software If the software is modified by someone else and passed on we want its recipients to know that what they have is not the original so that any problems introduced by others will not reflect on the original authors reputations
47. y the Fuji BAS5000 scanner grid grd This file type is produced and used by AGScan The format is kind of XML compliant experiment Alignment zaf This file type is produced and used by AGScan quantification It s an archive file that contains a al file experiment data file a manifest file and may contains the image s The ali format is kind of XML compliant s batch bzb This file type is produced and used by AGScan The format is kind of XML compliant Notice that AGScan provides also non openable export formats like a txt output easily usable with your favorite spreadsheet 11 3 Image quality Even if AGScan proposes methods for automatically align grids on image the result depends of a lot of things like the quality of the image the grid structure and parameters used for the alignment 1 Be aware that the image files have to be a 16 bits format to be processed by AGScan 2 Be careful BZScan and AGScan grd grid files and bzb batch files are incompatible 3 Never try to open a zaf file ali files are not alone support 4 Be careful BZScan and AGScan grd grid files and bzb batch files are incompatible AGScan User Manual page 7 46 Sigenae February 2006 Edition II 4 Installing AGScan on your Computer IA a Download AGScan is freely available at http mulcyber toulouse inra fr projects agscan 6 M L zs Gs x A http mulcyber toulouse inra friprojects agscan
48. yPlugin 24 11 2005 09 15 SMemoryPlugin zip 6 KB 22 386 zip Plugins download page On the website you can click on the name of the plugin to watch its presentation and on the zip file to download it IV 2 Example the Memory Plugin For example we will present you how to download install and run the SmemoryPlugin This plugin allows to add to AGScan a little window that give you the current memory consumption At first click on the SMemoryPlugin zip file to download it The zip file contains sources classes and compile notes version of javac and AGScan used of the Memory Controler plugin Note that there are one source file SMemoryPlugin java and 3 class files caused by embedded classes To use this plugin just put class files into the plugins directory of AGScan A Memory Controler submenu is added into the tool menu AGScan User Manual page 37 46 Sigenae February 2006 Edition MEMORY CONSUMPTION Total Available 417M Memory plugin window Notice that different categories of plugins exist So each plugin menu appears into its menu alignment tool quantification There are also other plugins like format plugins that allow to read save other images formats and that doesn t create menu into the application You know that they are installed by showing the different type of files you can open All these inforamtion are available int o the note provided with the plugin AGS
49. yze After opening an image you have to load a grid on it and therefore you must have already define it Note that you can use the same grid for all your experiments that use the same spotting scheme So this part will show you how to create open and modify a grid At first choose the creation grid menu found in the File gt Create gt New Grid menu or in the Grid gt New Grid menu A new tab and the structure editor will appear The main window is divided into two parts the grid the graphical representation of the grid the table a table with all the characteristics of the grid spots number name position It will be describe later AGScan User Manual page 16 46 Sigenae February 2006 Edition N ie January 27 2005 Version File Edition Display Image Grid Alignment Quantification Batch Tools About GRID_1138974828336 grd OS a EC EYES anh a als ah PS EA EN a a a Structure editor ae x R C Bloc KEE 1 1 1 Level 1 Level 2 H Humber of columns columns width um Number of lines Height of lines um spots diameter pr Starting position Tap Left Spot names 8 CLO number By default the editor appears on a one level grid So we have to introduce you the notion of level in the grid A grid can have two levels representing the block and the grid leveli the block level In fact the spotting is usually made using a block pattern This is usua
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