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1. 2D Gel Analysis Software sg f ez P User Manual Version 7 0 Geneva B oinformatics SA Swiss Institute of Bioinformatics Contents 1 Getting started 11 1 2 1 3 14 1 5 1 6 1 7 Aboutthe software eessen 7 System requirements ssssssssssnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn 7 Install Ve TC UC 8 LC ENGNO E 8 Launch the software 2 ss2 ss2 s22snusurnuzaznazzzzuzzznsaB 11 PESODICE EE 12 Mat S neW siari 14 2 Graphical user interface 2 1 About the interface sssssssnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn 15 2 2 VT E 15 Cp E 18 24 SAUS D r ebessen Eege EEN 19 2 5 Display 20N E 20 20 MWOISDOCE WEE 23 Sech EE eege 24 2 8 Dockable windowsS 12 s s ssszssannznnnszazaknnaaazazakkKKEE0Z 29 29 ee e 30 3 Image Pool CN Wl a TE 33 3 2 Start with good IMAGES 1 1 1 s sss nssszsasnsznnnzzaazkknnanzzEa 33 3 3 NEVEWIMAJES i GGGGiaGiiGibGGiciii iii 34 EWEN E 37 ee TEE 40 Al EN EC 45 4 Projects 4 1 INGOJUCKON siisii 49 AZ CIEGIE a DIOJECE EEN 52 4 3 Create match hierarchy ssusssnnssaaazananazzazknananzzaa 54 4 4 Create satcz i GEE doc tC RE 59 4 5 Handle project Reims u ssssssasssznnszzaznnananzzazzkknnaSESEE 59 46 SAVE e eh E 60 4 7 Manage PIOJECIS egkeegsusbueetesenkasskuerieggenbsk eg GLi ws 61 5 Gels Melanie UserManual Edition AC 5 IMMOJUCGON unicki siada 63 5 2 Manipulate imageS 1 s 1 ssssssssrnzssza
2. 78 Melanie User Manual Edition AC Gels 5 Figure 5 10 Dual color view 5 4 4 Spots overlapped Chooose View gt Sheet gt SpotOvenap once spotshave been detected and the visible spots on the sheet reference will be shown in blue on the image Figure 5 11 Thus you can easily compare the posttion and size of the red spots in the current gels with the blue spots on the sheet reference shown in Crossed or Outlined mode You can change the default colorto be used forovenapped spots by going to the Display tab in Tools gt Options and clicking the Overlapped box Rememberthat you can change the sheet reference by choosing View gt Sheet gt Set Reference E a b Figure 5 11 Spots overlapped with a outlined and b crossed spotshapes 5 5 Grid lines 5 5 1 Display grd lines Choose View gt Global gt Gnd Lines gt Show to display grid linesoveryour images Figure 5 12 Gnd linescan be used to evaluate distances between spots in terms of pixel coordinates pl MW units or real word units cm orinch Grd linesare also a helpful way to visualize deformations in aligned gels because the gnd linesare warped in the same way asthe image 5 5 2 ZH Editgrid lines Choose View gt Global gt Grid Lines gt Editto change the grid properties The software trys to partition the visible area into the Number of subdivisions entered by the user Figure 5 12 The graduationscan be attached to the Gelorthe Screen Thismea
3. 413 413 a b Figure 8 20 Class Analysis Histograms with a unsorted valuesand b values sorted in ascending order e Adaptive Gradations to adjust the histogram gradations according to the valuesin each histogram Deselectthisoption to display an identical gradation in all histograms Figure 8 21 abcdefzhijki abcdefshijki abcdefshijki 57 32 53 3 53 b Figure 8 21 Class Analysis Histograms with adaptive gradations set a individually foreach histogram and b according to the minimum and maximum values in all histograms e Show Labels to display a table with the gelor class na mes Altematively when you place the mouse overa letter in the Melanie UserManual Edition AC 141 8 Data analysis 8 4 Analyze classes histo gra ms a screentip displays the full image orclassname and the value in that image orclass Displayed value The displayed statistical descriptororoverlapping measure needsto be selected in the drop down list in the toolbar You can display the Center central tendency Dispersion Gap Ratio orNomalized values forthe different classes CAUTION The Gap Ratio and Nomalized options are not available for DIG E experiments when Vol Ratio isselected asthe quantification measure 142 Melanie User Manual Edition AC Annotations 9 9 Annotations 9 1 Introduction Individual pixels and spotsin a gelimage can be labeled with annotations Annotations are u
4. 4 Projects 4 3 Create match hierarchy 56 window and give a name forthe new match set Figure 4 9 A hierarchy Figure 4 2 can be created Workspace 73 Image Pool lige UM Project S ra a E Match pe ATI pa ATZ Classes Display Merge Match5et h Export Copy Ctrl C Cut Crl x Paste Ctrl V REMOVE Dei Properties Figure 4 9 Match sets BTI and BT2 are selected for merging Match sets AT1 and AT2 were already merged this way into a match setA 4 3 3 Setreference Within each match set the gelormatch set that hasa red markerand appears first in the list isused asthe reference in the matching process To change the match reference drag the desired gelormatch set onto the name of itsparent match set so that it moves into the first position CAUTION You can change the match reference aslong asthe imagesin your match set have not been matched Once they are the reference image can no longerbe changed The reference foreach match set must be carefully chosen This is because automatic matching comparesthe spots in the reference to those in the other images If a soot isabsent from the reference it cannot be matched automatically although it can be matched Melanie User Manual Edition AC Projects 4 manually with spotsin othergels The rule of thumb isto choose the gel or match set with the most and the best quality spots asthe reference 4 3 4 Use existing match sets You may wish to ca
5. Analyse Gels gt Table to display the Gel Analysis Table Figure 8 5 This report descnbes foreach match e The spot values from each gel e The Max value that is the highest of all these spot values e The Central Tendency and Dispersion over all the gels in the sheet regardless of whether they belong to different populations or not e The Coefficient of Vanation Coef Variation which isthe dispersion divided by the centraltendency It measuresthe relative varia bility of the sootsin a match by corecting forthe magnitude of the data values thus giving a measure that hasno units When you choose the Median and Mean Absolute Deviation statistics this measure is also known asthe Coefficient of Dispersion e The Range Rato which isthe maximum value divided by the smallest value in the sample specified To specify the sample click the Statistics icon in the Gel Analysis Table toolbar and suppress outliers by setting the percentage sliderin the Range Ratio section to the desired value e The Separability which isthe highest difference between two consecutively sorted values in the whole sample It measures the greatest gap that you can have if you want to split the spot values in a match into two separate classes e The Match Count which isthe number of gelsin which the spot is present and matched Gel Analysis Table Mean 100 and M 5 D Match ID Central Tendency Dispersion Coef Variation Range Ratio Separabilit
6. BT2 Anova Anova p lt 0 001 Ratio gt 2 00916512 0 0641169 0 0287126 6 11207e 5 ke ke 50 0 26883 0 137531 0 26883 0 0945454 0 0782478 6 30621e 5 ke 1191 0 0860342 0 0860342 6 4855e 5 vi Figure 6 4 Spot sets Anova p lt 0 001 and Ratio gt 2 in a Class Analysis Table New spot setscan be created by clicking on the Create Spot Set icon Spot setscan also be created by clicking the Create SpotSeticon ina reporttoolbar Allcurrently selected spotsare automatically included in the newly created spot set Delete a spot set by choosing Edit gt Spot Sets gt Delete and selecting the spot set s to be deleted Combine You can combine two spot sets using logical operators in orderto create a new spot set orchange the curent selection Figure 6 5 Four operatorsare available s And Keepsspotsthat belong to both spot sets e Or Keepsspotsthat belong to eitherone or both spot sets e Notequal Keepsspotsthat belong to only one of the two spot sets e Exclude Keeps spots that belong to the first soot set and do not belong to the second spot set Combine 5pot Sets TENN Operator Anova Anova bet DI ze O01 oi And Curent Selection Ratio gt 2 Figure 6 5 Combine Spot Setswindow To combine two spot sets 1 Choose Edit gt Spot Sets gt Combine Melanie UserManual Edition AC 91 6 Spots 6 5 Display spots 2 Fromthe Inputs drop down lists select the spot sets to be combined The cu
7. By default the toolbariconsin the software are small 16x16 pixels You can choose to display large icons 24x24 pixels for better visibility In addition when you move the mouse overan icon a screentip appears Perhaps you prefer to hide all the screentips or just remove the keyboard shortcuts forthe tools from the screentips To modify the toolbar format 1 Choose Tools gt Customize 2 Clickthe Optionstab in the Customize window 3 Selectthe desired options in the Other section 4 Click Close Custom toolbars You can create your own toolbars with icons for the functions you use most commonly To create a custom toolbar 1 Choose Tools gt Customize 2 Clickthe Toolbars tab in the Customize window 3 Click Newto create a toolbar Entera name forthe toolbarand click OK An empty toolbariscreated below the existing ones 4 Clickthe Commandstab in the Customize window Select the menu from the Category list Then select the command forwhich you want to create an icon and drag itto the empty toolbar 6 Repeatstep 5to add iconsto the toolbar Click Close in the Customize window 2 4 Status bar The Status Barat the bottom of the Display Zone isan important resource It indicates the total number of gels spots matches and annotations that are selected in the current sheet If you move your mouse overa gel image the Status Bar also indicates the X and Y coordinates at the cursor position aswell asthe image intensity Th
8. C Program Files GeneBio License server Licenses Figure 1 2 Starting the server from the Start Stop Reread tab in LMTOOLS 1 4 5 Testthe license file In orderto test that the license file is correctly placed start Melanie on allcomputers where the software is installed Fora floating license a FLEXnet License Finderwindow displaysasking to specify the License Server System or License File Choose the first option and enterthe name of the computer where the license server is installed Click OK 15 Launch the software Double click the Melanie icon Figure 1 3 found on yourcomputers desktop to start the software Altematively go to All Programs gt Melanie 7 0 in the Windows Start menu A splash page appears while the software is loading It will disappear automatically Figure 1 3 Melanie icon Melanie UserManual Edition AC 11 1 Getting started 1 6 Resources 1 6 Resources 12 16 1 Help Tutonals Two tutonalsare provided with Melanie The Non DIGE Tutonal and DIGE Tutorial documents are available from the Help menu in the software from the Windows Start menu and from the folder Doc in the software installation directory The comesponding image filescan be found in the folder Tutonals Images in the software installation directory Non DIGE Tutorial The 12 sample gelsused in thistutorial come from an experimentthat aimsto study protein expression changesbetween fourconditions Cells were grown on two dif
9. Increased memory therefore enhancesthe performance when many and orlarge imagesare analyzed e A high quality display To take full advantage of the software including the 3D View feature the color resolution should be set to 24 bit 16 7 million colors However a color resolution of 8 bit 256 colors isgenerally sufficient It isrecommended to use a screen resolution of at least 1024 x 768 pixels Melanie UserManual Edition AC 7 1 Getting started 1 3 Install the software Microsoft Intemet Explorer 6 or Mozilla Firefox 3 orhigher versions A browserallowsyou to print reportsand to access scientific databaseson the Web 1 3 install the software 14 licensing You can install Melanie from a CD ROM orby downloading the installation package overthe Intemet When the CD ROM is inserted into the appropnate drive on yourcomputer the Setup Wizard starts automatically and givesa senesof on screen instructions Altematively you can double click on the icon of the installer file msi or exe file to launch the Setup Wizard The Melanie installercreatesa default directory on yourhard disk called Program Files GeneBio Melanie 7 0 in which the program files are placed If you want to save the default directory in a different folder then browse and select the location before continuing the installation Once installation is complete it is recommended to restart your computer Upon installation Melanie is fully function
10. Ravier F Pasquali C and Hochstrasser DF 1997 Melanie Il a third generation software package for analysis of two dimensional electrophoresis images Featuresand user interface Electrophoresis 18 2724 2734 Appel RD Vargas R Palagi PM Walther D and Hochstrasser DF 1997 Melanie II a third generation software package for analysis of two dimensional electrophoresis images Il Algonthms Electrophoresis 18 2735 2748 Appel RD and Hochstrasser DF 1998 Computer analysis of 2 D images In nk AJ ed Methodsin Molecular Biology Vol 112 2 D Protocols for Proteome Analysis pp 363 381 Totowa NJ Humana Press ExPASy Molecular Biology Server 2003 The Melanie 2 DE analysis software Online http www expasy org melanie Miller MJ Olson AD and Thorgeirsson SS 1984 Computer analysis of two dimensional gels automatic matching Electrophoresis 5 297 303 Pun T Hochstrasser DF Appel RD Funk M VillarsAugsburger V and Pellegrini C 1988 Computenzed classification of two dimensional gel electrophoretograms by correspondence analysisand ascendant hierarchical clustering Applied and Theoretical Electrophoresis 1 3 9 VargasR 1996 Two dimensional gel electrophoresis computer analysis systems from image acquisition to protein identification Ph D thesis Faculty of Science Geneva University Wilkins MC Hochstra sser DF Sanchezj C Bairoch A and Appel RD 1996 Integrating two dimensional gel databases using th
11. in the case of ClassA A value smallerthan 1 indicates overlap Forexample 0 25 implies that 25 of the current class range isnot recovered by one of the other classes In the same way a value of 1 5 indicates that there isa gap equivalent to 50 of the current class range to the furthest other class The nomalized ovenapping isnot symmetncal the value from Class A Melanie UserManual Edition AC 131 8 Data analysis 8 4 Analyze classes 132 compared to ClassBisnotthe same asthe value from ClassB compared to ClassA m 0 2 4 6 8 EE WA A lt 1 5 e O ClasA a b S ClassB c d fl lt 2 a Class A Class B gt O gt Gap 2 0 c b 2 0 o Ratio 14 c b 1 4 Nomnalized 15 c a b a 2 0 0 2 4 6 8 H_a H 0 75 A gt lt ClasA a b O ClassB c d AE lt ClassA Class B sa LLI A Gap 1 0 c b 1 0 Ratio 0 8 c b 0 8 Nomnalized 0 75 c a b a 0 5 Figure 8 14 Scheme demonstrating how the Gap Ratio and No malized valuesare calculated fortwo non ovenapping classes upper part and two ovenapping classes lower part Amowsabove orbelow each class range illustrate how the Nomalized measure relates to this class range Please note that the formulasmentioned above only apply to ClassA in the current example Their purpose here isto illustrate the pnnciples of overlapping measures Many different cases and therefore formulas exist A value of 1e6 100000
12. one label from each category If one spot contains several proteins it may need to cany different labels from the same category You can do this by linking additional annotations to the spot 9 2 3 Link annotations to spots The software allows you to link an annotation to a spot This is helpful when you want to link an additional annotation to a spot to attach multiple labels of the same category orwhen you missed a spot to which you wanted to link a newly created annotation You may also decide that finally an annotation should not be linked to a given spot To link an annotation with a spot click onthe annotation basisand drag itto a spot If forsome reason an annotation already existswithin a spot 144 Melanie User Manual Edition AC Annotations 9 butisnotyetlinked youcanalso selectthe annotation and choose Edit gt Annotations gt Link with Spot To unlink an annotation from a spot select the annotation and drag it outside the spot or choose Edit gt Annotations gt Unlink from Spot 9 3 Create label categones 9 3 1 Predefined label categones The software offerssome predefined label categones e Ac Thiscategory is provided to hold the protein s accession number AC taken from a user defined database and canbe the linke to Melanie sremote database query engine When sucha link is defined double clicking on a label of this category displays the corresponding protein entry in the selected database with the default
13. staining Staining isa standard Gel Description This information is simply informative i e it is not used by the software except when you specify the cy dye for DIGE imagesthat do not have this information in their file name Each image can only have one description of a given category For example a gel description category Treatment could contain the description Drug 1 forcertain imagesand Drug 2 forother images Figure 3 6 You can define any numberof categonesforan image or a given set of images Gel Table FileName omm kt Staining Sub iption Treatment ESET A TL Gelz silver A TL Gel silver A T2_Gell Silver A_T2_Gel2 Silver A_T2_Gela Silver RTL Gell Silver RTL Gel2 Silver Figure 3 6 Gel descriptionsin a Gel Table using the Descriptions template Melanie User Manual Edition AC 45 3 Image Pool 3 6 Descnbe images 3 6 2 Display gel descriptions Geldescnptionscan be displayed ina Gel Table Select the predefined Descnptionstemplate in thistable to display only the columnsforthe gel description categories To display gel descnptons 1 Choose Reports gt Gel Table 2 Click the Settingsicon 3 Inthe Gel Table window select the predefined Descriptions template from the Load icon drop down list The boxes for the FileName and all gel descnption categones by default only Staining and Comment are checked 4 ClickOK 3 6 3 Edit gel descriptions You can edita gel description forone
14. 6 Workspace The Workspace plays an important role in the software It allows you to organize your gels into projects to specify how the gelsare to be matched together and to define your classes or groups for statistical analysis A briefdescription of the Workspace window ispresented here The utility of the Workspace is discussed in another chapter The Workspace window isa dockable window and hastwo main parts the Workspace toolbarand the Navigator Figure 2 4 Workspace B E F Image Pool MatchSet Class Path Creation Date Modificatio Type Size kB Ce ia i e A Control UM Proj C Doc 28 03 2008 08 04 2008 Class 66 2 LIM old Gelo1 Cy3 Control UM Proj UMProj C IDoc 28 03 2008 08 04 2008 Gels 438 A AB Gel 02 Cy5 Control UM Proj UM Proj C Doc 28 03 2008 08 04 2008 Gels 477 E E Match Gel 03 Cy3 Control UM Proj UMProj C Doc 28 03 2008 08 04 2008 Gels 451 ri A Gel 04Cy5 Control UM Proj UM Proj C Doc 28 03 2008 08 04 2008 Gels 417 p ATI Treated gt UM Proj C Doc 28 03 2008 08 04 2008 Class 66 S EA Si Gel 01 Cy5 Treated UM Proj UM Proj C Doc 28 03 2008 08 04 2008 Gels 439 g Ca Classes eg 02 Cy3 Treated UM Proj UMProj Cr Doc 28 03 2008 08 04 2008 Gels 477 ata DIGE Control vs Treated Gel 03 Cy5 Treated UM Mis UM Proj C Doc 28 03 2008 08 04 2008 Gels 451 C Match Ge
15. Analysis Histograms are very useful Y Glider To see the matchesdisplayed on additional pages use the sliderin the toolbar When a match isselected on an image orin another report it is automatically highlighted in a histograms window ave Statistics Set the statistics to be used in the histograms These settings are common to the Gel Analysis Table and Gel Analysis Histograms Settings Click the Settings icon to select one of the following options s Sorted Values to sort the spot valuesin ascending order Figure 8 7 Melanie User Manual Edition AC 121 8 Data analysis 8 3 Analyze gels 122 a Figure 8 7 Histogramson matcheswith a unsorted valuesand b values sorted in ascending order e Adaptive Gradations to adjust the histogram gradations according to the spot valuesin each match Deselectthisoption to display an identical gradation in all histograms Figure 8 8 Figure 8 8 Histogramson matcheswith adaptive gradationsset a individually foreach histogram and b according to the minimum and maximum values in all histograms e Show Labels to display a table with the gel names Altematively when you place the mouse overa letter in the histograms a screentip displays the full image name and the spot value in the image Nomalization You can display noma lized spot valuesto simplify the compansons acrossmatches These nomalized valuesare particularly useful in combination with the histogra
16. Cy5 Control Ev Gel 03 Gel 03 Cy Standard Gel 03 Cys Control Gel 03 Cyh Treated E Gel 04 Gel 04 Cy Standard Gel 04 Lui Treated Gel 04 Cy5 Control Gels 13 Spots 0 Matches 0 Annotations 0 Figure 3 2 Image Pool folder in the Workspace and Image Pool sheet 36 Melanie UserManual Edition AC Image Pool 3 3 4 Process images The Image toolbarprovidesthe basic toolsto process images Rotate Flip Crop and Invert Gray Levels which can also be found in the Edit gt Gels menu These tools are only available when the Image Pool isthe Curent sheet They are also optional and should only be applied when needed Processing your images in Melanie doesnot affect youronginal image files Copiesare saved ina temporary folderuntilthe imagesare added to a project and saved in the conesponding project folder Please note that these operationsare simultaneously camed outon the two orthree imagesina DIGE gel 341 JAX EZ Rotate Selected imagescan be rotated by 90 CCW counterclockwise or 90 CW clockwise Free rotation the third icon can also be applied although it should be avoided since it modifies the onginal data To freely rotate an image 1 Select the gel image to be rotated in the Image Pool sheet 2 Clickthe Fre Rotate icon 3 Ared grd appearson the image The bold horizontal grid line plays the role of landmark to help you visualize the rotation It becomes the new honzontal in your rotated image 4 Clic
17. Intemet browser s pl MW Thiscategory contains the known isoelectric point pl and molecular weight MW information which is subsequently used to compute approximate pl and MW valuesforany point in a gel You should enter the pl value first and MW value second separated bya space By replacing one of the values by 1 you indicate to the program that no value isset probably because you do not know it e Comment Thiscategory isan example of a general category where users may store their comments 9 3 2 Create new categones When you create a new category dunng one of the procedures described in Section 9 2 you must enter the constraints and attributes forthe new category in the Create Category window Figure 9 2 This section explains the different options CAUTION User defined categonesare only available from the category list aslong asthere isat least one label of thiscategory in the open gels in any of the open sheets To create categonesthat are permanently available you must define them in the Annotations tab of the Options window available by choosing Tools gt Options The same category constraints must be defined Atany moment you can change the category constraints by choosing Edit gt Annotations gt Categories gt Edit Atibutes or rename a category by choosing Edit gt Annotations gt Categories gt Rename a Category Melanie UserManual Edition AC 145 9 Annotations 9 3 Create label categones
18. Note that you can select files from multiple DIGE gels 3 You are prompted to s Fornon DIGE images Spec ify the staining If the staining cannot be found in the list you can type a new one The staining entered isonly informative and doesnot influence the analysis unless you assign the Cy2 Cy3 or Cy5 stains in which case the imagesare treated asDIGE images Click OK e ForDIGE images Select the imagesthat are part of the same DIGE gel Figure 3 1 By default the software proposes image combinations based on the file names Click Add to confirm one DIGE gelata time or Add All if the proposed combinations are allcorect The suggested namesforthe created DIGE gelsmust be edited and confimed individually Create DIGE Gel Select the images that are part of the same DIGE gel Gel 01 Cue Standard Gel 01 Cys Control Gel 01 Cu Treated Gel U2 Cy Standard Gel U2 Cys Treated Gel 02 Cu Control Gel 03 Cye Standard Gel 03 Cys Control Gel 03 Cy Treated Gel 04 Cue Standard Gel 04 Cya Treated Gel 04 Cu Control Figure 3 1 Create DIGE Gel window The first list contains the Cy2 images to be opened the second the Cy3 imagesand the third the Cy5 images The software proposesthe combination of images based on the file names Please note that the software checksthe image resolution before opening a file If the resolution istoo low you geta waming message If it is higherthan 225 dpi you are able to scale the image in order to reduce i
19. PDB http www rcsb org pdb explore explore do structureld 1BM T The required query format for Melanie is http www rcsb org pdb explore explore do structureld 9 4 3 Query the database The database accession number for example P02649 isentered asa label of the particular category linked to a spot When you subsequently double click on the label while the Selection tool is selected Melanie opens your default Intemet browser and launchesan HTIP query that takes the form of a Web page address Asa result the entry forthe protein with the given accession number opensin your browser Figure 9 4 In the case of the above example this would be the entry for Human Apo E Gels in the SWISS 2DPAGE database SWISS 2DPAGE Mozilla Firefox File Edit View History Bookmarks Tools Help gt E authors name SWISS 2DPAGE P02649 spot ID serial number identification methods pl Mw range P02649 combined fields Maps experimental info View entry in simple text format Entry name APOE_HUMAN Primary accession number P02649 integrated into SWISS 2DPAGE on February 1 1995 release 1 2D Annotations were last modified on December 1 2000 version 1 Query Remote Interfaces General Annotations were last modified on January 31 2006 version 11 World 2DPAGE Portal C Name and origin of the protein protein list graphical interface McAfee SiteAdvisor Figure 9 4 The SWISS 2DPAGE
20. The selected spot isabsent from groups AT and ATZ including the sheet reference A_T1 Gell Since the spot is present in the referencesB Tl Gell and B T2 Gell itismatched to coresponding spotsand tums up in the analysis Gel and Class Analysis Tables 7 2 Display a match hierarchy In the example below the match hierarchy AB was displayed by nght clicking on itsname in the Workspace and selecting Display In the resulting sheet only the lowest level ATI AT2 BTL BT2 and highest level AB itemsare specifically visualized in the panesand sheet respectively To select intermediary levels A B you can use the corresponding icon in the pane tab of the reference e g ATL forthat level A 102 Melanie User Manual Edition AC Matches 7 Atthe same time the red markersin these iconsindicate whatitem ina given match level is used asthe reference in the matching File Edit View Select Reports Tools Help Ph O 2 Aa GO OG cht AB 73 Image Pool Sige UM Project E ha AB aAA EN Cal Classes PO ATI H a AT GG BT GP BT2 lt 2 Gels6 Spots0 Matches DO Annotations 0 Figure 7 2 Match hierarchy AB displayed in a sheet Note thatthe layoutof the sheetand paneswaschanged to optimize the use ofspace and to geta better overview of the hierarchy The match level A was selected by clicking on the coresponding icon in the pane tab of match setATL 7 3 Define landmarks Melanie isdesigned to match gels with minimu
21. UserManual Edition AC 167 11 Undo redo and history 11 2 History 11 2 History 168 If you are not satisfied with your latest undo oryou canceled too many operations you can obviously reapply the actions To redo specific actions 1 Choose Edit gt Redo 2 Select the action to be redone 3 Click OK The selected action and any preceding actionsare redone You can display a history of the modifications that have been camed out dunng the present work session on the imagesor layout of the Current sheet Choose Edit gt History gt Show to open the History window Figure 11 2 History 22 10 43 D LEG Sheets Settings Create Bookmark Load Bookmark Create Bookmark Load Bookmark oon Image oom Image Move Image Create Annotation by Click Figure 11 2 History window Please note that the action names displayed in the History window are the same asthose used in Undo Redo As for Undo Redo you can choose Only Editfrom the Filter drop down list to restrict the displayed actionsto those that bing about permanent modifications to the image data Some actions are preceded bya or node allowing the item to be expanded orcollapsed Once an action isexpanded by clicking on the plus sign you see parameters and values that further describe the action Modificationsto the Workspace and functionalities linked to opening and saving files including image rotation flipping cropping and inversion of gray le
22. You can click once ortwice in a grayed check box category that takes different visibility states in the varous selected images to hide or show the coresponding label category in the selected gels s Ghow All Makesall the annotations on the selected gels visible including the labels that were hidden e Hide All Hides all the annotations on the selected gels including the cyan cross or square that remains visible when all labels but not annotations are hidden When you click on an annotation that has hidden labels all of its la bels are displayed on the screen dunng the time it remainsselected Figure 9 8 The hidden labels disappear when the annotation is deselected Label from Hidden Category Figure 9 8 a Unselected annotation with two labels b Unselected annotation with 1 label hidden c Unselected annotation with all labels hidden d When the annotation is selected the hidden labels become visible 9 8 Editannotations and labels Youcanadd and modify annotations and labelsin different ways The Selecttool is helpful if you want to add or modify a single labelorjusta few labels The menu options are more adapted to the creation of a large numberof annotationsorlabels simultaneously Reports are useful for editing existing annotations Melanie UserManual Edition AC 157 9 Annotations 9 8 Edit annotationsand labels 158 9 8 1 R Select When double clicking on a label while the Selecttool i
23. a pick list This list containsthe location in pixels of the center of each spot you wish to pick aswell asthe pixel coordinates forthe centers of the two reference markers To exporta file with soot coordinates for use by the spot picker you first have to open the image files and perform image analysis spot detection ismandatory You should then annotate the reference markers Ifa reference marker is well detected during the automatic spot detection process nice round spot perfectly centered on the marker you can jut add an annotation on the marker spot The basis of such an annotation isdisplayed asa smallsquare and itscoordinates correspond to the center of the spot Ifa reference marker is not well detected imegular shape orconsisting of several spots it is better to delete the existing spot s and create an annotation on the pixel that is in the center of the marker This kind of pixel annotation hasa crossed basisand its coordinates corespond to the pixel it isattached to Note that the two optionscan be used ina single gel one markerwith a spot annotation and the other with a pixel annotation aslong asthe annotations are centered on the markers To create a pick list 1 Identify the two reference markers on your image 2 Zoomthe image to better see the left reference marker 3 Checkifthe markerisdetected asa nice round spot If it is not selectthe spot s on the markerand delete by choosing Edit gt Spots gt Delete 4
24. all yourdata in the coresponding project folder on the hard disk 4 6 3 Copy Youcanmakea copy ofa project by selecting Copy in the Projecticon drop down list in the Workspace toolbar This allows you to make modifications on the copy without altering the original project 4 6 4 Share project Ifthe project data are saved ina folderona shared network colleagues having accessto this foldercan open and work with the project The project must be added to the colleague s Workspace using the procedure descnbed below Melanie User Manual Edition AC Projects 4 4 7 Manage projects 4 7 1 Add remove projects Remove You can remove a project from your workspace by selecting Remove in the Pojecticon drop down list in the Workspace toolbar Selectthe project s to be removed and confim Thisdoesnot delete the project folderfrom yourhard disk To permanently remove a project folder you must delete it from your hard disk Make sure that this folder isnot accessed by colleagues in your network before proceeding Add A previously removed project can be re inserted at any time Another usercan also add it to his her workspace To insert an existing project 1 Select Add in the Projecticon drop down list in the Workspace toolbar 2 Inthe Add Ales window browse the directory where the project file prj islocated select its name and click Open 4 7 2 Add files to project Please note that Add in the Projecticon drop down list
25. analysis and not colorimages The extra color information one intensity value foreach colorchannel isof no value Melanie UserManual Edition AC 33 3 Image Pool 3 3 Preview images 3 2 3 Calibration Some imaging devicescan measure more gray levels e g 100000 or more than can be stored in the availabe image formats These instruments encode the gray valuesusing a nonlinear calibration curve generally encoding low intensity values with higheraccuracy than the high intensity values to conserve asmuch information aspossible in the saved files Melanie uses the calibration curve contained in these image files to recalculate the measured gray valuesand use them for display and quantification Other devices convert raw pixel values into real world units generally optical density or OD This means that the range of gray values isthe Same no matter what the onginal image depth There are image capture devices such asthe GE Healthcare ImageScanner in conjunction with the LabScan software that even allow you to perfom this type of calibration When this is the case Melanie takes into accountthe conversion tablesorcalibration formulasstored in the files You can also calibrate such an image capture device within Melanie using calibration step wedgesor calibration stnps 3 2 4 Image editing General purpose graphicssoftware such asAdobe Photoshop ignore oreven remove calibration infomation Therefore you should not use them t
26. attractive documents If you are familiarwith XMLand XSL you can even create personalized templates for pnnting Note that XSL stylesheetscan also be used to convert an XMLfile into another XML file Melanie User Manual Edition AC Data integration 10 The main interest in XMLfomat isthat extemal applications can easily extract necessary data Moreover the filescan be converted to other user defined formats CAUTION Because the XSL stylesheets are specific to the browser you use you will find that different versions both for printing reports and the history are installed with the software in the Template Report and Template Scnpt folders of the installation directory Melanie will therefore ask you to choose the appropnate XSLtemplate each time you print a report or history Look at which template works with your browser and delete the other one In this way the software automatically opensthe remaining file and doesnot ask you to make a choice 10 3 3 Gel and report identfiers Melanie allocatesa unique identifier ID to each project match set gelimage etc This is useful to assure data consistency allow reliable identification of objects acrossa computer network and enable database integration The IDsin Melanie are UUIDs Universal Unique IDentifiers which are 128 bitnumbersthat are guaranteed to be unique through combinationsof hardware addresses time stampsand random seeds These IDs allow the Melanie objectst
27. automatically locating all the spots in your image To detect spots automatically 1 Display the gelsto be detected by selecting a match set in the Workspace Right click and select Display in the contextual menu Choose View gt Spots gt Outlined to visualize the spot borders Click the Region tool Draw preview regionson one ormore gels Note that you can still draw resize ormove regionswhile setting the detection parameters 4 Selectthe gel imagesfor spot detection Choose Edit gt Spots gt Detect 6 The Detect Spots window appearson screen and the spots in the preview regions of the selected imagesare detected with the default parameters If you do not want to recalculate the spots in the preview regions foreach parameterchange tum the Auto Preview option off To manually refresh the preview regions simply click the Preview button 7 Adjust the detection parameters Figure 6 1 In particular optimize Smooth to detect all real spots and split the overlapping ones Subsequently filterout the noise by changing the Saliency and Min Melanie UserManual Edition AC 83 6 Spots 6 2 Detect spots in non DIGE gels Area values See below formore detailson spot detection parameters 8 When you are satisfied with the preview click OKto detectallspots in the selected gelsusing the specified parametervalues Note that you can still change your gel selection at this point 9 The spot shapesare displayed on the images File Vie
28. axesforthe current Factor Projection Report were selected Factor projection table Show orhide the Factor Projection Table cormesponding to the displayed plot Factor projection plot Show orhide the Factor Projection Plot comesponding to the displayed table Melanie UserManual Edition AC 125 8 Data analysis 8 3 Analyze gels 126 s Ges M Displayed items ch the itemsto be displayed from the drop down list in the toolbar of the plot You can choose matches crosses gels blue vectors or both Y Matches displayed Enterthe numberof matchesto be displayed inthe plot Interpret a factor analysis How to interpreta factoranalysisisexplained with an example of sixgels run to compare the effect of two treatments T1 A_Tl Gell A_ Tl Gelz A_Tl_Gel3 and T2 A_T2 Gell A_T2 Gelz A_T2 Gel3 on bactena cultivated on substrate A The gels were detected and matched and a Gel Analysis Table displayed The Factor Analysis Table Figure 8 9 summarizes the vanance accounted for by successive axes or factors expressed asa percent of the total vanance Thus factor 1 accounts for 91 8 of the vanance factor 2 for 5 4 and so on The coordinates foreach gel projected on these axes is also listed The numberof factorsequalsthe numberof gelsbeing analyzed Factor analysiscannot be perfomed with lessthan two gelsand so atleasttwo factors are always calculated Of course the more gels you use the more reliable the results a
29. can match composite spots that are treated as unique entities in the quantitation e Exploit the capability to propagate allorselected spots from one image to the other images CAUTION Both solutions require pror matching Therefore before doing any spot editing first match your images and only then considerto use one of the options described below 6 6 1 G Manual editing You must enter the special spot editing mode to manually edit spots Choose Edit gt Spots gt Edit Enabled or click the comesponding icon in Melanie UserManual Edition AC 93 6 Spots 6 6 Edit spots 94 the Detect And Match Spotstoolbar The Edit Spotstoolbardisplays Selecting the Edit Enabled option again disablesspotediting CAUTION For edited spots the Saliency value becomes zero This can be used to quickly check which spots have been edited The following spot editing tools are available L Create spots Clickthisicon and draw the outline ofthe new spot Altematively double click the desired location in yourimage and set the disc radius forthe circularspotto be drawn in the Create Spot window Q Delete spots Click this icon select the spot to be deleted and confim M Split spots Clickthisicon selecta spotto be split and draw a line through the spot at the position where the separation should occur Make sure you start and finish outside the spot vi Merge spots Click this icon select two or more spots to be mer
30. can then rename the copied match sets see below To copy a match set in the same project hold down the Ctrl key while dragging the match setonto the projectname 4 3 5 Export importa match set The easiest way to provide otherswith accessto the data isto exporta match hierarchy the match setthat isthe parentofthe project containing the folders Match and Classes Simply nght click on the parent match set and choose Export MatchSet The entire match set including images matches spots annotations spot sets is compressed into a single exp file A exp file can be imported into a project by nght clicking on the project name and choosing Import MatchSet Note that if the project data are saved ina folderona shared network colleagues having accessto this foldercan open and work with the project and therefore the match sets 58 Melanie User Manual Edition AC Projects 4 44 Create classes 44 1 Create a class In the Match folderofa hierarchy select all images that should be added to a Single class group of biologically related images Youcan select match sets as well However only one type of item images or match sets should be selected ata time e Option 1 Drag the selection onto the Classes folder of the same hierarchy Entera name forthe new classand click OK You can also use the Copy and Paste options in the contextual menus e Option 2 Right click on one of the selected itemsand choose Add In Class Entera na
31. choose Help gt About Melanie to obtain product system and license information The product name version number and version date that are displayed will be requested in technical support issues You can view system and license information by clicking on the corresponding buttons The Copy to clipboard button in the License Information window canbe used to paste license data into an email In the System Information window find factsabout yourcomputer or choose Ale gt Exportto save allthe information in a text file We listen The GeneBio team is attentive to your suggestions Many of the enhancements to the software are a direct result of conversations with our customers We truly appreciate any comments cnticisms or ideas that would help usto improve the software Please do not hesitate to contact us by email at melanie supportaQgenebio com Melanie UserManual Edition AC 13 1 Getting started 1 7 Whatsnew 1 7 Whatsnew The graphical interface and user interaction modeshave been entirely revamped The streamlined analysis in Melanie now offers enhanced usability and speed Features have been redesigned to help minimize manual spot editing and repetitive match editing Ultimately tasks are simplified and the reliability of the results are increased The most important new featuresin Melanie version 7 0 are listed below 14 Fully dynamic tables histograms plotsand 3 D viewsin which both content and selection are continuo
32. content 153 C calibration 33 apply 43 control 43 copy to clipboard 43 create 40 fitting table 43 images 40 MW and pl 96 open 43 print 43 remove 43 save 43 Melanie UserManual Edition AC table 43 tablet file 40 categones 159 center 137 centraltendency 114 119 137 CGlscrpt 147 choose matchset 110 Classes analysis 131 analysis histograms 139 analysistable 137 create 59 folder 51 co detection 87 coef variation 119 coefficient vanation 119 colorpalette 74 colors 71 combine spot sets 90 command prompt 9 common labels 153 compare images 76 composite spots 94 concunrent license 8 contact information 13 us 13 contextualmenus 24 contrast 70 73 controlcalibration 43 copy annotations 158 formula to clipboard 116 labels 158 to clipboard 26 Cor 116 corelation coefficient 116 create annotation categones 159 annotations 143 calibration 40 geldescrptionscategory 45 labelcategones 145 labels 143 match hierarchy 54 pick lit 164 projects 52 specific link 150 spotset 120 138 spotsets 90 spots 93 toolbar 18 crop 37 area 37 Melanie UserManual Edition AC 181 exportarea 37 importarea 37 Current sheet 24 cursorinformation 75 98 customersupport 13 customize reports 27 toolbars 18 D data analysis 113 type 146 database options 30 DD 40 DeCyder2D algorithm 83 default mapping 69 define landmarks 103 delete annotations 158 labels 158 matches 109 spotsets 90 spots 93 d
33. contextual menu It allows you to select the soot where your cursor is positioned and to quickly access one of the image manipulation tools described below The pixel gray levelsare overlaid on 3D views the top of the peaksare therefore always darker File Edit View Select Reports Tools Help OPEL OP B OO O S PRRs SMA ATL AT2 Figure 5 6 3D views of the regions defined in A_T1 Gell and A_T2 Gel2 72 Melanie User Manual Edition AC Gels 5 File Edit View Select Reports Tools Help h l o OL P ALCI OP ea Figure 5 7 3D views forthe selected spots The following toolsare available in the 3D View toolbar ba Tools To manipulate 3D views with your left mouse button you can switch to one of the following options e Rotation Rotate the images using your mouse e A Zoomy Contrast Zoom in or out by dragging the mouse honzontally Adjust the contrast orheight of the spots by dragging the mouse vertically on the view 4 gt translation Choose one ofthe suboptions Z Y X XY to move the views along the desired axis e Auto Rotate Let the images rotate automatically around the Zaxis so that you can view the spots from all sides e Show Default View Retum to the orginal view Melanie User Manual Edition AC 73 5 Gels 5 3 View signal intensity 74 Bi sack Stack Display the imagesone on top of the other instead of using the default tiled
34. corresponding to the background occurvery often whereas the high gray levels on the right of the histogram cormesponding to the darkest spots are much less frequent The red line indicates that the minimum and maximum gray levels 3524 and 28530 respectively are remapped by the default linearmapping The vertical axisforthe red remapping graph comespondsto the 256 screen gray levels Melanie User Manual Edition AC Gels 5 Adjust Contrast Contrast A TI Gell k Unit Intensity W Region Whole Image W Colors Gray w z Invert Apply Figure 5 4 Adjust contrast function Change the default mapping The software offerstwo complementary waysto change the default mapping function in orderto improve the visual display ofthe gels Figure 5 5 e You can define the minimum and maximum gray levels i e look at only the light orthe dark regionsin the images Do this by decreasing the size of the gray level range that isto be remapped linearly to the screen gray levels Move the left ornght borders of the sliderthat isfound below the histogram function Once the size interval of the slider is decreased you can also displace the interval to the left or right Altematively you can type valid numbers in the boxes at the lower left and right comers of the histogram Melanie UserManual Edition AC 69 5 Gels 5 3 View signal intensity 70 Adjust Contrast Contrast Modified A 3524 13300 Unit In
35. designs Figure 4 2 and Figure 4 3show an example ofa matchhierarchy orrootmatch set ABand submatch sets A B ATI ATZ BTL and BIZ Gelsor match sets with a red markerare used asthe reference in the matching and alwaysappearfirst in the list Workspace O 1 F Image Pool Sige UM Project Gp AB E _ql Match Geh A Geh AT A Ti_Gell amp T1_Gel2 A T1_Gels Geh AT A_T2 Gell A_T2 Dei A_T2 Dei EN Classes Figure 4 2 The Match folder In thisexample experiment a setofsamplesfrom bacteria were cultivated with either substrate A orwith substrate B Underboth growing conditions two treatments were tested Therefore 4 different populations exist A gel wasrun foreach of the 3 samplesin a population giving a total of 12 gel images Melanie User Manual Edition AC Projects 4 nk W PT z d 4 n d W Si B T2_Gel3 B T2 Gel2 B T2 Gell A Tl Gel A T2 Gel BTL Gel Figure 4 3 A tree view of the example match hierarchy des ribed above An important advantage of this type of match hierarchy is that it minimizes the number of difficult match combinations Instead of having to match 9 images those belonging to ATZ BTl and BT2 to a global reference e g A_Tl Gell that isnot belonging to the same population only 3 slightly more complex match combinations must be performed AT1 versus AT2 BT1 versus BIZ and A versus B This significantly reducestime spent on match editing
36. filled on the gelsfrom the View gt Spots menu Melanie User Manual Edition AC 6 6 Editspots Spots 6 6 5 2 Spotcolor By default enabled spotsare displayed in red disabled spotsin yellow selected spotsin green and overlapped spotsin blue Youcan change these default colors in the Display tab in the Options window accessible by choosing Tools gt Options Click the colored box you want to change and the Color window opens Choose the preferred color from the spectrum and click OK 6 5 3 SpotID You can display Spot IDs forselected spots with View Spots Show ID To hide all Soot IDs again choose View Spots Hide All ID CAUTION Except fordeleting spots spot editing isnot allowed on DIGE gels Quantitative protein data and in particularthe spot volume are highly dependent on an optimal and reproducible definition of the spot bordersand a corect splitting of partially overlapped spots To guarantee reproducibility of quantitative work it istherefore recommended to create spots by using the automatic spot detection algonthm in the software and to avoid manual editing asmuch as possible However spot detection differencescan till occur In particular some spots are differently split in gelsto be compared The software offersthe following solutions to deal with detection vanations between gels without calling for spot editing e Exploit the capability to create multiple matches In practice this meansthat you
37. finished a single click in any cell quitsthe editing mode Please note that annotation categones of the data type Setare displayed ascheck boxes A checked boxmeansthat the item belongs to the Set An empty box means that it doesnot belong to the Set The same istrue for spot sets represented in any of the tables a checked box meansthat the spot belongsto the set An empty box means that the spot doesnot belong to the set To check several boxes i e lines simultaneously Melanie User Manual Edition AC Graphicaluserinteface 2 1 Use the Shift or Ctr keysto select the rowsin which you want to check oruncheck the box 2 Clickinthe boxof one of the selected rows Allrowsare now either checked orunchecked 2 8 Dockable windows The Workspace reports and Adjust Contrast window are dockable windows Dockable windows make it easier for you to work with numerous windowsat the same time These windowscan be docked Le fixed in place against the left right top or bottom edgesof the Melanie window and alwayslie on top when visible A visible window can be in one of three modes Pinned The pinned mode E enables dockable windowsto be locked into position around the edgesof the Melanie window Once a window Is pinned you can move itto a different location by dragging the title bar Figure 2 6 Guides indicate where the window may be docked By moving the cursoroverthe guides a shaded blue boxappearsshowing where the window will
38. geland click A validated landmark symbol bold orange circle appearson the spot Figure 7 3 3 Inthe otherimages drag the non validated symbols green circle with orange plussign onto the corresponding spots If the symbol is already ona good spot double click to validate tt 4 Repeatsteps2 and 3to add more landmarks In certain gels the symbolsonly become visible once the landmark has been validated in the reference Inthe example the landmark must be validated in the image B T2 Gell before any symbols appear in the imagesB T2 Gel2 and B T2 Gel3 Sometimes you may want to move orzoom your images during the landmarking process When you click the Move or Zoom tools the orange landmark symbolsdisappear and only labelswith the landmark numbersare left When you click the Landmark tool again the symbols are reactivated and you can continue defining landmarks To delete landmarks you must delete the comesponding annotations Choose Select gt Annotations gt By Category and select Landmarks in the list Then choose Edit gt Annotations gt Labels gt Delete Melanie User Manual Edition AC Matches 7 File Edit View Select Reports Tools Help CEIR SOI PB COLO PG PAR Validated landmark ageds Hani Gels 12 Spots 7 Matches 0 Annotations 7 4 51 cm 2 3 cm 4274 Figure 7 3 Placing of landmarks Note the difference between the validated and non validated landmark symbols In general itisgood to validate
39. gels into which you want to paste the annotations and choose Edit gt Annotations gt Labels gt Paste Adjust the annotation positions using the Selecttool Copy paste labels Instead of copying entire annotations you can also copy distinct labels from one gel to selected spots or annotations in a senes of gels Select the labels you would like to copy and choose Edit gt Annotations gt Labels gt Copy Then select spotsorexisting annotationsinto which you wantto paste the labelsand choose Edit gt Annotations gt Labels gt Paste Propagate to matched When gels have been matched labels selected in one gelcanbe copied to their coresponding spotsin other gels by choosing Edit gt Annotations gt Labels gt Propagate to Matched This is particularly useful Melanie User Manual Edition AC Annotations 9 when you have annotated one image during analysis and wantto propagate the labelsto allmatched gels Duplicate labels You can copyselected labelsto another category by choosing Edit gt Annotations gt Labels gt Duplicate Since the selected labelsmay belong to different categones thisoption can be used to merge several categonesinto a new one However only one label perannotationcan be duplicated ata time HA Annotation table You can edit labels via the Annotation table available from Reports gt Annotation Ta ble Make sure you have displayed the desired categones for editing by clicking the Settings icon Cre
40. one ortwo landmarkson allthe images in the hierarchy so that they are used formatching atall levels Ifittums out afterthe matching that the gels within the lowest hierarchies e g ATI AT2 Bll and BT2 have been propery matched but that the higher level matches e g A and B are not satisfactory you can add additional landmarks to the higher levels only Do this by validating the landmark only on the reference images 7 4 Automatic matching An entire hierarchy can be matched automatically To match the gels in a match hierarchy 1 Selectthe gelsto be matched in the sheet In principle allthe gels even ina multilevel hierarchy can be selected Melanie UserManual Edition AC 105 7 Matches 7 5 Select matches 2 Choose Edit gt Matches gt Match Gels Orclic LP Match Gelsin the toolbar 3 If several match sets are selected you are asked to choose the onesto be matched in the Match Gels window Figure 7 4 Items preceded bya sign still require matching Use the Ctrl or Shift keys to make multiple selections All match setscan be selected at the same time Click OK Match Gels Please choose the Matchs et AE A B 4 All ATZ E EN eh 2 ATI 4 71_Gell 4_T1_Gel3 4_T1_Gel2 ATZ 472 Gell A_T2 Gels A_T2 Gelz BT1 DT Gell B_T1_Gel3 B_T1_Gel2 BT2 B 72 Gell B T2 Gel B T2 Gell Cancel Figure 7 4 Match Gels window In thisexample all seven match sets were selected for matching The items pre
41. reports 15 159 3D view 24 71 analyze classes 24 analyze gels 24 annotation 159 annotations 156 columns 27 customize 27 Melanie UserManual Edition AC 189 190 edit labels 159 export 162 factorprojection 123 gel 80 gelanalysishistograms 121 ID 163 import 162 labels 156 159 match 110 spot 98 toolbar 26 resolution 33 restore 61 review matches 109 rotate 37 rotation 73 S saliency 84 save 26 81 projects 60 spots 92 scatter plots 116 table 116 scrollbars 21 select 15 90 annotations 152 by category select all 153 by content 153 by value 26 89 120 138 common labels 153 enabled spots 92 labels 152 matches 106 menu 153 sootsets 92 tool 106 152 selected region 71 separability 119 set 146 extemal protein databases 148 reference 55 74 76 spots 92 settings 26 120 121 138 141 setup wizard 8 Sheets 20 align images 76 close 20 Curent 24 free 20 Melanie User Manual Edition AC image pool 35 layout 20 one column 20 one row 20 print 82 reference 76 selection 20 stacked 20 tiled 20 to clipboard 82 to file 82 Shortcuts 171 annotations 173 gels 172 matches 173 redo 171 spots 173 tools 172 undo 171 show annotations 156 default 3D view 73 dualcolor 78 ID 108 labels 121 156 overview 64 profile 74 vectors 107 shrink spots 93 signal intensity 67 single detection 87 sider 116 121 129 141 smooth 84 software references 175 sorted values 121 141 specific links create 150
42. reside if the left mouse button is released If you move the dockable window to a non predefined location itbecomes a floating window Moving a docked window may affect the location and size of otherdocked windows Un pinned A visible window in un pinned mode EJ automatically collapses when not in use to become a tab at the edge of the Melanie window in Figure 1 7 the Workspace isin thismode When you clickon a docked tab the window slides back into view and isready foruse You can also click on Minimize Fl to minimize a window in un pinned mode Hoating A dockable window in floating mode willalwaysappearon top Itcan be dragged to any position within the software or even outside the Melanie window You can switch in and out of floating mode by double clicking on the title barof a dockable window Tabbed groups Dockable windowscan be organized into tabbed groups This feature extends your ability to maximize the use of limited screen space by combining multiple dockable windows into one window In order to form a tabbed group drag the title barof a dockable window into the center of another You will see the nested tabsat the bottom of the docked window In orderto separate a tabbed group drag a tab away from the docked window ordouble click on the tab Please note Melanie UserManual Edition AC 29 2 Graphical user interface 2 9 Options that tabular reports and graphical reports cannot be grouped together File Edit View Se
43. serverjlmgrd log Browse View Log Close Log Iw Start Server at Power Up M Use Services 10 E Figure 1 1 The Config Servicestab in LMTOOLS 1 4 3 Place a license file You will receive the license file with a lic extension from GeneBio once you have sent the physical address of your computer Fora node locked license the file must be placed in the Melanie installation folder C Program Files GeneBio Melanie 7 0 by default Fora floating license the file must be placed in the Licenses subfolder of the License server installation folder C Program Files GeneBio License server Licenses by default 14 4 Startthe license server This section only pertainsto floating licenses If you have a node locked license then skip to the next section To start the GeneBio license server 1 Select the Stary Stop Reread tab in LMTOOLS 2 Click Start Server Figure 1 2 Melanie User Manual Edition AC Getting started 1 3 Close LMTOOLS LMTOOLS by Macrovision Corporation http iwww macrovision com i m x File Edit Mode Help Service License File System Settings Utilities Start Stop Reread Server Status Server Diags Config Services Borrowing FLEXnet license services installed on this computer Segen Stop Server ReRead License File I Force Server Shutdown EE NOTE This box must be checked to shut down a license serverwhen licenses are borrowed Using License File
44. several databaseson the Intemet It isthe responsibility of the user to acquire the database licenses if needed In particular the PROSITE and SWISS 2DPAGE databasesare copynghted and allcommercial users of these databasesare required to purchase a database license from GeneBio No license fee ischarged to academic users for non commercial use For questions about obtaining a license subscrption forthe PROSITE and SWISS 2DPAGE databases please contact GeneBio www genebio com Melanie takesadvantage of the fact that virtually alldatabaseson the Web and in particular those containing 2 DE and other protein data use CGI scnptsto enable data queries A CGI Common Gateway Interface script isa program or script file executed on a Web server in response to a userrequest The CGI script transmits information suchas a database accession number or object identifier from the client to a database engine receives back the results and displays them to the Client Melanie UserManual Edition AC 147 9 Annotations 9 4 Connect to protein databases 148 9 4 2 Setthe database To query a remote database through the Intemet you must send HTIP queries for instance http www expasy org swiss 2d page P02649 to a CGI scnpt ona server The HTIP query must be composed of e The database HTIP address http www expasy org e The database query engine swiss 2dpage e The database accession number P02649 In Melanie the databa
45. small spots are hardly visible To emphasize these very faint spots you can adjust the contrast of the image and ordisplay images using pseudo colors Melanie User Manual Edition AC 67 5 Gels 5 3 View signal intensity 68 Choose View gt Gels gt AdjustC ontrastand select a region of interest on one or more images using the Region tool These regions will allow you to preview the contrast and pseudo colorsettings before applying them to the selected images The size and position of the preview regionscan be adjusted atany time Afterhaving adjusted the settings asdescnbed below click Apply The new contrast settings are saved with the image file CAUTION Any Adjust Contrast changes only influence how the image is displayed on yourscreen and do not affect the undenying data spot detection and quantitation Gray level mapping Some scannersare able to scan 2 DE imageswith 100000 gray levelsor more Because common computerscreensare only able to display 256 gray levels mapping must be undertaken between the image gray levels and the 256 screen gray levels By default the software usesa linearmapping function where the lightest point in the image is mapped to 0 white and the darkest point ismapped to 255 black This is illustrated in Figure 5 4 The histogram displays the frequency with which each gray level from 3524 to 28530 occurs in the first image of the sheet The low gray levels on the left of the histogram
46. sssss xzssssss 147 9 5 Create specific links ssssssnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn 150 9 6 Selectannotations and labels ssssssnnnnnnnnnnnnnnnnnnnnnnnnnn 152 Melanie User Manual Edition AC 9 7 Display annotations and labels 1 1 ss s2sxnssss 156 9 8 Editannotations and labels s sssszsszsssssssssszuse 157 9 9 Annotation table enee KKK KREE KER KKK KREE KEREN KbREk Ken 159 10 Data integration 10 1 Convert projects from earlier software versions 161 10 2 Acquire images from Twain compatible scanners 161 105 Bpon data ui Ea id ii 162 10 4 Export to Spot EXCISION robofS 1 s2ssnsssssssnsszsssxn2 163 11 Undo redo and history SEL Undo edO EEN 167 TE SOMA GC AOC EE 168 Appendix A Shortcuts AL SNONCUERCYS osi 171 A 2 Tool MOI US wasi AE 172 A3 Gel eil E 172 AA Soton uls siiani 173 A 5 Annotation shortcuts ssssssssnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn 173 A 6 Match SOM UG uassa iao wawacianianiawiwk oda 173 Appendix B References BL SOMaIE brrconnin ah 175 B 2 Statistical methodS sssssnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn 175 BS FURNELITEAONIOJ sca kciz anaa 176 Melanie UserManual Edition AC 5 Melanie UserManual Edition AC Getting started 1 1 Getting started 1 1 Aboutthe software Melanie offers a unique and flexible solution forthe comprehensive visualization exploration and analysis of 2 D gel data This version of Melanie
47. staining by considenng the total volume overall the spotsin the image This means that in an image where globally the soots are darker than in another image the majonty of soot volumes is higher However the bulk of Vol should be similarto those in the compared image at least for gels with similarspot pattems Vol VO x 100 n K Vols V l SCH where Vol isthe volume of spot Sina gel containing n CAUTION When you look ata detected spot in the 3D View you will notice that the bordersare not localized at the base of the spot especially for intense spots This is nomal because the spot contours displayed effectively reflect the quantified parts of the spot and they do not conespond to the whole spot which is difficult to define 6 3 Co detect spots in DIGE gels The co detection algonthm is designed to simultaneously process one two orthree imagesdenved from a single gel e Single detection one image e Double detection two images e Tiple detection three images Single detection is performed on images of fluorescently post stained gels used for picking a case where there isa single image associated with the gel Double and tnple detection takes advantage of the inherent co migration benefits of the CyDye DIGE Fluor dyes A set of co run imagesare merged together thereby incorporating all soot features in a single image Spot detection and spot boundary definition isthen performed using pixel data from al
48. the other annotations Figure 9 6 152 Melanie UserManual Edition AC Annotations 9 Figure 9 6 a Annotation hidden by some otherannotations b Same annotation displayed in front of all other annotations after selection Ifthe annotation isattached to a spot the spotisalso selected Simila ry if you select a spot the attached annotation is selected To selecta label clickon the label You can selectmore than one label by using the Shift key To select an annotation click on the annotation basis To select more than one annotation select the first one then hold down the Shift key and select additional annotations To select all annotationsin a region position the cursorat the top left position of the desired region hold down the left mouse button and then drag the cursorto the bottom nght position All annotations in the defined region are selected To select annotations in more than one region hold down the Shift key while selecting additional regions To deselect all annotations click on a gel not on an annotation 9 6 2 Selectmenu You can select specific labels and annotations with the options in the Select gt Annotations menu Please note that this also selects hidden annotations e By Content This feature enables the selection of labels belonging to one orseveral categonesthat must be selected based on their content When the Regular Expression box in the window isnot checked the entered string of
49. the same results as the t test for independent samples One way ANOVA tests the null hypothesis that all populations have identical means It generatesa P value that answers this question If the null hypothesis is tue what isthe probability that randomly selected samples vary as much or more than actually occured It isbased on the same assumptions as the t test e The samplesare randomly selected from orat least representative of the larger populations e The two samplesare independent There isno relationship between the individuals in one sample ascompared to the other e The data are sampled from populations that approximate a Gaussian distnbution If you are notable to assume that yourdata are sampled from Gaussian populations then non parametnc tests like the Mann Whitney or Kolmogorov Smimov tests can provide a better analysis Please note that these test only allow you to compare two samples at the same time Mann Whitney or Wilcoxon test The Mann Whitney U test or rank sum test isthe non parametric substitute forthe two sample t test when the assumption of noma lity Gaussian bell shaped distnbution isnot valid It is equivalent to the Wilcoxon rank sum test It should only be used for companng two unpaired samples The assumptions of the Mann Whitney U test are Melanie User Manual Edition AC Data analysis 8 e The variable of interest iscontinuous not discrete and the measurement scale is at least ordinal Th
50. the step tablet with the red calibration step overlay The Create Calibration window showsthe calibration curve and the OD valuesfor the different steps on the left When selecting a step in the list the cormesponding step becomes automatically highlighted in green on the step tablet overlay and in the calibration graph 6 Atthe left of the Create Calibration window you see the theoretical optical density OD values of the different steps in the tablet the values you entered in the Calibration Tablet File Some stepsare automatically deselected grayed out because of their unreliable values You can deselect additional onesif you estimate that they should be excluded from the calibration process At the 42 Melanie User Manual Edition AC Image Pool 3 right of the Create Calibration window you see the calibration curve between the loganthmic transmittance valuesonthe X axis and the OD intensities on the Y axis Note that the measured intensity values foreach step are calculated as median intensities overallthe pixels in the small rectangle area foreach step on the step overlay The honzontal dispersion intervals in blue orgray for deselected spots represent the intensity ranges when 10 of the less intense and 10 of the most intense pixel values are removed The calibration formula and erorare given below the graph 7 You can display some reports to judge the quality of yourscanner calibration see below for more details 8 Yo
51. to cancelany unwanted modifications to the images or layout of the current sheet by choosing Edit gt Undo in the menu Figure 11 1 Undo List Select commands to undo Ze UA Ab Create Annotation by Click Zi UL Zb Create Annotation by Click 20 45 47 Create Bookmark 20 45 37 oor Image 20 45 37 oom Image 20 45 32 Delete Selection Box 20 45 31 Delete Selection Box 20 45 30 Delete Selection Box 20 45 29 Delete Selection Box 20 45 29 Delete Selection Box 20 45 26 Delete Selection Box 20 32 12 Load Bookmark 20 31 52 Create Bookmark 20 20 51 New Selection Box Soa Se ge Figure 11 1 Undo window Each action in the Undo Redo list is preceded by the time at which it wascamed out This information helps you to identify the particular sequence of actions that you would like to cancel By default allactions are displayed If you specifically want to track permanent changes that have been made to the image data essentially the commands available under the Edit menu then choose Only Editfrom the Filterdrop down list All operations performed can be reversed except for modifications to the Workspace and functionalities linked to opening and saving files This includes image rotation flipping cropping and the inversion of gray levels To undo specific actions 1 Choose Edit gt Undo 2 Selecta pnoraction to be undone 3 Click OK The selected action and allfollowing actionsare undone automatically Melanie
52. wasdeveloped by a team of top researchers from the Swiss Institute of Bioinformatics SIB in collaboration with Geneva Bioinformatics GeneBio SA and GE Healthcare Professor Hochstrasser s group at the Geneva University Hospital contributed their real world expenence The application has advanced to its current level primarily due to the input of users wondwide There are two modulesof Melanie 7 0 available for purchase e Melanie 7 0 DIGE To be used with conventional 2 DE and DIGE gels It isfully functional enabling you to add and import DIGE gels directly in the workspace You can also co detect DIGE gels using the algonthm created by the GE Healthcare DeCyder software development team aswell as match report plot histograms and perom statistical analyses on DIGE gels e Melanie 7 0 To be used with conventional 2 DE gels All menu commandsrelated to DIGE are not functional and are grayed out in the graphical user interface 12 System requirements In orderto install and run Melanie yourcomputer must satisfy the following requirements Microsoft Windows XP Vista 7 or8 operating systems e Administrative pemission to install Melanie e Atleast500 MBof RAM forMelanie recommended Intel dual core processor with 1GBof RAM and 768 MB RAM for Melanie DIGE recommended Intel dual core processor with 2GB of RAM The amount of memory required isdetermined by the numberand size of image filesto be processed simultaneously
53. 0 122 131 137 number 146 Melanie UserManual Edition AC 187 188 O OD 40 one column 20 21 row 20 21 one wayANOVA 134 openimages 34 options 30 113 print 82 overlapping measures 131 spots 79 overview option 64 P page setup 82 panes 20 close 21 free 21 layout 21 one column 21 one row 21 selection 21 stacked 21 tied 21 paste annotations 158 labels 158 physicaladdress 8 pl 96 pick list 164 pinned 29 pixel distances 66 place a license file 10 plots empincal distribution 136 scatter 116 PNG 81 populations 51 preview images 34 previousin selection 26 print 26 81 images 82 options 82 sheets 82 processimages 37 product infomation 13 profile 74 projectname prj 60 projects add 61 backup 61 convert data to new version 161 create 52 Melanie User Manual Edition AC display 59 folder 60 importolderversions 161 manage 6l move 59 properties 59 remove 59 61 restore 61 save 60 propagate spots 95 properties 59 purchase software 7 8 Q quantification options 30 value 113 query extemal protein databases 149 quick start manual 13 R range ratio 119 ratio 120 122 131 137 raw images 60 recycle bin 60 redo 167 reference 114 match 55 set 76 sheet 76 references 176 software 175 sta tistic al methods 175 region adjust contrast 71 applyto allgels 66 edit 66 tool 65 regularexpressions 153 relative 120 122 remove calibration 43 projects 59 spots 92 rename category 159
54. 0 269066 0 044982 0 178557 0 561399 0 677918 4 0 871153 0 669511 0 589342 0 0975621 0 104748 0 235228 0 358619 2 0 59571 0 46165 0 444102 0 688504 0 243621 0 205315 0 11878 6 0 312768 0 176669 0 104868 0 27321 0 742061 0 554295 0 21687 Figure 8 9 Factor Analysis Table The Factor Projection Plot Figure 8 10 displays the projection of each match cross and each gel blue vector on the two factonal axes The blue curve representsa part of the corelation circle itsform is linked to the scale of the axes When matchesare selected on the plot they are automatically highlighted on the gels and or any open reports The Factor Projection Table Figure 8 10 displays the contnbution of each match to the two axesdisplayed in the Factor Projection Plot The Quality measures whether the projection of the match is well represented on the factonal subspace Melanie User Manual Edition AC Data analysis 8 Factor Projection Report Mean 100 and M 5 D GH tv Match ID Contrib Axel Contrib Axe Quality 483 0 531088 0 0350992 0 998527 228 0 098352 0 077144 0 98901 237 0 0495339 0 09425 0 984883 48 0 0141454 0 086142 0 991989 181 n nn 4n347 0 0557AN7 n 9945 Figure 8 10 Factor Projection Report includes the Factor Projection Plot top and the Factor Projection Table bottom You can use the following toolsin the Factor Projection Report HH Factor analysis table Display the FactorAnalysis Table from which the two
55. 0 in the Class Analysis Histograms characterizes the case where the protein iscompletely absent from a class The software cannot compute ranges A value of 0 forthe Ratio or Nomalized measures indicates that the particular class is entirely covered by another one CAUTION The Gap Ratio and Nomalized options are not available for DIG E experiments when Vol Ratio isselected asthe quantification measure Melanie User Manual Edition AC Data analysis 8 CAUTION As indicated in their definition the Gap Ratio and Nomalized values always calculate the MAXIMUM difference ratio or percentage with respect to ANY of the other classes Ihismeans that when the Ratio values forthree populations e g A B C are compared the software calculates the ratio of A with respect to B and of A with respect to C but only displays the highest value in the column forclassA The number shown doesnot indicate with resoectto which classthe value was obtained The idea isto quickly enable you to find a match i e protein marker that distinguishes the current class from any of the other classes Once such protein markers are found the Class Analysis Histograms can be used to study the match in more detail 8 4 3 Fold change Instead of using overlapping measures between the classintervals you may simply want to look at the fold change between the central tendency of one classand the centraltendency of one orseveral other classes ignoring the d
56. 00 To estimate the saliency range to use with your images you can display the Cursor Information window or Spot Table and look at the saliency value given fora spot that you would like to suppress Enter this value in the Saliency field in the Detect Spots window The spot detection algonthm then discardsall spots with saliencies smaller than the specified threshold e Min Area Aftersetting an appropnate Saliency to filter out all noise spots there may stillbe noise in yourgel that cannot be eliminated without suppressing real spots This often happens with dust particles that consist of a few very dark pixels Get nd of these artifa cts by using the Min Area parameter It eliminates spots that have an area smallerthan the specified threshold expressed in number of pixels 6 2 3 Spot quantification The software automatically computesthe amount of protein present in each spot Figure 6 2 illustrates the pnnciples of spot quantification in the Melanie algonthm Measunng the protein quantification values in Melanie UserManual Edition AC 85 6 Spots 6 2 Detect spots in non DIGE gels this way hasthe advantage of being more robust and reproducible when calculating protein expression vanations relative quantification File view Edit Show Select Analyze Reports Tools Window Help Ia ole L l4 y 11095 83EdS 10 Intensit CL 0 75 Intensity ZB La 1326 W Intensity J 10214 877
57. 1 4 DD Create calibraton You can create a Calibration once you have scanned the step tablet and have a corect Calibration Tablet File To create a Calibration 1 Open the step tablet image file If necessary rotate the image so that the light stepsare displayed at the top Choose Tools gt Calibration Tablet gt Create In the Load Step Tablet Definition window browse to the folder where you saved the Calibration Tablet File tab specifically tailored to yourstep tablet see above select the file and click Open Melanie User Manual Edition AC 41 3 Image Pool 3 5 Calibrate images 4 A red calibration step overlay appearson the image and the Create Calibration window isdisplayed Figure 3 5 5 Adjust the position of the steps by dragging the overlay while holding the left mouse button The size of a step on the red overlay the distance between two short horizontal lines should comespond exactly to the size of a step on the image If thisisnot the case you must adjust the height of the tablet in the Calibration Tablet File File Edit View Reports Tools Help GAN Sa Co ld 2h A ES HR t Add Files to Project 3 Image Pool Create Calibration S E Ess SR bei IZ A Fitting Table S 8 Calibration Table step tablet no 2 21 3 050 OD 0 0489 log t 2 0 843 log t 0 00687 mean error 0 027 Gels 1 Spots0 Matches 0 Annotations 0 Figure 3 5 Image of
58. 146 Create Category Define constraints for category Test Category attributes Url Exe Display properties Color C By default Hidden Figure 9 2 Create Category window Data type By default the labelscan contain any character However to ensure coherent annotation data the label contentscan be constrained to one of the following Data Types e Text Can contain any character e Number Can only contain numencal values s Auto Numberng An incremental number per gel isentered automatically asthe new label e Set Use this data type to mark spots with common properties The labelsin such a category do not contain specific information They only display the name of the category to which they belong Note that labels of this category are displayed in the form of check boxes in tables A checked box indicates that the spot belongs to the category Is unique When you check the Is Unique box you indicate that each labelona gel within the new category should be unique The software will not accepta new label when an identical one already exists You are asked to entera new label Extemal engine Melanie offersthe possibility to link spots on gel imagesto protein data in 2 DE or other databases All you have to do is input the appropriate query format database addressand query engine in the Extemal Engine field of the new label category and enter valid database accession numbers AC aslabels This fun
59. 4 0 322517 A_T2_Gel2 607 715 1180 631 861 1036214 0 149291 A_T2_Gel2 606 713 47 627 370 404 663287 9 0 00911814 A T2 Gel 605 1333 400 627 356 330 56666 1 0 0081641 A_T2_Gel2 604 217 1256 630 5404 5 1725404 0 248586 Deg Gel 603 2727 9 6m0 19270 1146 9619569 1 38593 E Ss ma AEN ia zwi aa GEN iwo Gel Table Gel Analysis Table Mean 100 and M 5 Dj Spot Table Gels 12 Spots 12 Matches 1 Annotations 0 Figure 2 1 The Melanie window a Menu Bar b Toolbar c Status Bar d Display Zone e Sheet f Pane g Workspace and h Reports 2 2 Menu bar You can choos actionsto be perfomed during your analysis from the Menu Bar The menus are context related This means some of the commandsmay not be available allofthe time and eithergo away or Melanie UserManual Edition AC 15 2 Graphical user interface 2 2 Menubar 16 are grayed out Forexample the Select menu allows you to select spots matchesand annotations for detection and matching Therefore thismenu isneitheravailable nordisplayed while viewing and editing images in the Image Pool Meru Descipfon File Close save import export print and otherbasic operations You can also exit Melanie Edit Undo redo the last operations show a history of operations oredit add modify delete specific gels spots annotations or matches You can also edit spot sets and enable spots View Modify the settings for grid lines profile or overview i
60. 8 display 156 duplicate 159 hide 156 modify 159 paste 158 report 156 159 select 152 show 156 landmarks 77 define 103 tool 103 launch software 11 software first time 52 license file 10 information 13 server 9 licensing 8 LMTOOLS 9 10 load spots 92 log volume ratio 129 M machine license 8 MAD 115 manage projects 61 manipulate images 63 Mann Whitney 134 manual editing 93 mapping default 69 gray levels 68 match count 106 119 137 ID 108 match sets existing 57 export 58 import 58 merge 55 matches add 109 create hierarchy 54 delete 109 display 107 display hierarchy 102 displayed 125 edit 109 gelstool 105 hierarchy 49 101 Melanie User Manual Edition AC multiple 109 reference 101 report 110 review 109 shortcuts 173 statisticstable 110 vectors 107 matching 101 automatic 105 reference 55 max 119 137 intensity 130 Slope 130 volume 130 mean 114 absolute deviation 115 squared deviation 115 measure 66 129 measures overapping 131 median 114 menu bar 15 select 153 merge match set 55 spots 93 midrange 114 min area 84 minimize 29 modify labels 159 move altematives 63 apply to allgels 63 double click 63 projects 59 tool 63 MSD 115 multiple matches 109 MW 96 N navigator 23 contextualmenus 24 icons 24 next in selection 26 next previous 74 node locked license 8 non DIGE 7 83 detectspots 83 spot detection parameters 84 spot quantification 85 normalization 120 121 normalized 12
61. 9 4 Projects 4 6 Save projects 4 5 4 Move Youcanreamange yourimages match setsand classesto change their position in the list or move them into another match set or class J ust drag your image match set orclassto the desired position It is generally inserted after the item you drop it on Whenever there isa possibility to insert it inside orafteran entity you are asked to specify 4 6 Save projects 60 4 6 1 Project folder When you create a project you are asked to specifya name anda location on your hard disk Allthe data related to the project images spots matches annotations spot sets are saved in this location ina folder with the name of the project All users that have accessto this folderare able to open view and work with the project The project folder contains the following files and folders e Projectname ph This project file isthe link between all the other data filesin the project folder If you want to add an existing project to your workspace you must search for and open e pn file s Raw Images This folder contains the raw image filesin mel format e Mather This folder contains the gel gda and match mda data in subfolders The gda files are used to store the spot information The mda files contain the match information 46 2 Save The software automatically saves your work when you close a sheet or exit the software You can also save your work by choosing File gt Save This saves
62. Clickonthe Selecttool Double click on the markerspot orif such a spotisnot present on the pixel that isin the middle of the marker Select the Commentcategory Enter IRL asthe label text 8 If an annotation attached to a pixel isnot in the center of the reference marker you can move it to the appropriate position Do this by clicking on its basis cross and holding down the left mouse button while dragging the annotation to its new position 9 Move yourgelto see the second right reference marker 10 Repeat the procedure but enter the label text IR2 this time Melanie User Manual Edition AC Data integration 10 11 Once the two markers have been annotated select spots to pick Figure 10 2 12 Choose File gt Export gt Spots to Picker gt GE Healthcare Ettan 13 Foreach gel you will be asked to save a pick list in text or XML format only the text file can be read by the Ettan Spot Picker File Edit View Select Reports Tools Help Goa wwo bi PROB OPPER Close H Save Ctrl 5 Import Be ee mp on FE Image to File a Fage 5etup Print EH Sheet to Clipboard LOD Sheet to File Spots to Picker Bruker Proteineer SP GE Healthcare Ettan Exit Genetix GelPin Genomic Solutions ProPic Gels 1 Spots 14 Matches 0 Annotations 14 Figure 10 2 Reference markers IR1 and IR2 IR1 isattached to a pixel cross basis IR2 isattached to a spot Both optionscan be u
63. Dispersion Gap Ratio or Nomnalized values foreach class orfold change versusa selected reference class X versus Class X depending on the selection made in the drop down list see below By default the Center values are displayed e The Max value that is the highest from all these class values The Match Count which isthe number of classes in which the spot Is present and matched e The results from the statistic al tests the ANOVA probability P the Wilcoxon U statistic and the Kolmogorov D statistic e The Fold change between the class with the lowest Center value and the class with the highest Center value e The columns conesponding to spot sets GA 44 VY Match ID Max MatchC ATI AT ATi BT2 Anova Wilcoxon ATL Kolmog Indu 46 0 1577 4 0 086471 0 156624 0 0685141 0 1577 1 25988e 4 Id E 47 0 282926 4 0 170495 0 0809451 0 237309 0 282926 2 08238e 4 LE g 0 d i 2 982635 4 188822 0 57478 2 56263 2 060975 6 14695e 5 0 1 ke 2 0 666667 0 w 49 0 320064 3 0 320064 0 262944 0 1854 10 0115026 amp Figure 8 17 Class Analysis Table In addition to the standard functionalities for saving printing copying content to the clipboard and navigating in the report the following tools in the Class Analysis Table are particularly useful Y Select by value Select itemsin the table based ona numericalsearch cntenon Choose the measure i e column to be used for refinement and set the lower a
64. Melanie User Manual Edition AC Annotations 9 indicated in the labels must then contain the name of the subfolder e g AA composition AA P10413 xls Altematively you can link labels with files located anywhere on your system You should then include the complete file path when referencing the file Forexample you can link a protein spot to an Excel file containing the amino acid composition of a protein Figure 9 5 The label should then consist of the string file followed by the file name with its extension 9 5 3 Textlink In some cases you might want to associate a long text comment with a specific protein spot but without overnoading the display The solution isto create a text link ratherthan a very long annotation label Double clicking on the linked label issufficient to disolay a window containing the entire text Figure 9 5 Textlinksare particularly usefulforattaching bibliographic referencesto a spot forinstance oranyothercommentsuchasthe one in Figure 9 5 Please note that the string text must first be inserted followed by the text you would like to associate with the spot To connect general information about the gel other tools are better adapted Commentscan be attached to projects match sets and classesin the workspace Additionally you can specify gel descriptions Melanie UserManual Edition AC 151 9 Annotations 9 6 Selectannotationsand labels ExPASy Proteomics Server Mozil
65. See below for more details on XML Graphical reportscan be saved in PNG TIFF or BMP formats 10 3 2 XML XML standsforeXtensible Markup Language and wascreated asa cross platfom software and hardware independent tool to structure store and exchange information It allows the creation of customized tags enabling the definition transmission validation and interpretation of data between applications and organizations XML files can be viewed in the latest versions of Web browsers such as Intemet Explorer Netscape and Mozilla Firefox However as AM was designed to describe data and not to display data it doesnot look like a Web page An XMLdocument contains color coded rootand child elements A plus or minus sign to the left of the elements can be clicked to expand orcollapse the element structure If you wantto view the raw XML source you must select View gt Source from the browser menu XMLdoesnotuse predefined tags asisthe case forHIML Therefore the browser doesnot understand the meaning of the tagsand doesnot know how to display the XMLdocument Therefore XSL eXtensible Stylesheet Language stylesheets must be used in addition to the XML document to transform the XML into the sort of document that is recognized by the browser This isthe case when tabular reports or the history are printed from Melanie The software uses the XSL stylesheets located in the Template folder of the Melanie installation directory to print
66. Special character gt 8 R Table 9 2 Orderof precedence from high to low for regular expressions 9 6 3 Reports You can select annotations by selecting their comesponding lines in the Annotation Table available by choosing Reports gt Annotation Table 9 7 Display annotations and labels 156 You can change the way annotations and labels are displayed 9 7 1 Annotation flag position Sometimes you may wantto move an annotation flag because you are prepaning an illustration or just want to see what lies undemeath To interactively change an annotation s flag position click on one of the labelsand drag the flag to the desired position Figure 9 7 ODO2_ ECOLILTD ECOLI a b Figure 9 7 a Default and b modified annotation flag position ODOZ ECOLILTD ECOLI 9 7 2 Visibility of annotations and labels You can quickly coverentire gel imageswith a considerable number of annotations and labels which are not always necessary at any given moment in the analysis Therefore you can hide all annotationsor Melanie User Manual Edition AC Annotations 9 certain label categories in selected gels These options are available from the View gt Annotations menu e Visible categories Sets the visibility state of the various categones on the selected gels To hide a category make sure its box is unchecked On the other hand select an empty check box to show the corresponding category
67. There is another important reason why it is useful to adopt hierarchical population matching instead of matching all images against a unique arbitrary reference image Only spots matched with a spot in another gelare included in Geland ClassAnalysis Tables all soots are of course presented in the Spot Table The likelihood of a spot being matched is much higher when matching with a gelfrom the same biological population Spots that are represented in a single population submatch set are therefore included in the analysis even if they are not in the global match reference Thisconsiderably reducesthe number of spots missed in the analysis CAUTION DIGE gelsare inherent match sets A DIGE gel the entity with its composing images istreated asany other non DIGE gel when setting up a match hierarchy Once created in the Workspace you can display a complete match hierarchy e g ABin Figure 4 2 ina sheetand cany out spot detection on allthe included gels Afterdefining one ortwo landmarks the entire experiment is matched ina matter of seconds and matchesare automatically propagated at each level of the match hierarchy 4 1 2 Classes In the Classesfolder you state yourbiological questions Thismeansthat you define a classforeach set of gelsthat you want to compare with other such entities Your goal by companing classes and therefore the Melanie UserManual Edition AC 51 4 Projects 4 2 Create a project gels within those cla
68. ab delimited text format txt asa Microsoft Exc el Workbook xls orin XMLformat xml Graphicscan be saved in PNG HEET or BMP formats GA Print Normal pnnt options are available when printing graphical reports When printing tabular reports the table is first displayed in your default Web browser This is because the XSL stylesheet located in the Template Reports folder of the Melanie installation directory is used to transform the XMLreport into an attractive table You can then use the print option in your browser to geta printout Ba Copy to clipboard Export your data directly into another application Firs select the desired linesin a table orthe desired graphicsin a window using the Shift or Ctrl keys Then copy the selection to the clipboard by choosing the Copy to Clipboard icon Paste directly into the prefered software tf Previous selection Click the Previous Selection icon to skip to the first selected item encountered when scrolling towards the top of your table When only one row isselected this selects the previous row in the table Melanie User Manual Edition AC Graphicaluserinteface 2 Ka Next selection Click the Next Selection icon to skip to the first selected item encountered when scrolling towardsthe bottom of yourtable When only one row isselected this selects the next row in the table Settings Click this icon to change the display settings for your report In most cases thismeanssetting the
69. abedeferhijk 174 175 0 2 0 1 o EN Fri H H 0 page 20 217 03 0 2 l abedeferhijkl 0 3 0 2 0 1 0 abkedefghijkl abedefshijEkl abedefgchijkl 17 178 179 Figure 8 18 C la ss Ana lysis Histog ra ms with C enter values displayed Class Analysis Histograms Mean 10034 and M 5 D Gata a pe M 1 0 page 52 163 Figure 8 19 Class Analysis Histograms with Ratio displayed 140 Melanie User Manual Edition AC Data analysis 8 The histogramscan be selected to highlight the comesponding spotson the gelsand in any open reports The following tools in the Class Analysis Histograms are very useful y Slider To see the matchesdisplayed on additional pages use the sliderin the toolbar When a match isselected on an image orin anotherreport it is automatically highlighted in a histograms window CH Statistics Set the statistics to be used forcalculating the Centerand Dispersion value of each class and consequently the Gap Ratio and No malized values These settingsare common to the ClassAnalysis Table and Class Analysis Histograms Settings Click the Settings icon to activate one of the following options e Sorted Values to sort the classesaccording to their central value and sort the spot values inside each class Figure 8 20 Thiscan simplify the search fornon overapping intervals 0 7 0 7 0 6 0 6 0 5 0 5 0 4 0 4 0 3 0 3 abcdefzhijki fedhgibackij
70. able Displayed value The displayed statistical descnptororovenapping measure canbe selected from the drop down list in the toolbar You can display the Center centraltendency Dispersion Gap Ratio or No malized values forthe different classes aswell asthe ratio versusa selected reference Class versus Class X CAUTION The Gap Ratio and Nomalized options are not available for DIG E experiments when Vol Ratio isselected asthe quantification measure 8 4 6 Class analysis histograms You can visually investigate the statistical and overlapping descnptors of populations by displaying Class Analysis Histograms J ust choose Reports gt Analyse Classes gt Histograms When the Center central tendency value isselected fordisplay in the drop down list the Class Analysis Histograms display all the individual spot values in each match separated foreach class by vertical gray lines Figure 8 18 The classesare charactenzed by their central tendency blue horizontal line and dispersion interval bounded by the outer red lines The Match IDs appear below each histogram When displaying the Dispersion Gap Ratio or No malized values Figure 8 19 each class is represented by a single value red bar Melanie User Manual Edition AC 139 8 Data analysis 8 4 Analyze classes Class Analysis Histograms Mean 100 and M 5 D GB 24 Berle Sorted Values w Adaptive Gradations show Labels 0 06 0 04 abedefghijkl
71. age Pool 3 6 Descnbe images 48 Melanie User Manual Edition AC Projects 4 4 Projects 4 1 Introduction A project includes all gels spots matches annotations spot sets and other information produced and analyzed during the course of a specific gel study You can create oradd many projects in the Workspace A project in the Workspace can include one ormore match hierarchies eg AB in Figure 4 1 each of which containsa Match folderand a Classes folder The Match folder describes how gels or populations of gels called match sets should be matched together The Classesfolder is where the biological question is stated through the definintion of classes of gelsto be compared Workspace KC E 7 Image Pool Sige UM Project Gat AB E el Classes a ATI a AT HE BTI E GP eH T2 Spe Test 080411 Figure 4 1 Project structure The nextsectionsfurtherexplain the principlesof match hierarchiesand classes and how they can be used 4 1 1 Match hierarchies Allimagesin an experiment are not equally easy to compare even when the gelsare run ina highly controlled way Typically gels belonging to the same biological group are easierto match i e corresponding spots are easier to find than images from different biological populations Melanie User Manual Edition AC 49 4 Projects 4 1 50 Introduction It istherefore recommended to use hierarchical match structuresto create more efficient match
72. agesare opened through the Image Pool To further analyze images detect match cany out statistical analysis etc they must be transferred to a project You can workon many projectsin the Workspace each of which will contain one ormore root structures with Match and Classes subfolders Curent sheet Any item in bold in the Navigator comespondsto items displayed in the Current sheet Contextual menus A contextual menu containing relevant options appears when you right click an item in the Navigator Navigator icons Each foldertype in the Navigatorhasa specific icon Image Pool Project Match Class In addition imagesand match set folders have icons that indicate their status You can distinguish DIGE gels from non DIGE gels know which gelshave been detected and matched and recgonize gels used asreferences for matching Undetected image reference image Detected image reference image Matched gel reference gel Table 2 1 The Navigatoricons inform you about the type and status of the im age Reportsare highly practicalfororganizing and descnbing yourgeldata They make it much easierto processall of the information Melanie User Manual Edition AC Graphicaluserinteface 2 Reportsare notnecessariy in table format Graphicalrepresentationsof data such ashistogra ms scatter plotsand 3D viewsare treated as reports aswell All reports are dockable windows Report Description 3D View A three dimensiona vi
73. al when analyzing the tutonal images but it doesnot allow spot detection and matching on other gels To analyze yourown images you must purchase and install an electronic license orrequesta tral license The availability of the license file is venfied each time the software is launched There are two types of licenses e Node locked Machine license o be used ona single computer It is practical when only a few computers are used for working with Melanie A node locked license file must be placed on the computer running Melanie s Foating Concurrent license To be used by all computers networked to a a license server It is useful when many users but not all atthe same time need accessto the software The number of computers that can simultaneously work with Melanie depends on the license and isadministered via the license server A floating license file must be placed on the computer running the license server This server can either be installed on a computer running Melanie oron any other network computer recommended install ona network computer that is continually running Read onto leam how to obtain and place a license file 1 4 1 Find the physical address of the computer In orderforGeneBio to create a license file the physical addressof your computer isneeded Thisaddressidentifiesthe computerand isused by Melanie User Manual Edition AC Getting started 1 the licensing system If purchasing a node locked license
74. alysis 7 8 Match reports 7 8 1 Match statistics table Choose Reports gt Analyze Gels gt Match Statistics Table to display the numberand percent of matches found foreach of the images By default the numbersare calculated based on All MatchSets in the 110 Melanie User Manual Edition AC Matches 7 hierarchy But you can selecta particularmatch setby clicking the Choose MatchSeticon in the toolbarofthe Match Statistics Table Figure 7 7 Match Statistics Table All MatchSets G A BI Geane noth Count Percents REN l A_T1 Geli 755 a8 Please choose one matchSet All MatchS ets AB A B A ATI ATZ B ETI BT2 ATI 4_T1_Gell 4_1T1_GelS 4_T1_Gel2 ATZ 4_T 2_Gell 4_T 2_Gels 4_T2_Gel BT1 B6_T1_Gell B_T1_Gel3 B_T1_Gel ET2 B T2 Gell B T2 Gela B T2 Gelz Co Figure 7 7 Match Statistics Table Melanie UserManual Edition AC 111 7 Matches 7 8 Match reports 112 Melanie UserManual Edition AC Data analysis 8 8 Data analysis 8 1 Introduction To study the vanations in protein expression among a senesof gels the gels should be matched together be part of the same match hierarchy Data analysiscan be camed out at two different levels e Analyze Gels Study protein expression changeswithin a set of gels without taking populations into consideration This type of analysis can be camed out on both MatchSet and Classes sheets The analytical methods used include scatter plots des
75. analysis 8 Gel Analysis Histograms Mean 100 and M 5 D SREL Je sg 3 RK 1 1 5 1 5 1 1 5 0 5 05 0 0 0 abede abede 180 181 abede abede 338 483 Caption Name A Ti Gell A_T1_Gel2 A_T1_Gel3 A_T2_Gell A_T2_Gel2 A_T2_Gel3 Figure 8 12 Gel Analysis Histograms for the ten matches ranked highest for their contribution to the second axisin the factor analysis example Matches 48 123 180 181 and 248 are charactenstic of population Tl matches 237 and 338 are characteristic of population T2 8 3 5 DIGE histogram A DIGE Histogram can be displayed when detected DIGE gels are selected It shows foreach non reference image data associated with detected spots in the image plotted against Log Volume Ratio on the X a xis Figure 8 13 It hastwo different Y axes e The left Y axis displays the spot frequency The blue curve represents the frequency distnbution of the log volume ratios e The nght Y axis represents the Measure parameter see below selected from the Measure tool in the DIGE Histogram window A plotted single data point on the histogram represents an individual protein spot Melanie UserManual Edition AC 129 8 Data analysis 8 3 Analyze gels 130 The name of the DIGE reference forthe current image used forthe calculation of the Log Volume Ratio appears at the lowernght comer of the DIGE Histogram window The DIGE Histogram isdynamic You can click on the data points representing sp
76. and 0 9 is any numeral from 0 Matches any characterexcept P bd matches Pa Pc those listed between the Pe but not Pb or Pd brackets Matchesthe preceding element P0 1 matches P1 and zero orone times POL Matchesthe preceding element PO 1 matches PO1 one or more times P001 POOO1 Matchesthe preceding element PO 1matches P1 POL zero ormore times P001 POOO1 n Matchesthe preceding element POBH matches PO001 exactly n times but not PO1 or POO1 n Matchesthe preceding element POQ l matches POO1 at least n times P0001 but not P0O1 Matchesthe preceding element PO L 3 1 matches PO1 atleast n times but not more POO1 and P0001 but than m times The charactersbetween parentheses fom a subexpression 154 not P1 or POOOO1 P 24 matches P24 P2424 P242424 Melanie UserManual Edition AC Annotations 9 Matchesthe expression before P ab cd 1 orafterthe vertical line Mostly matchesPabland used within a subexpression Pcdl A circumflex outside a bracket ec matchesiecoli expression anchorsthe element ecoli_eftu but not it starts with to the beginning of eftu_ecoli a string such an element can match only a sequence starting at the first character of a string A dollarsign outside a bracket ecoli expression anchorsthe element matches ecoli it terminateswith to the end ofa eftu_ecoli but not string such an elementcan ecoli eftu matc
77. atch Spotstoolbar File Edit View Select Reports Tools Help MJP R 0 2 B CO 0 G PAR ATI AT2 BT1 BT2 Add Match Cti Shift 5 zk a GR T Gels 12 Spots 22 Matches 4 Annotations 0 Figure 6 6 Composite sots can be defined through multiple matches 6 6 3 Propagate spots You can propagate allorselected spots from one image to the other matched images Figure 6 7 Thisallowsyou to quantify identicalareas on all gels e Format hed spots the spot in the destination image is replaced with the shape of the spot in the source image e Fornon matched spots the spot from the source image iscopied to the equivalent location in the destination image This position is extrapolated from the sumounding match vectors Melanie UserManual Edition AC 95 6 Spots 6 7 MW and plcalibration Matchesare automatically created between the original and the propagated spots File Edit View Select Reports Tools Help HP CC eo Oooh P ARF dap A ATI AT2 BT1 BT2 zh m a e a Gels 12 Spots 12 Matches 1 Annotations 0 Figure 6 7 All spots from gel A_T1 Gell have been propagated to the other images To propagate spots from one matched image to another 1 2 3 4 Select spots on one image Choose Edit gt Spots gt Propagate Select one or more images you want to copy the spotsto The new spots are added to the selected images 6 7 Mu and pl calibration If you have a gel with pl MW s
78. ate labels and label categones Click on the Add Label icon in the Annotation Table to create new labels forselected spots During this process you can create a new label category which will be inserted asan extra column in the table Add or modify labels You can directly add new labelsto the appropnate cells of the Annotation Table or edit existing ones Double click in a cell to start typing your label ormodifying an existing label When finished a single click in any cell quits the editing mode Please note that categonesusing the Data Type Set are displayed inthe form of check boxesin the table These boxes show whether the corresponding item belongsto the category checked box ornot 9 84 fe Import annotations You can import annotations from a file into open gels Choose Edit gt Annotations gt Importto import annotations from an Annotation Report ora tab delimited text file containing the required columns SpotlD X Y and a column foreach category to be imported If the Spot ID isnot known use 1 in thisfield orremove thiscolumn and the software will position the label in the coresponding Xand Y positions of the gel If X and Y positions are not known use 1 in these fields and the software will position the label on the spot with the comesponding Spot ID 9 9 Annotation table The Annotation Table Figure 9 9 availble by choosing Reports gt Annotation Table providesspecific information about annotations and con
79. azknnnanzzzakkAEESSEEA 63 5 3 View Signal intensity 1sssssssrnzsszzasznananzzaakkkAaSSSEE 67 5 4 Visually compare IMAGES Luu 2ssssss nssszsaznnzanzzzazknnznn2 76 3 GNA INES si EG a 79 5 6 EEGEN 80 5 7 Save exportand print IMAGES 1 111ssssss xr sssszssxrrnus2 81 Spots 6 1 Intoduc on eege 83 6 2 Detectspots in non DIGE Gels ssssssssnnnnnnnnnnnnnnnnnnnnnnnnnnn 83 6 3 Co detectspots in DIGE gelS ssssssssnnnnnnnnnnnnnnnnnnnnnnnnnnnn 87 GA LE 90 65 Display PO E 92 0 6 ER e EN 93 6 7 Mu and pl Calibration 1 sssssssnssssssnsszsasznaszzasnnz 96 6 8 SPOtreponsS eege 98 Matches ZE IKGOJIK EE 101 7 2 Display a match hierarchy 2ssssavssznnszzaaznznnnnz 102 7 3 Define landmarks seesrreEERESSKKEEEEEEERRKEEEEEEEKEKKEEEEEERRKKEE 103 7 4 Automatic matching ss sssssasssnnnaszzaaznananazzakkznE00Z 105 75 SEIECEMAKNES wisi cd Giwi W 106 7 6 Display matches 1 1112sssss nnssssssznnnaszzaskkananzazakkKKE0BZ 107 ZZ Edt MAWES oiriin 109 78 MATE el EE 110 Data analysis Sik IRGOJUC DO eene Eege EG araea 113 8 2 General E e E 113 8 3 Analyze E 116 8 4 Analyze classes sssssssnnnsnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn 131 Annotations 9 1 doten 143 9 2 Create annotations and labels 2 1112ssss x11ss3s11 144 9 3 Create label categoreS 11 1112ssssssxsnnnzzzzszznnanzzaa 145 9 4 Connectto protein databases 1 11
80. by the timmed mean ortimmed midrange where a predefined numberof outliersare removed from the sample The trimmed measures are more robust than the mean or midrange but are more sensitive than the median The percentage slider in the Statistics window allows you to remove outliers and obtain the different central tendencies A 100 value Melanie User Manual Edition AC Data analysis 8 meansthatallthe spot values available ina match are used to calculate the statistics no outliersare suppressed With a value of 80 forexample 10 of the minimum valuesand 10 ofthe maximum valuesare discarded from the sample and the timmed measure is calculated with the remaining values e Select Mean and the arithmetic mean iscalculated that is the sum of all the sample values divided by the sample size e Select Midrange and the midpoint of the sample value is calculated that is the middle location between the two sample extremes e Obtain a Thmmed Mean or Timmed Midrange by selecting Mean orMidrange and discarding the desired percentage of outliers with the percentage slider e Obtain the Median by selecting Mean and discarding 50 of the values at each of the extremities that is select 0 in the percentage slider e Select Reference and the value from the spot in a specified reference gel istaken asthe central tendency This option is only available for Gel Analysis Reports Dispersion The dispersion measures the vanability o
81. ceded by a sign are not yet matched Match vectors are displayed in blue The vector pattem is proof of consistency If there isa mismatch the vectorhasa different length and ororientation If the matching did not work propeny you can rematch a particular match level afterhaving added additional landmarks to the appropnate images 7 5 Selectmatches 106 7 5 1 Select When you select a spot with the Selecttool the matched spots are automatically selected Altematives There are other ways to select matches e Choose an option in the Select gt Matches menu All Inverse Selection Multiple Matches e Select matches in reports 7 5 2 Match count The software allowsyou to select spots present Melanie UserManual Edition AC Matches 7 e Ina certain number of gels the Match Countin a Gel Analysis Table givesthe numberof gelsin which the spot ispresent detected and matched e Ina certain numberof classes The Match Countin a Class Analysis Table givesthe number of classesin which the spot is present detected and matched Clic kthe Select By Value ic on in these tablesto refine yourselection For example select only spotsthat are present in at least X out of Y gels or X out of Y classes 7 6 Display matches You can visualize matches by selecting them The software also provides the following tools to display matches 7 6 1 np Show vectors Right after matching the software automatically displaysthe
82. chSet Cl Cols Rows MaxValue MaxGray Modified Comment Staining SeEtungs 1412 28530 28530 03 05 2008 17 02 31 Silver 1409 1412 25282 25282 03 05 2008 17 02 31 Silver 1409 1412 31424 31424 03 05 2008 17 02 31 Silver 1409 1412 96315 03 05 2008 17 02 31 Silver Figure 5 13 Gel Table You can customize the Gel Table to display only the columnsof interest Four predefined report templates are already available from the Load icon in the Settings of the Gel Table toolbar e Properties Shows varous properties of the gel images File Name image height and width in pixels maximum gray levels before and after calibration Modification Date aswell asthe MatchSet and Classto which it belongs e Files Shows information on the image files File Name ID Path Size Creation and Modification Dates aswell asthe MatchSet and Classto which they belong e Descnptions Showsthe user defined gel descriptions e Calibration Provides calibration related information File Name ID and Path aswellasCalibration Formula Name Creatorand Date Click the Add Description icon in the table toolbarto add oredit the Comment Staining or other user defined gel description fields for selected gels 5 7 Save exportand pnntimages 5 7 1 Save The software automatically saves yourimagesand all associated data as part of the project when you close a sheet or exit the software You can also save your work by choosing File gt Save 5 7 2 Export Rath
83. charactersis taken literally and the program selects all labels containing this string By activating the Regular Expression option regular expressions can be used in the search field see below for details Forexample to selectalllabels containing the string POO you can eithertype the expression P00 and choose the Regular Expression field in the window ortype POO and deselect the Regular Expression field e By Category This feature enables the selection of all labels belonging to one or several categories Use the Shift and Ctrl keys to pick several category namesata time s All This highlights all annotations in the selected gels Melanie UserManual Edition AC 153 9 Annotations 9 6 Select annotationsand labels e Common Labels This option allows the retneval of all sets of identical labels within a geloramong a seres of gels forall the categones chosen in the window Regular expressions Regularexpressions provide a mechanism to select specific stingsfrom a set of character strings Regular expressions use symbols and syntax elements to describe a generalized pattem Melanie invokes the standard Extended Regular Expressionsto search pattemsin labels the essentials of which are summanzed in Table 9 1 Smax Dam rom Matchesany one character e oli matches eaoli eboli ecoli Matchesany character listed Pla d matches Pa Pb between the brackets a z Pc Pd indicates the range of characters betweenA and Z
84. ches 237 and 338 on the otherhand are charactenstic of the T2 population with higher spot valuesin the T2 gels In ourexample the second axis appears very important for separating the gels into two classes It isrelated to the ratio between the mean spot valuesin each population of gels The matches in the upper part of the graph have ratiosthat favorthe 2 population while those below the honzontal axis have ratios that favor the T1 gels Comments on factor analysis Factor analysis is used to examine the protein expression pattem within each match The quality of the output dependson the quality of spot matching Hence it may be useful to exclude spots that are not well matched acrossall gels using Match Countin the Gel Analysis Table Nevertheless in cases where a majority of spots were properly matched the inclusion of all matches in the factor analysis can yield good results with no preliminary match filtering necessary This statistical method based on data vanation and their standard deviations highlights the natural formation of populations among the gelsand allows identification of matches i e matched spots that are characteristic of these classes However one should be very critical when analyzing factor analysis plots since the resultscan be greatly influenced by outliers bad matches and so on Factor analysis can provide valuable indications in some cases but not in others 128 Melanie User Manual Edition AC Data
85. come froma calibrated image file mel such asthe calibrated step tablet image obtained in the section above 4 Nextload the definition ofthe controlstep tablet ThisC ontrol Tablet File must be specifically adapted to this new step tablet That is it should have been edited with a tool such as Windows Notepad so that it contains the appropnate OD values height width and number of steps cornesponding to the control step tablet Melanie User Manual Edition AC Image Pool 3 5 The red calibration step overlay appearson the image of the control step tablet and the Control Calibration window isdisplayed 6 Selectthe Spotor Annotation tool in the Melanie toolbarand adjust the position of the steps 7 You should now venfy that the calibration curve is passing through the data points comectly and with minimum dispersion intervals If this isnot the case try to find out why yourcunent calibration does not seem to work propery 3 6 Descnbe images 3 6 1 Gel descnptions A gel description isa kind of label forthe gel image You can enter information about your gel images to be used for later reference by yourself orany colleagues This information can include sample type gel running protocol date of the experiment operatorname pH range SDSgelpercentage oranything else youcanthinkofthatwould be useful to know aboutthe images Allofthisdata isentered asGel Descriptions When opening images in the software you are asked to specify the
86. criptive statistics histograms and factor analysis e Analyze Classes Find significant protein expression changes between classes of gels For this type of analysis images must be placed in classesand opened in a Classes sheet The analytical methods used include descriptive statistics per class histograms overlapping measures and statistical tests 8 2 General settings The following conceptsand settings are used in the different analytical methods described later in this chapter 8 2 1 Quantification value The quantification value found in vanoustables graphsand plotsisa software option Choose Tools gt Options and go to the Quantification tab to set e Value To be used forthe analysis of conventional 2 D gels You can choose between Intensity Area Vol or Vol The default value is Vol e DIGE Value To be used forthe analysis of DIGE gels You can choose between Intensity Area Vol Vol or Vol Ratio The default value is Vol Ratio Note Although Vol Ratio are displayed in the tables graphsand plots the intemal calculation of the statistical values such ascentral tendency dispersion and Anova isbased on the Log Vol Ratio 8 2 2 GH Statistics Central tendency and dispersion are the most frequently used descriptive statistics They are calculated in Gel orClass Analysis Tables and Histograms Melanie UserManual Edition AC 113 8 Data analysis 8 2 General settings 114 These statistics a
87. ctionality is further described In Section 9 4 Melanie User Manual Edition AC Annotations 9 When you subsequently double clickon a labelofsuch a category the softwa re opensyourdefault Intemet browserand launchesan HTTP query that takesthe form of a Web page address Please note that you can define a different query engine foreach label category and therefore you can link one protein spot to different database entnes Display properties User defined categones use a gray background colorby default You can change the default color by clicking on the Colorbox The new background coloris used forall labels of that specific category It isoften not desirable to have all annotation categones displayed because thiscan clutter the display You can therefore choose to hide the category by default by clicking the By default hidden box In this case you mus choose View gt Annotations gt Visible Categonesto make the category visible by checking its box 9 4 Connectt protein databases 9 4 1 Introduction Melanie can link spots on gel imagesto protein data in 2 DE or other databases Such databases contain information on proteins identified on 2 DE images such aspl and MW values bibliographical references to protein related literature information on protein functions etc If your computer has Intemet access you can remotely query and retneve protein data related to spots on your gels CAUTION The Melanie software providesaccessto
88. d spot isselected and vice versa Melanie UserManual Edition AC 143 9 Annotations 9 2 Create annotationsand labels 9 2 Create annotations and labels 9 2 1 Create annotations The creation of annotations essentia lly consists of taking three steps 1 Select the spots or pixel on which you would like to create an annotation Choose the Selecttooland double clickon a spotora pixel he Select several spots make sure to select only the imageson which you want to create new annotations and choose Edit gt Annotations gt Add 2 Choose one of the existing categonesorcreate a new category The creation of new categones is described in Section 9 3 3 Enterthe desired label content 9 2 2 Add labels to existing annotations Labelscan be added to an existing annotation The procedure isalmost identical to that described above with some slight differences in the first step 1 Select the annotation to which you would like to add a label Choose the Selecttool and double clickatthe basisofthe annotation to which your label should be added Select several annotations make sure to select only the imageson which you want to create new labels and choose Edit gt Annotations gt Labels gt Add 2 Choose one ofthe existing categoresorcreate a new category The creation of new categoriesisdescrbed in Section 9 3 3 Enterthe desired label content Note One annotation may have many labels but itcan only contain
89. d to the borders It is important to identify such problems as they will lead to incorect spot quantitation The Profile function activated ordeactivated by choosing View gt Global gt Show Profile can help in such cases Red curvesrepresent the intensity vanations of the gel in the vertical and honzontal directions at the position of the mouse cursor Figure 5 8 The Profile can clanfy the intensity changesin a geland assist in making editing decisions without the need to open an additional window asisthe case with the 3D View tool Melanie User Manual Edition AC Gels 5 Figure 5 8 Profile feature Red curvesrepresentthe intensity variationsofthe gel in the vertical right and horizontal bottom directionsatthe position of the mouse cursor Green lines indicate the exact position of the cursor whereas the numbers indicate the minimum and maximum gray levels in a specific profile view 5 3 4 Cursor infomation At any given time the Cursor Information window can be used to display information on pixels such asthe pixel intensity and the Xand Y coordinates expressed in pixels in pl and MW units if available and in cm orinches Note that if spots are detected the Cursor Information window also displays spot information The Cursorinformation window isavailabe by choosing View gt Global gt Cursor Information or by clicking the corresponding icon in the Display toolbar J ust place the cursoroverthe pixel for which y
90. data isstrictly smallerthan 0 064 and so on The same procedure can be followed forthe second sample classY in our example the dashed line in Figure 8 16 The Kolmogorov Smimov test statistic D isthen defined asthe maximum distance between the empincal distribution functions EDF forthe two samples In the example D is 0 63 0 83 0 20 If D isgreaterthan a particular decision limit critic al value found in a Kolmogorov Smimov table there isa statistic ally significant difference between the two samples However the test provides no insight asto what causes the difference Empincal Distibution Plot Fraction of data points to the left of Spot value z 0 02 0 04 0 06 0 08 0 1 0 12 0 14 Spot value z Figure 8 16 The Empirical Distribution Plot forthe spot values of match 613 Figure 8 15 in Classes X and Y The Kolmogorov Smimov statistic D corespondsto the maximum distance between the two empincal distnbution functions 8 4 5 Class analysis table The Class Analysis Table provides valuable data forfinding significant protein expression changes between populations of gels Using this data itispossible to differentiate one classfrom the othersbased on just a few matches spots Choose Reports gt Analyse Classes gt Table to display the ClassAnalysis Table Figure 8 17 Foreach match there isa description of Melanie UserManual Edition AC 137 8 Data analysis 8 4 Analyze classes e The Center centraltendency
91. displayed Figure 5 3 The units of the disolayed coordinatescan be changed by choosing Tools gt Options and looking under the Display tab 66 Melanie User Manual Edition AC Gels 5 Figure 5 3 Measured distance between pixelson an image In the example the horzontal and vertical distancesbetween the starting left and end right points are 2 51cmand 1 11 cm respectively 5 2 5 Bookmarks You can bookmark a view of yourimagesin a sheet This saves the currently visible areas Bookmarks allow you to retum to the same area on yourgelsata latertime orto show a similararea on the same images opened ina different sheet TE Create Choose View gt Sheet gt Bookmarks gt Create and entera name W Load To go backto previously visible areas choose View gt Sheet gt Bookmarks gt Load and select the name of the coresponding bookmark Selectthe name of anothersheet in the listto reproduce the visible areas of that sheet in the current sheet x Delete To remove a bookmark from the list choose View gt Sheet gt Bookmarks gt Delete 5 3 View signal intensity The digitized image iscomposed of individual pixels each of which is characterized by its X and Y coordinates and its signal intensity raw pixel value This section descnbes different approachesto explore the Signal intensity and adapt the way tt is visualized 5 3 1 Adjust contrast Sometimes the gray levels displayed by default are so low that
92. dmarks should be keptto a minimum There is no point in putting a landmark on each spot e Landmarks should be well distributed overthe whole images covering both the Xand Y directions To corect for local distortions it is recommended to define landmarks around the distorted regions rather than within those regions e Landmarks should only be defined on spotsthat clearly represent the same protein fom Protein vanants definitely should not be used aslandmarks e Landmarksshould be placed on small sharp spots of similararea ratherthan on large diffuse ones which may differ considerably in size because in the latter case the enorin the position is much more substantial e Landmarks should be added gradually so that you can monitor their individual influence on the alignment automatically updated aftereach landmark addition Immediately remove landmarks that decrease the alignment quality e When a spot is missing on a gel sometimes happensto border spots you should not puta landmark Le validate a landmark in a hypothetical spot position Missing landmarks are not an issue The Align Images feature isa purely visual tool It isnot used in and will not improve the matching process The only reason to align your imagesisto ease their visual companson possibly in combination with the Dual Colormode Altematively double click in an image with the Move tool selected to locally superimpose the different images Melanie Us
93. e Projecticon drop down list in the Workspace toolbar Figure 4 6 Again enter the Project Name Location and Comment Figure 4 5 Workspace JE B 7 Add Remove Backup Restore Display Figure 4 6 Project icon drop down menu in the workspace 4 2 3 Add files to project Use the Add Files to Projecticon in the Image toolbarto add gelsfrom the Image Poolto a project Figure 4 7 There isthe possibility to create Melanie UserManual Edition AC 53 4 Projects 4 3 Create match hierarchy a project in the Add Filesto Project window An existing ornew match set name must be entered aswell Add Files to Project Choose a destination project and matchs et Project MatchSets REES SE bk Figure 4 7 Add Filesto Projectwindow A projectcanbe created bychoosing lt New gt from the Project list 4 3 Create match hierarchy 54 4 3 1 Create a match set The easiest way to create a match hierarchy isby adding gelsfrom the Image Pool to a project The idea isto select only the gels that should be added to a particular match set e g gels from the same population orsame expenmental batch and then to create thismatch set Different options exist e Select gelsin the Image Pool folderand drag them onto a project name Entera name forthe new match set e Select gelsin the Image Pool sheet and drag them onto a project name in the Workspace Entera name forthe new match set e Select gelsin the Ima
94. e delete them before proceeding Multiple matches Melanie enablesthe creation of multiple matches Figure 7 6 In contrast to a single match where only a single spot isselected pergel a multiple match implies that one or more spots in one image canbe matched to several spots in other images All the spots from such a multiple match ona given gel image are considered asa single spot in the subsequent data analysis The calculated quantification values forthis composite spot reflect the size intensity and abundance of the combined spots Therefore this isan excellent solution to avoid spot editing Melanie UserManual Edition AC 109 7 Matches 7 8 Match reports File Edit View Select Reports Tools Help HARE OLA CTO O Pea 5 z GR T Gels 12 Spots 6 Matches i Annotations 0 3 9 cm 3 33 cm 4768 Figure 7 6 Multiple matches The selected spot was corectly matched throughout match set A and within BT2 But due to spot splitting in B TL Gell it wasnot matched within match sets BTL and B Ratherthan merging the two spots in BIL Gell the spots can both be included in the match and treated asa single entity in the spot quantification Review matc hes To review matching choose Select gt Matches gt All and specify the hierarchical level at which you want to select the matches Matched spots are highlighted in green The matching of any red spots should be scrutinized However thiscan also be done dunng data an
95. e Melanie II software Trendsin Biochemical Sciences 21 496 497 BA Statistical methods Armitage P and Beny G 1987 Statistical methods and medical research Oxford London Blackwell Scientific Publications Melanie UserManual Edition AC 175 B References B 3 Furtherreading 176 Kim JO and MuellerCW 1978 Introduction to factoranalysis What is and how to do it Newbury Park Sage Publications Tabachnick Band Fidell LS 1996 Using multiva fate statistic s 3rd edition New York HarperCollins College Publishers B 3 Further reading Appel RD Bairoch A Sanchezj C Vargas R GolazO Pasquali C and Hoc hstra sser DH 1996 Federated 2 DE database a simple meansof publishing 2 DE data Electrophoresis 17 540 546 Appel RD Sanchez C Bairoch A GolazO Ravier F PasqualiC Hughes GJ and Hochstrasser DF 1996 The SWISS 2DPAGE database of two dimensional polyacrylamide gel electrophoresis Nucleic Acids Research 22 3581 3582 Binz PA Mueller M Walther D Bienvenut WV GrasR Hoogland C Bouchet G Gasteiger E Fabbretti R Gay S Palagi P Wilkins MR Rouge V Tonella L Paesano S Rossellat G Ka mime A Bairoch A SanchezjC AppelRD and HochstrasserDF 1999 A molecularscannerto automate proteomic research and to display proteome images Analytical Chemistry 71 4981 4988 Binz PA Wilkins MR GasteigerE Bairoch A Appel RD and Hochstrasser DF 1999 Intemet resourcesfor protein identification and charactenzat
96. e by an appropnate imaging device During digitization a gel isresolved into a two dimensional matnx of squares or pixels Each pixel in the generated image file is charactenzed by its X and Y coordinates and its signal intensity or gray value To make any analysis meaningful it isimportant to start with good quality image files The following paragraphs give some helpful tips on what resolution depth and image formatsshould be used to obtain the best possible results 3 2 1 Resolution The scanning resolution ofa geliscntical asit influencesthe amount of visible detail in the image A low resolution comespondsto a large pixel size ora small number of pixels ordpi dots perinch When the image resolution is too low individual spots cannot be distinguished On the other hand when the scan resolution is too high the image file becomes very large This sows down the gel analysis significantly A resolution between 150 and 300 dpi is generally sufficient for gel analysis 3 2 2 Image depth The range of potential gray levels in an image vanes according to the image depth In the case of an 8 bit image one pixel has 256 2 possible gray values 0 to 255 Images scanned with a higher image depth contain more information A 16 bitimage Ee 65536 gray levels will reveal more subtleties We strongly recommend an image depth of at least 12 bits for gel analysis 16 bits is preferred Please make sure to use gray scale images for your
97. e land Davison M 2001 Validation and development of fluorescence two dimensional differential gel electrophoresis proteomicstechnology Proteomics 1 377 396 Melanie UserManual Edition AC 177 B References B 3 Furtherreading 178 Melanie UserManual Edition AC Index Symbols 70 v0l 85 bmp 81 gda 60 mda 60 png 81 Dn 60 tif 81 Numerics 3Dview 71 animate 74 auto rotate 73 colorpalette 74 disolay options 74 next previous 74 rotation 73 setreference 74 show 73 spotshape 74 stack 74 tools 73 translation 73 transparency settings 74 transparent mode 74 visibility 74 zoom contrast 73 A accesscode 10 adaptive gradations 121 141 add filesto project 53 61 labels 159 matches 109 spots 92 adjust contrast 67 region contrast 71 algonthm 85 matching 101 align images 76 all 153 analysis class 113 gel 113 analyze classes 24 113 131 gels 113 116 Melanie UserManual Edition AC 179 180 animate 74 annotations 143 copy 158 create 143 create categores 159 delete 158 display 156 flag position 156 hide 156 import 159 MW and ol 96 options 30 paste 158 report 156 159 select 152 shortcuts 173 show 156 table 24 tool 152 apply Calibration 43 move to allgels 63 region to allgels 66 zoom to allgels 64 area 85 130 auto rotate 73 automatic matching 105 autonumbering 146 B backup 61 BMP 81 bookmarks create 6 7 delete 67 load 67 Bruker Proteineer SP spot picker 163 by category 153
98. e of Bioinformatics GeneBio owns the worldwide exclusive distribution rights on this intellectual property No part of this User Manual may be reproduced or trans mitted in any fom orby any means electronic or mechanical including photocopy recording orany information storage or retrieval system without pemission in writing from the Swiss Institute of Bioinformatics or GeneBio 2004 2014 Swiss Institute of Bioinformatics All rights reserved Swiss Institute of Bioinformatics CMU 1 Rue Michel Servet CH 1211 Geneva Switzerland Melanie providesaccess to several databases on the Intemet It is the responsability of the userto acquire the database licenses if needed In particular the PROSITE and SWISS 2DPAGE databases are copyright and all commercial users of these databasesare required to purchase a database license from GeneBio Please contact GeneBio at license genebio com for more information Geneva Bioinformatics GeneBio SA c o Swiss Institute of Bioinformatics CMU 1 rue Michel Servet 1211 Geneva Switzerland Melanie usesthe DeCyderco detection algorithm 2008 General Electric Company All rights reserved Melanie uses the TIFF library 1988 1999 Sam Leffler and 1991 1999 Silicon Graphics Inc All rights reserved Melanie uses software developed by the Apache Software Foun dation http www apache org 1999 2007 The Apache Software Foundation All rights reserved Cy CyDye DeCyder Ettan Ima
99. e unitofthe coorinatescan be changed in Tools gt Optons under the Display tab Melanie UserManual Edition AC 19 2 Graphical user interface 2 5 Display zone 2 5 Display zone File Edit Gel imagesare opened in the Workspace and viewed in sheets and panes in the Display Zone Figure 2 3 Their layouts can be amanged according to your requirements Select Reports Tools Help WPzkRksojPAWOOGERAL soedsylo Rap DR Image Poo Stacked Gels 3 Spots3 Matches 1 Annotations 0 20 Figure 2 3 The Display Zone There are two sheets open here including the Image Pool The current sheet AB isin front and contains four panes ATI ATZ BIL and BIZ Only the pane AT1 isselected green tab By clicking the Layout icon forthe pane different options Stacked Tiled One Row One Column Free are displayed Similarly click the Layout icon in the upperrightcomerto re arrange a sheet 2 5 1 Sheets When no dockable windows Workspace reports are open sheets occupy the entire Display Zone Each sheet hasa tab with itsname and an icon representing its type E Image Pool This sheet contains images for viewing and basic processing rFhMatc hSet This sheet is opened by nght clicking on the name of a match set in the Workspace Spot detection and matching must be camed out on this type of sheet l3Class This sheet isopened by nght clicking on the name of one or
100. eet reference You can change the default color forthe match vectors by choosing Tools gt Options and going to the Display tab 7 6 2 Show ID Choose View gt Matches gt Show ID to display Match IDs for selected spots on selected gels Obviously the gels must be matched To hide the Match IDs again possibly in selected gels only choose View gt Matches gt Hide All ID 108 Melanie User Manual Edition AC Matches 7 7 7 Edrtmatches Sometimes it may be necessary to manually add ordelete matches afterthe automatic matching procedure Use the optionsin the Edit gt Matches menu orthe conesponding iconsin the Detect and Match Spots toolbar Note that matches were created ina hierarchical manner This means that soots may be propeny matched in one match set level but not in another To delete your matches it istherefore important to select the appropnate images panes R Delete match Select spotsthat were incorrectly matched make sure the appropriate match level gels panes isselected and clickthe Delete Matchicon to remove the spots from the match A Add match Select allspootsto be matched and clickthe Add Matchicon to add the spots to the match While selecting spots you may see that some spots were already matched When selecting one of them the others are automatically selected This makes it very easy to add matches Nevertheless you must make sure that the existing matchesare correct If this is not the cas
101. entry for Human Apo E Entnes from this database contain full protein names bibliographic references annotations such asprotein function orpathologicalvanations and the pland MW ofthe related spotson the 2 DE maps It also includes cross referencesto numerous other databases 9 4 4 Connextto an executable Analogousto an HTIP query where the content of a label istransmitted to a CGI scnptona server Melanie allows you to passon the content of a label asthe first parameterto any executable When you double click ona label that hasan executable defined in the Extemal Engine Melanie UserManual Edition AC 149 9 Annotations 9 5 Create specific links field of the label category the executable runs with the label content asa parameter To define an executable in the Extemal Engine field click on the Exe button and locate the exe file 9 5 Create specific links 150 Asseen above it is possible to link labelsto remote database entriesby defining an extemal search engine fora particular category By using specific keywords in the labels of any category you can also create links to Web pages files and text Figure 9 5 To create a specific link you should add an annotation to a geland include the following items in the label field s A shortdescnptorthat will be the visible part of the label e A keyword indicating the type of link http file text s A link content which contains the information necessary to estab
102. er than saving yourgel imagesin the Melanie file format you may wantto export them to a different file format TIFF BMP or PNG The gel imagesare exported as 8 bit flat rastenzed images without any Melanie UserManual Edition AC 81 5 Gels 5 7 Save export and print images 82 structure This means that gel components such as spots and annotations are saved exactly asthey appearon the screen butare no longerrecognizable asMelanie objectsand therefore become part of the image Consequently exported gel images should only be used for presentation puposesand not for further analysis with any software package Please note that you can adapt the size of the exported image by zooming in orout Ifa region isselected on the image you are given the choice to export the entire image orthe selected region only Altematively you can export imagesto the clipboard fordirect pasting into another software The procedure is very similar to exporting images to files However you can only export one image ata time to the clipboard Finally you can also export a view of the current sheet The following export options are available in the File gt Export menu e E Image to Clipboard Image to File Sheet to Clipboard Ze Sheet to File 5 7 3 Pnntimages and sheets Print options The software provides varous printing options in the File gt Pnnt menu Images Print selected imagesorimage areas if defined with the Region tool This o
103. erManual Edition AC 77 5 Gels 5 4 Visually compare images e Ifthe imagesare matched the image positions are synchronized based on the surounding matches e Ifthe imagesare not matched but have landmarks the image positions are synchronized based ona simple interpolation between the two nearest landmarks e fno matchesorlandmarks are present the image positions are synchronized based on the same image location X Y coordinates 5 4 3 Show dual color Choose View gt Sheet gt Show Dual Colorto display each of the images the sheet reference and the current image in one of two colors red and cyan Figure 5 10 Remember that you can select the sheet reference by choosing View gt Sheet gt Set Reference When the pixel colors of the two supenmposed gelsare added e Overlapping spotsappearasshadesof gray e Cyan spotsare present only in the curent gel e Red spotsare present only in the sheet reference e Halosof cyan orred around dark spots indicate that the protein is over orunder expressed respectively compared to the sheet reference The less color you see in the dual color mode the more similarthe gels Of course this is only true if the gels are corectly aligned see above and supenmposed CAUTION It isrecommended to perfom any operations related to gel alignment especially the definition of landmarks before entering the Dual Color mode to avoid a Sow down due to the recalculation of the overlaid images
104. escribe images 45 descrptions 45 desktop shortcut 11 DIGE 7 83 co detectspots 87 exclude spots 89 file naming convention 33 histogram 129 spot detection parameter 88 spot quantification 88 value 113 disable spots 92 dispersion 115 119 137 display annotations 156 calibration information 40 geldescrptions 45 labels 156 match hierarchy 102 matches 107 options 30 74 projects 59 properties 147 spots 92 zone 15 20 displayed items 125 value 138 141 dockable windows 20 floating 29 182 Melanie UserManual Edition AC minimize 29 pinned 29 tabbed groups 29 un pinned 29 double detection 87 dualcolor 78 duplicate labels 159 E EDF 136 edit 15 annotations 159 enabled 92 geldescrptions 45 grid lines 79 matches 109 region 66 spots 93 table cells 28 empincal distribution function 136 plot 136 enabled spots 92 ExPASy 150 export 81 162 croparea 37 match set 58 spot coordinate file 163 spot excision robot 163 extemalengine 147 extemal protein databases query 149 set 148 F factoranalysis 123 131 comments 128 interpret 126 table 120 125 factor projection plot 125 table 125 files 15 format 33 link 150 names 33 first time software launched 52 fitting table 43 flag position 156 FLEXIm license finder 11 flip 37 floating 29 license 8 free 20 21 Melanie UserManual Edition AC 183 184 rotate 37 furtherreading 176 G gap 131 137 Gaussian distribution 134 GE Healthcare Ettan spot picker 163 gel
105. esof one gel variable X and the cormesponding values in the sheet reference variable Y Rememberthatyou can change the sheet reference darkergel name by choosing View gt Sheet gt Set Reference The lineardependence isdefined asthe best fit line through the data points The best fit line isdescnbed by a slope and its offset from the equation y slope xx offset The goodness of fit for this approximation is given by the correlation coefficient Com This coefficient can vary between Land 1 where an absolute value near 1 indicatesa good fit The spot values of one gel canbe predicted to some extent by the valuesof the othergel On the Melanie UserManual Edition AC 117 8 Data analysis 8 3 Analyze gels 118 other hand a low value indicates that the data could not be approximated by a straight line The types of conclusions that can be drawn from the regression line equations and the correlation coefficients are 10xx 0 and Cor 1 indicates that the spot values forall matched spots are the same in the two gels 12xx 0 and Cor indicatesthat almost all spot valuesare 0 95 approximately 20 higher in the sheet reference 10xx and Cor indicatesthat almost all spot valuesare 0 2 0 95 shifted by 0 2 with respect to the sheet reference In general when the data are highly corelated Corrclose to 1 butthe best fit line isfa rfrom identity 1 0 xx 0 you should search for possible reasonsto explain why yourvaluesare sy
106. etween these two points Since Melanie doesnot have any information about the experimental possibly non linear pl scale the calculated values are only approximate In the case of MW of the spots the procedure issimilar except that Melanie searches for the closest spots above and below the spot for which the MW will be determined and it makesa loganthmic interpolation Extra polating pland MW values is more complicated For example if the plofa spotonthe extreme nght side of yourgelisto be determined the software looks forthe two closest spots to the left of the spot in question If these two spotsare sufficiently distant from each other in orderto decrease the enor the value forthe spot in question can then be extrapolated Noma lly the pland MW valuesin the Spot Report or Cursor Information window should be the same asin the defined pl MW annotations However this is only the case if the annotationsare attached to actual spots and not just to pixel positions in the image If an annotation is attached to a pixel the pl and MW values forthe spot that lies closest to it will be slightly different from that of the pixel to which the annotation is attached You can solve this ambiguity by linking the annotation to the spot Melanie User Manual Edition AC 97 6 Spots 6 8 Spot reports 6 8 Spotreports 6 8 1 O Spot Table The Spot Table Figure 6 8 obtained by choosing Reports gt Spot Table displays summarized information abo
107. ew of selected gel regionsor areasaround selected spots Analyze Gels Information about each selected spot match such as its Match ID value foreach spot in the match and chosen statistical measures calculated on all spots in the match Scatter Plots also provide information slope offset conelation coefficient fitting emor thatcompare the spot values fortwo gels The Match Statistics Table displays the number of matchesand percentage of matches foreach gel at the selected level of the match hierarchy The DIGE Histogram displays the frequency distribution of the DIGE volume ratios Analyze Central tendency dispersion and overlapping Classes measures for classes of gels computed forall selected matches Differences between the spot values in several classes can also be quantified with Sta tistical Tests such as ANOVA Mann Whitney U test and Kolmogorov Smimov test Gel Table Summanzed information about the selected gels such as Gel ID file name and path gel calibration data gel resolution and size number of detected spots userdefined properties such assample type staining ordate of the expenment and much more Predefined templates Properties Files Descriptions Calibration allow you to quickly display a specific subset of fields You can also create custom templates Spot Table Specific information about selected spots such as Spot ID and coordinates quantification values attached labels etc Annotation Inf
108. f the sample data asindicated by how clustered orscattered the data pointsare around their center value There are numerous measures of varia bility standard deviation range interquartile range and so on Like the statistics for central tendency these measures make use of all the available sample data and can be heavily influenced by outliers Therefore you can also restnct the sample to the central values by timming out the extreme values with the percentage slider e Select Mean Squared Deviation M S D and the square root of the average squared difference of each sample value to the center location iscalculated e Select Mean Absolute Deviation M A D and the mean of the absolute difference between each value and the central value is calculated It isnot affected asmuch by outliersasthe Mean Squared Deviation because the differences are not squared e Select Half range Size and the difference between the largest and the smallest values divided by 2 iscalculated Examples e he Mean 100 and the Mean Squared Deviation 100 are the most commonly used statistics Figure 8 2 a Note that the standard deviation isthe Mean Squared Deviation multiplied by JN N 1 where N isthe sample size This difference comes from the fact that the standard deviation should be an unbiased estimator Melanie UserManual Edition AC 115 8 Data analysis 8 3 Analyze gels e he Median Mean 0 and Mean Absolute Deviation 100 are much more rob
109. ferent substrates substrate A and substrate B and underwent one of two treatments treatment 1 and treatment 2 Three biological replicates have been run foreach condition Condition Substate Treatment A Il Substrate A Treatment 1 A_T2 Substrate A Treatment 2 B T1 Substrate B Treatment 1 B T2 Substrate B Treatment 2 DIGE Tutorial The sample gelsused in thistutonal come from anexperimentthat aims to study protein expression changes between control and treated groupsofbacterial cultures In this tudy eight sample lysates were prepared four were denved from treated bactenal culturesand four were derived from un treated control bactenal cultures Aliquots from each sample were taken and pooled to prepare a standard sample pooled intemal standard The Samples were run on fourgels The control and treated samples were altematively labeled with Cy3 orCy5 dye swap to avoid dye bias The standard waslabeled with Cy2 and run on each gel in the experiment Gel Cy2 Cy3 Cy5 Gel0l Pooled intemalstandard Control A Treated A Gel02 Pooled intemalstandard Treated B Control B Gel03 Pooled intemalstandard Control C Treated C Gel04 Pooled intemalstandard TreatedD ControlD A preparative gel including reference markers wasrun on the pooled sample and post stained with a Fluortotal stain to pickspotsand identify them by massspectometry MS Tutorial projects When the software isstarted forthe very first time two tutorial projects Tutor
110. file 150 http 150 text 150 specify classes 131 spot coordinate file 163 excision robots 163 ID 98 spot pickers 163 Bruker Proteineer SP 163 GE Healthcare Ettan 163 Genetix GelPix 165 Genomic Solution ProPic 165 spots 83 add 92 co detection parameter 88 color 92 Melanie UserManual Edition AC 191 192 composite 94 create 93 delete 93 detection algonthm 83 DIGEco detection 87 disable 92 display 92 edit 93 enabled 92 frequency 129 grow 93 load 92 manualediting 93 merge 93 non DiGE detection 83 ovenapped 79 propagate 95 remove 92 report 98 Save 92 select 90 sets 90 Shape 74 92 Shortcuts 173 shrink 93 table 24 98 stack 74 sacked 20 21 start the license server 10 statistical methodsreferences 175 statistics 113 120 121 138 141 tests 132 133 statusbar 15 19 step tablets 40 student st test 132 133 134 suspend synchronization 26 switch order 21 system information 13 requirements 7 T tabbed groups 29 tables annotation 24 calibration 43 Classanalysis 137 edit cells 28 factoranalyss 123 fitting 43 gel 24 80 gelanalysis 119 match statistics 110 scatter 116 118 Melanie User Manual Edition AC spot 24 98 technicalsupport 13 tests ANOVA 132 133 134 Kolmogorov Smimov 132 133 136 Mann Whitney 132 133 134 student st 132 133 Wilcoxon 134 text 146 link 150 TIFF 81 tied 20 21 toolbar 15 18 create 19 format 19 position 19 tools 15 add filesto project 53 adjust cont
111. ge Pool sheet and click Add Fles to Projectin the Image toolbar Set orcreate the destination project and then click lt New gt in the MatchSets field Figure 4 8 Entera name for the new match set Altematively create an empty match set by nght clicking on the project name and selecting Create MatchSetin the contextual menu Drag imagesfrom the Image Pool folder orsheet into the new match set Gels selected in the Image Pool sheet can also be added to an existing match set by clicking the Add Files to Projecticon Melanie User Manual Edition AC Projects 4 File Edit View Reports Tools Help DJA E 8 CEET AA Oh Sh A e kW E AddFies b s Workspace Be 3 Image Add Files to Project CG ER Image Pool ATI Del 4_T1_Gel2 4_T1_Gel3 4_T2_Gell 4_T2_Gel2 4_T2_Gel3 1 B_T1_Gell B_T1_Gel2 B_T1_Gel3 B_T2_Gell KSE E_T2 Del MatchSets B_T2_Gela gpw EF UM Project Project R Gels 3 Spots 0 Matches 0 Annotations 0 Figure 4 8 Add Filesto Project The three imagesof population A_Tl are selected in the Image Pool sheet Afterhaving clicked the Add Fllesto Project icon the destination project and new name forthe MatchSet can be set 4 3 2 Merge a match set Once match setshave been created they can be merged into higher level match structures for further matching Select the match sets right click on one of them choose Merge MatchSetfrom the contextual Melanie User Manual Edition AC 55
112. geMaster Immobiline and ImageS Cannerare trademarks of GE Healthcare companies GE isa trade mark of General Electric Company All third party trademarks are the property of their respective own ers Allgoodsand servicesare sold subject to the termsand conditions of the license agreement communicated to you by GeneBio GeneBio reservesthe right subject to any regulatory and contractual approval if required to make changesin specifications and features down herein or discontinue the product described at any time without notice orobligation Contact GeneBio at license genebio com for the most curent information
113. ged and draw a trajectory through the selected spots by making sure to start and finish in the same spot Grow spots Click this icon select a soot to be grown and outline the area you would like to add by making sure to start and finish within the selected spot G Shnnk spots Click this icon select a spot to be shrunk and outline the area you would like to suppress by making sure to start and finish outside the selected spot 6 6 2 Composite spots Asshown in Figure 6 6 the software allows you to match several spotsin one gel with multiple spotsin othergels In the figure the three selected spotsin gelA_Tl Gell are matched to the single green spot in A_T2 Gell and the two selected spotsin B T1 Gell and so on Once the match has been effectively created the three spots in A_Tl Gell forexample are treated asa single entity in the quantification The quantification value forA_ Tl Gell displayed in the different reports isthe one obtained after combination of the values from the three individual spots This is an efficient solution for dealing with spot detection differences without subjective and time consuming spot editing When selecting a spot on a gel any matched spots on the other imagesare automatically selected as well Asa result defining a multiple match is Melanie User Manual Edition AC Spots 6 very easy and quick You just need to selectallspotsto be matched and click the Add Match icon in the Detect And M
114. h only a sequence ending atthe lastcharacterofa string Matchesany one character e oli matches eaoli eboli ecoli Table 9 1 Regular expressions available to search pattems in labels Please note that the term element used in the description indicatesa characterora subexpression The characters 1 have a special meaning in certain contexts You must exclude them from the expression with a backslash if you want them to be taken literally This means that if your labels contain any of these special characters you must precede them with a backslash if you want to include them asnomal characters in your search expression Note that you must also release the backslash character itself from the expression Forexample the search pattem R retums the result R 3 24 but not R 2 87 Nevertheless bracketed expressionsare an exception to the rule Inside bracketed expressions all soecial characters including the backslash lose their special meaning for instance A matches exactly any of the characters inside the brackets The orderof precedence forthe regularexpressionsdescnbed above is as shown in Table 9 2 Forexample the regular expression abc 2 3de matches either the string abc2 orthe string 3de ratherthan the string abc2de orabc3de because concatenation hasa higher order of precedence than altemation Melanie UserManual Edition AC 155 9 Annotations 9 7 Display annotations and labels lt
115. he bottom right position Allspotsin the designated regionare selected To deselect all spots click in the gel not ona spot Surrounding box By default spot selectionsare surrounded by green boxes This makes it iseasierto localize selected spots especially when working atlow zoom factors To deactivate thisoption choose Tools gt Options and uncheck the comesponding box in the Display tab Altematives There are other waysto select spots e Choose a command inthe Select menu e Select spotsin reports 6 4 2 Spotsets It is possible to focus your analysis on particular spots by creating and saving spot sets for later selection or combination Create Select spots you want to include in a spot set either manually or by selection in a report Then choose Edit gt Spot Sets gt Create entera name forthe new spot set and click OK Spot sets can be visualized ascolumns in varous reports Figure 6 4 If they are not displayed by default you can add them by checking the cormesponding boxin the Settings of the report window only fortabular reports Once the column is displayed you will see a checked box for spots that belong to the set oran empty box for spots that do not belong to the set Click in a boxto change its state If several spots are Melanie User Manual Edition AC Spots 6 selected youcanchange the state by clicking in one boxwhile holding down the Shift key ss Analysis Table Mean 100 and M 5 D
116. he possibility to replace it with the archived project If the project is not present in yourworkspace you are given two optionsforrestonng a project By restoring to the original file path the files are put back in the original project folder By restoring to a new file path the project is replicated in a new location which must be specified Click Restore 4 7 4 Project visibility When there are several projects in the Workspace it can be useful to temporanly hide some of them Select Display in the Projecticon drop down listand check oruncheck the Visibility boxin front of the projects that should be hidden orshown 4 7 5 Project properties To quickly view details creator comments etc about a project nght click on the project and select Properties in the contextual menu You can also modify the Project Name and Comment 62 Melanie User Manual Edition AC Gels 5 5 Gels 5 1 Introduction This chapter presents operations that specifically relate to gel images You will leam how to manipulate images use different tools to view Signalintensities and discoverwaysto visually compare images The last section explains how to save export and print gel images 5 2 Manipulate images The main toolbar provides the following tools to deal with images 5 2 1 NW Move Select this tool click in the image and hold down the left mouse button while moving the cursor The image changes position Release the button at
117. he software computes ovenapping measures between the class intervals where classintervalsare defined by the ranges Central value Dispersion Central value Dispersion The overlapping measures quantify the overlap between these intervals and thusgauge how different the protein expression changes between the classes really are They take into account both the difference between the central tendency in each population and the dispersion The following overapping measuresare available e Gap Maximum difference between the range of the current class and the range of one of the other classes Figure 8 14 c b in the case of ClassA Negative values indicate overlap whereas positive valuesare non overlapping class ranges e Ratio Maximum ratio between the lower limit of one of the other classes and the upper limit of the current class Figure 8 14 c b in the case of Class A Absolute values smallerthan 1 indicate overlap whereas absolute values higher than 1 show that there is no ovenap In orderto easily distinguish matched spots that are over or under expressed in one of the classes the ratio value is preceded by a minussign when the protein spot is under expressed forthe class in question compared to the other class Positive valuesare attnbuted to the Ratio value in the over expressed class e Normalized Maximum percentage of the current class range not overlapping with the range of one of the other classes Figure 8 14 c a b a
118. hide columns in the table Nomalization In orderto simplify compansons across matches the spot values in the Gel Analysis Table can be nomalized relative to their gel analysis Statistics Select the desired type of nomalization in the drop down list in the toolbar The following optionsare available Raw spot value Relative Spot value Central tendency Spot value Central tendency Nomalized Spot value Central tendency Dispersion CAUTION The Relative and Noma lized options are not available for DIGE expenments when Vol Ratio isselected asthe quantification measure Melanie User Manual Edition AC Data analysis 8 8 3 3 M Gel analysis histograms Histograms are a way to look at matched spots The Gel Analysis Histograms window Figure 8 6 isdisolayed by choosing Reports gt Analyze Gels gt Histograms In the histograms the vertical orange barscorespond to the spot values the blue horizontal line represents the chosen centraltendency and the red linesdelimit the range defined by Central value Dispersion Central value Dispersion The Match ID isdisplayed atthe bottom of each histogram Gel Analysis Histograms Mean 100 and M 5 D Ga tv eg Se sorted Values o oe Adaptive Gradations show Labels i mm in k abedefghijkl 362 0 06 0 04 0 06 0 02 0 04 0 0 abedeferhijkl abedefghijkl abedefghijkl 363 364 365 Figure 8 6 Gel Analysis Histograms The following toolsin the Gel
119. hoosing Reports gt 3D View If spots are selected the area around these spots is visualized for all imagesin the sheet Figure 5 7 If only regions were defined on images in a sheet these regions are shown in the 3D View Figure 5 6 If both spots are selected and regions are defined the area around the spots is shown by default but you can choose to display the regions The layout of the imagesin the 3D View reflectsasmuch aspossible the layout of the imagesin the sheet Bold black linesseparate imagesthat are in different panes You can restnct the numberof imagesdisplayed Melanie User Manual Edition AC 71 5 Gels 5 3 View signal intensity in the 3D View by setting their Visibility in the Display Options see below CAUTION To ensure satisfactory performance the numberof images that can simultaneously be viewed in 3D view is limited to 24 If you selected more than 24 images you will be able to choose the visible images from a list that contains information about the match setsor classesto which the images belong By default 3D viewscan be rotated by dragging the mouse while holding down the left button If your mouse hasa scroll wheel itcan be used to zoom in orouton the view You can change to one of the other image manipulation tools avaible in the Tools icon drop down list see below for more details Please note that all views in the 3D View window are manipulated simultaneously Right clic king is reserved forthe
120. hould be satisfactory 6 3 3 Spotquantification Volume area intensity sope and volume ratio for individual spots are automatically calculated and included in the Spot Table and Cursor Infomation window e Vol Spot volumes sum of pixel intensities within the spot boundary are always expressed with background subtracted Background is subtracted on a spot specific basis by excluding the lowest 10th percentile pixel value on the spot boundary from all other pixel values within the spot boundary The spot volume isthe sum of these comected values e Vol ratio Volume ratios volume of current image spot volume of DIGE reference image spot indicate the change in spot volume between two images CAUTION When using single detection the volume ratio value is 1 0 for all spots since there isno second image Melanie UserManual Edition AC Spots 6 6 3 4 Exclude spots To exclude small non proteinaceous spots on DIGE images it is recommended to filter the spots based on the Volume More precisely spots that have a maximum volume in the two orthree images that is lower than a given threshold can be deleted To exclude DIGE spots based on maximum volume 1 Choose Reports gt Spot Table 2 Click the Select by Value icon in the toolbar of the table 3 Select Volume in the displayed list Make sure the gt boxischecked and entera value to be used asthe cutoff forthe volume Deselect the boxand click OK Figure 6 3 4 Alls
121. ial and Tutoria IDIGE are restored Each project containsthe three first images of the comesponding experiment described above The Melanie UserManual Edition AC Getting started 1 project can be completed with the remaining imagesthat can be found in the folder Tutonals Imagesin the Melanie installation directory In this way you first get a view of what the project structure looks like and can then leam to add imagesto a project Note If Melanie wasalready used on yourcomputer before you can rename the file Exoenment mws in Documents and Settings usemame My Documents Melanie Projectsto make sure the tutonal projects are restored User manual Melanie is intended to be intuitive and comprehensive In orderto exploit the software sfull potential referto the UserManual You will find detailed explanations of all the features and functionalities The chapters in this manual are generally organized according to the logicalsequence ofa 2 DEgelanalysis although expert users willagree that some stepscan be inverted orrepeated at some point 1 6 2 Customer support GeneBio providestechnical and scientific support for Melanie Please contact us if any problems arse during the installation or use of the software Our support team is happy to help you How to contact us By email melanie support genebio com By phone 41 22 379 50 50 By fax 41 22 379 58 58 Product system and license information Launch the software and
122. ies is true All you can do iscalculate the probability of observing a certain difference orlarger between sample meansin an Melanie User Manual Edition AC 133 8 Data analysis 8 4 Analyze classes 134 experiment of this size for populations that in reality have the same mean If the probability issmall you can conclude that the difference isnot likely to be caused by random sampling and assume instead that the populations have different means CAUTION You can display the desired statistical valuesforeach match in the ClassAnalysis Table These values should be considered as qualitative indications of the vanations in protein expression between two populations To draw quantitative conclusions you must venfy that the restnctive assumptions of the vanous tests are met In addition one should alwayscheck the results by visual inspection of the spots since the conclusions may be erroneous due to inaccuracies in detection or matching CAUTION The given statistical values are useless when the samples classes do not consist of more than two gels Your objective should always be to work with the largest possible sample sizes One way ANOVA Analysis of Vanance ANOVA isone of the most important statistica tests available for biologists It is essentially an extension of the logic of Student s t tests to those situations where the comparison of the means of several groups is required Thus when comparing two means ANOVA gives
123. igned to another menu command You risk destroying an existing shortcut However a given command can have several different keyboard shortcuts To add your own keyboard shortcut 1 Choose Tools gt Customize 2 Clickthe Keyboard tab 3 Selectthe menu in the Category drop down list and then selectthe command forwhich you wantto create a shortcut Figure 2 2 4 Checkthe Key assignments list to see if the shortcut is currently assigned 5 Clickin the Press new shortcut key box and then pressthe new combination on your keyboard 6 Click Assign The software wams you when the shortcut is already assigned to another command and asks if you want to re assign tt Click No and come up with a different shortcut 7 Click Close Customize Ss cmama Toolbars Commands Keyboard Options eit Commands Key assignments Matches Unmatch Gels Matches Add Match Matches Delete Match Reset al_ Annotations Add 3 Reset A Annotations Delete Annotations Link with Press new shortcut key Annotations Unlink fror 4 Etrl Shift M Description Figure 2 2 Defining keyboard shortcuts in the Customize window Melanie User Manual Edition AC 17 2 Graphical user interface 2 3 Toolbars 2 3 toolbars 18 2 3 1 Defaulttoolbars The standard toolbars provided are designed so that you can quickly manipulate yourgeldata and apply the most frequently used features Each of the tool
124. in one of the recommended input formats please contact ourtechnical support service 3 5 Calibrate images 3 5 1 Display calibration infomation It isrecommended to use calibrated images for your gel analysis To venfy if yourimages were calibrated and possibly view the calibration information e Choose Reports gt Gel Table click the Settings icon and select the Calibration template from the Load drop down list The Calibration Unit Formula Name Creatorand Date are displayed inthe report If nothing appears in these columns the gels were not calibrated e If yourimage wascalibrated with LabScan 5 0 or 6 0 you can select the image and choose Edit gt Gels gt Show Calibration Plotto view the calibration curve See below for details about the Calibration curve If you are digitizing yourimagesusing a flatbed document scannerand do not have calibrated images yet you can cany out and applya 40 Melanie UserManual Edition AC Image Pool 3 Calibration at this point before adding yourimagesto a project Use a scanned calibration step tablet to assign optical density OD valuesto the measured pixel intensities and ensure that darker materal is measured in the conect proportions to the lighter material It isgenerally recommended to perfom thistype of intensity calibration on the image capture device once every month 3 5 2 Create a calibration Calibration step tablet To calibrate the image capture device you need t
125. in the Workspace toolbaralso enablesyou to add projectelements instead of entire projects You can open the following file types e Project pn e Project Backup bkp e Export File exp e Alllmage Files mel tif gel img e Matcher Data mda e Gel Data gda 4 7 3 Backup restore project Itisgood practice to do regularbackupsso that you can recover your work at any time With the Backup function in the Workspace one or more project s can be archived by writing all project related data images spots matches annotations spot sets enabled spots into a single compressed file with the extension bkp This file can then be restored when needed To backup a project 1 Inthe Workspace select Backup in the Projecticon drop down list Melanie User Manual Edition AC 61 4 Projects 4 7 Manage projects 4 Select one ormore projectsto backup and confirm when prompted In the Backup Project window browse to the directory you want entera file name and click Save The backup file with the extension bkp isarchived To restore a project 1 2 In the Workspace select Restore in the Projecticon drop down list In the Restore Project window browse the directory where the backup file islocated select itsname and click Open Select the project s to restore from the list Use the Ctr or Shift keys to make multiple selections Click Restore Ifthe project already exists in the Workspace there ist
126. ing etc Select this tool place the cursorat the top left comerof the area you want to define hold down the left mouse button and move to the bottom right position a dashed box is displayed Release the mouse button at the end point Figure 5 2 Melanie UserManual Edition AC 65 5 Gels 5 2 Manipulate images Ke RW En ST wn O ergi e GA ES K J PER of ei d ed 26 5 GAM pes BW na Wf Figure 5 2 Region tool The left gel showsa region after selection the middle gel during selection and the right gel shows when the region hasbeen reduced to its minimum size Edit region You can move a region by clicking inside the boxand dragging it You can also change the size of the box by dragging a comeroredge To remove a region double click on the gel Please note that if the box is reduced to its minimum size then its appearance changesto a blue Circle Apply to all gels To define the same region on allgelsin the current sheet hold down the Shift key while drawing the boxon one of the gels or while clicking in an existing region 5 2 4 Measure Select this tool to measure pixel pl MW or real word centimeter or inch distances between two pixelsin an image Click on a pixel e g center of first spot hold down the left mouse button and move to the pixel e g center of second spot for which you want to measure the distance The honzontal and vertical distances between the start and end points are
127. ion In Kellner R Lottspeich F Meyer HE eds Microcharactenzation of Proteins 2nd ed pp 277 300 Weinheim Wiley VCH ExPASy Molecular Biology Server 2003 Online http www expasy org Hoogland C Baujard V Sanchezj C Hochstrasser DF and Appel RD 1997 Make2ddb a simple package to setup a 2 DEdatabase on the WWW Electrophoresis 18 2755 2758 Hoogland C Sanchez C Bairoch A Hochstrasser DF and Appel RD 1999 The SWISS 2DPAGE database what haschanged during the last year Nucleic Acids Research 27 289 291 Link AJ ed 1998 Methodsin molecular biology Vol 112 2 D Protocols for Proteome Analysis Totowa NJ Humana Press Lopez MF 2000 Betterapproachesto finding the needle ina haystack optimizing proteome analysis through automation Electrophoresis 21 1082 1093 Sanchez C WilkinsM Appel RD and Hochstrasser DF 1997 Identifying proteins for proteome studies In Creighton ET ed Protein Function a practical approach 2nd ed pp 1 27 IRL Press Wilkins MR Williams KL Appel RD and Hochstrasser DF eds 1997 Proteome research new frontiers in functional genomics Benin Heidelberg Springer Venag Melanie User Manual Edition AC References B Unlu M Morgan ME and Minden J S 1997 Difference gel electrophoresis a single gel method fordetecting changesin protein extra cts Electrophoresis 18 2071 2077 Tonge R Shaw J Middleton B Rowlinson R RaynerS Young J Pognan F HawkinsE Cum
128. is means that repeated values ties are not acceptable When ties are present in your data there isan approximation provided in the calculations but the exact results no longer hold e The distributions of the two samplesare identical although not necessarily nomal and differ only in location i e central tendency e The two samplesare independent To perfom the Mann Whitney test the software first ranks all the spot valuesfrom low to high paying no attention to which of the two classes e g Xand Y each value belongs Then each value is given a rank number The smallest number getsa rank of 1 The largest number gets a rankofN where N isthe total numberof spot valuesin the two classes If two values are the same then they both get the average of the two ranksforwhich they tie Finally the ranksin each classare summed thus giving WX and WY which are used to calculate the Mann Whitney test statistic U The formula for UX isas follows the formula for UY isobtained by replacing X by Y H ny ny 1 NN 2 The smaller of the two calculated U values corespondsto the number of shifts needed in orderthat the spot values from the two populations do not ovenap For the first example in Figure 8 15 no shifts are necessary since the spot values of classes X and Y are already separated On the contrary forthe second example the Mann Whitney test indicates that the first two values from Y orthe three last values from X have to be s
129. ispersion Melanie provides two options The Fold value displays the fold change between the class with the lowest central value and the class with the highest central value It is available in a separate column of the Class Analysis Table see 8 4 5 Class analysis table It is possible to display foreach class the fold change againsta selected reference classX That is the ratio of the central tendency for the class versus the central tendency forthe selected reference class To display these ratios select the option versus Class X in the Displayed value drop down of the Class Analysis Table that comespondsto the desired reference class X see 8 4 5 Class analysis table 8 4 4 Statistical tests The software provides three statistical tests ANOVA Mann Whitney U test and the Kolmogorov Smimov test These tests are used to analyze differences in protein expression between classes of gels The idea isto draw conclusions about the significance of the protein expression changesby extrapolating information from the data you collected For example when you have two samples classes with different means Le different means forthe spot valuesof a particular match you might want to know whetherthe data were sampled from populations with different means or whether the populations have the same mean with the observed difference being a coincidence of random sampling In fact there isno way to definitely conclude which of the two possibilit
130. k anywhere in the image and rotate the grid while holding the left mouse button Release the button when the bold line is parallel with what should be the new honzontal in your image Figure 3 3 You can also manually entera rotation angle in the Rotation Tool window Melanie User Manual Edition AC 37 3 Image Pool 3 4 Processimages File Edit View Reports Tools Help QP IP ROO JA 2h Sho AS KM D Add Files to Project K A a a a oo m Rotation Tool Tum the image Pick mel by rotating the grid za that the bald line defines the new horizontal reference Angle Figure 3 3 Rotation Tool window The grid isrotated until its bold line is parallel with what should be the new honzontal reference When the mouse button is released the gel image is rotated Da 342 ANE nip When gelimagesare scanned in the wrong direction you can Flip Horizontally or Flip Vertically to produce their corect minor image 3 4 3 4 Crop You can crop yourgel images with the Crop tool This creates new gels that only contain the selected area and removes the outer area When you crop a gel you get the choice to create a new image a numberisappended to the existing name orto overwrite the image in the Image Pool rememberthatgelsin the Image Poolare copiesofthe original image file i Crop area The crop area isa region than can have an anchorattached to it You can position the anchoron an easily recognizable protein sp
131. kOK To create and save your own report template 1 Click the Settings icon in a report toolbar 2 Inthe template window select box ischecked the attnbutes that you wantto show inthe report and deselect box isunchecked the attnbutesto hide columns 3 To save the template for later use click the Save icon Choose a name from the list or select lt New gt to create one Click OK Melanie User Manual Edition AC 27 2 Graphical user interface 2 Reports 28 4 Click OKin the template window To delete an existing report template 1 Click the Settings icon in a report toolbar 2 Inthe template window click the Remove icon and choose the template to be deleted Click OK 3 Click Yeswhen asked for confirmation Classes Report ita x Default All Classes Anova Wilcoxon Kolmogorov Figure 2 5 Report template window Column order You can reorganize table columnsdirectly in a report window To do so drag the column header to its new position You will see red arowsat the insertion point Column size You can enlarge or reduce the column size Drag the nght edge of the column headeruntil the column isthe width you want To resize columns so that theirwhole content isdisplayed double click on theirseparator The column to the left of the Cursor is resized 2 7 5 Edittable cells In the Spot table cells containing annotation labels are editable Double click in a cellto start typing orediting your label When
132. kl Gels D Spots o Annotations o Figure 6 2 Soot quantification The 3D View in Melanie reflectsthe spot shape and volume of what will effectively be quantified The spot outline corresponds to the area at 75 of the spot height when measured from the peak e Intensity The software first calculates the intensity of a spot The intensity is based on the highest calibrated pixel intensities in the spot from which the background hasbeen withdrawn The background isdefined asthe minimum pixel value in the spot neighborhood e Area he area of a spot isnot determined atthe spot base because the base isoften arbitrary and difficult to determine It is calculated atan intermediary height of the spot More precisely Melanie computes the area at 75 of the spot intensity as measured from the peak of the spot The spot outlines displayed in 86 Melanie User Manual Edition AC Spots 6 Melanie exactly encircle thiscomputed spot area expressed in mm e Vol The volume ofa spot iscalculated asthe volume above the spot outline which issituated at 75 of the spot height as measured from the peak of the spot In Figure 6 2 the measured volume of the spot ishatched Please note that the volume values like the intensities depend on pixel intensity calibration e Y WVol The relative volume of a spot iscalculated as indicated below Itisa normalized value that remains relatively independent of vanations due to protein loading and
133. l 04 Cy3 Treated ee Gels 418 ee Gel 01 Gel 01 Cy Standard Gel 01 Cys Control Gel 01 Cy5 Treated py Gel 02 fp Gel 03 fe ht Gel 04 E Cal Classes Eia Control Us A 01 Cys Control tal 2 Cy5 Control Gel Gel 03 Cod Control d Gel 04 Cy5 Control EI ma Treated img Gel 01 Cy Treat d Gel 02 Cya eg Figure 2 4 The expanded view of the Workspace window The Workspace toolbarand Navigator at the left are always visible The file details in the expanded view at the right are only displayed when desired 2 6 1 Toolbar Commandsto create new projects add filesto backup and restore projectsare found underthe Workspace toolbar Clickthe EE Expanded View icon to enlarge the Workspace window to include details of all files selected in the Navigator The filescan be sorted in ascending ordescending order by clicking on a column header Clickagain on the Expanded View icon to hide the file infomation iacts remove Projecticon in the Melanie User Manual Edition AC 23 2 Graphical user interface 2 Reports 2 Reports 24 2 6 2 Navigator The Navigator displays all files and folders you can view Image Pool and analyze projects The look and feel issimilarto Windows Explorer There isa hierarchical structure of folders subfolders and filesthat can be expanded orcollapsed dragged and dropped ina new location copied and pasted etc When launching the software forthe first time there isan empty Image Pool folder Im
134. l the individual raw imagesand the merged image The resultant spot map isovenaid backonto the original Melanie User Manual Edition AC 87 6 Spots 6 3 Co detect spots in DIGE gels 88 image files Since the soot boundanesand the detection areasare identical for all images the spots are effectively already matched This process results in highly accurate volume ratio calculations 6 3 1 Procedure To perfom spot detection on DIGE images 1 Display the gelsto be detected by selecting a match set in the Workspace Right click and select Display in the contextual menu 2 If desired select a subset of gelsto be detected Choose Edit gt Spots gt Detect 4 Inthe DIGE Spot Detection window enteran estimation see below of the Number of Spots present in the images Click OK 6 3 2 Spotdetection parameter When detecting DIGE images you must enter an estimation of the Number of Spots present in the images It isrecommended that this value be overestimated to compensate forthe detection of non proteinaceous spots on the image e g dust particles which can subsequently be excluded from the analysis using spot filtering If allthe spots are not identified the soot detection processcan be repeated with a highernumber of estimated spots Forexample fora mammalian lysate run on an 24cm pH 4 7 Immobiline DryStrip and a large format gel such asthe Etta n DALT Gel 20cm x26cm a value of 2500 forthe estimated Number of Spots s
135. la Firefox File Edit View History Bookmarks Tools Help gt a fit La dh http fwww expasy org ME Glaso L g R Search ExPASy Search Swiss Prot TrEMBL sforj Create Annotation by Click Il gt ExPASy Proteomics Server Enter label text for category Link The ExPASy Expert Protein Analysis System IS ktos proteomics server of the Swiss Institute of http fw expasy ord Bioinformatics SIE is dedicated to the analysis of protein sequences and structures as well as 2 D PAGE Disclaimer References Linking ta ExPASy P10413 his is a link to ExPASy a AA composition B 12 5 Show Label 7 14 1 Ki s Edit Label H show content of text link G Enter label text for category Link Ldditional info T AA Composttion text A WW FUNCTION Molecular chaperone Has ATPase activity file AA P1041 3 xlg SUBUNIT Homodimer Oligomerzes at 65 degrees Celsius P SUBCELLULAR LOCATION Cytoplasmic 11 PTM Phosphorylated Sg sk 7 r HE NE w SIMILARITY Belongs to the heat shock protein 90 Family Figure 9 5 The annotation on the spot containing protein P10413 includes linked labelssuch asa linkto a Web site top a link to a file bottom right and a text link bottom left 9 6 Selectannotations and labels 9 6 1 Select Annotationsand labelscan be selected with the Selecttool The selected labelsorannotationsare highlighted in greenand displayed in front of
136. ld always be available in the software Quantic ation e Specify the spot quantification value to be used fornon DIGE Value and DIG E DIGE Value experiments This soot quantification value is used in Gel and Class Analysis Reports e Compute the spot areasin mm based on gel resolution default or pixels General e Specify whetherthe raw image data of newly opened files should be kept in memory With the option Keep image in memory selected the image data is Continuously loaded and the software will require more memory With the option Keep image in memory deselected the image data isonly loaded by the software when specific operations Detection Adjust Contrast 3D view Profile require the use of raw image data This gives you the possibility to optimize the use of memory available to the software but some functionality Pixel Intensity in the C ursor Infomation Window and Status Ban willnotbe available unless you activate Adjust Contrast or Profile at the same time Melanie User Manual Edition AC 31 2 Graphical user interface 2 9 Options 32 Melanie UserManual Edition AC 3 3 1 Image Pool 3 Image Pool Intoduction Thischapterisabout opening and processing imagesin the Image Pool You must be familiar with the concepts descnbed in the last chapter the Workspace sheets panesand images in orderto get the most out of it 3 2 Start with good images Your gels must first be converted into an image fil
137. lect Reports Tools Help MARSO FB CO O0 GG ez Image Pool SE AT1 AT2 BT1 BT2 Mx Class Analysis Histograms Mean 100 a O A X SR tem m ji abedefghijkli Eeer ageds HOR 0 1 0 08 0 06 0 04 0 02 0 abedefghijkl SE m2 SH ASYW wm i Max ATI d 02 0317969 0 ep i abedefghijkl W aron Gels 12 Spots3 Matches i Annotations 0 Figure 2 6 The Class Analysis Histograms window previously docked at the right edge will now be docked to the right of the Class Analysis Table window in the lower left comer of the Melanie window 2 9 Optons You can set varous para meters that influence your work in Melanie These settings are accessed by choosing Tools gt Options in the menu More detailed information about the options are provided in the related chapters of the User manual However an overview of the settings pertab isgiven below ey Indicate the default soot colors for enabled disabled selected and ovenapped spots and the color forthe match vectors e Choose the unitsto express coordinates in the Status Bar and Measure tool 30 Melanie User Manual Edition AC Graphicaluserinteface 2 e Indicate whether spot selections should be surounded by boxes for easier localization Annotation e Define the annotation categonesthat should always be available in the software and set their attnbutes and display properties Gel descriptions e Define the gel descriptions that shou
138. lish the link orthe content of the link in the case of a text link To indicate that a label is linked its short descriptor is followed by three dots When you double click on such a linked label you do not enter the typical editing mode but the link Web page file or text Is automatically opened with the appropnate software Altematively to open any linked label choose View gt Annotations gt Linked Data in the menu Note that you can define links in any label category but you can only have one link per label 9 5 1 Http link You can link spotsorpixelsto specific Web pages A double clickonan http linked label will launch your Intemet browser and automatically callthe coresponding Web page You can forexample create a direct link to the ExPASy Proteomics Server Figure 9 5 In this case the label content should contain the sting http followed by the address of the Web page 9 5 2 be link You can link spotsorpixelsto software files Double clicking ona file linked label launchesthe specified file with the default system application associated to the file extension The linked filescan be placed in a specific directory which isdefined by choosing Tools gt Optionsin the menu and by setting the Annotations folder in the Annotations tab In this case you only need to give the name and file extension when creating the link You can create subfolders in the Annotation folderto arrange yourfiles The file names
139. lysis Basically the image matching algorithm compares gel imagesto find matches between related spots that is spots representing the same protein in the gels A match istherefore composed of spot n tuples S1 S2 Sn where S1 is a Spot ID in the first gel and Sn a Spot ID in the last gel The matching algonthm always starts from the reference image or match set and looks foreach spot in this reference if comesponding spots in the other images are found If a soot isabsent from the reference it cannot be matched automatically However if you have several match sets in your hierarchy there isa good chance that the spot ison the reference of at least one of them If so the spot will tum up in the analysis Subsequently additional spotscan be matched to it manually Figure 7 1 Matches are propagated at each level of the hierarchy Thismeans that once all match sets are effectively matched spots from one gel can be directly compared with those in any of the other gels CAUTION A DIGE gel isan inherent match set for which the co run imagesare automatically matched To subsequently match different DIGE gels proceed like any other match sets Melanie User Manual Edition AC 101 7 Matches 7 2 Display a match hierarchy File Edit View Select Reports Tools Help QP LYRIS OPA Gah OG az oh AB 5 SC m KS m Q o Gels 12 Spots6 Matches 1 Annotations 0 1 83 cm 9 87 cm 5062 Figure 7 1 Reference and matching
140. m user intervention Nevertheless when the gels are very distorted or different you may need to help the matching process by specifying a few landmarks Landmarks are points that relate corresponding spotsin each of the gelsto be matched We recommend trying to match yourgelswith only a single landmarkor possibly none atall In some cases no landmarks are required More often a single landmark is sufficient for quick and efficient matching If the matching resultsare notsatisfactory you can repeat the automatic matching procedure using additional landmarks The following rules should be considered when defining landmarks Melanie UserManual Edition AC 103 7 Matches 7 3 Define landmarks 104 e Keep the number of landmarksto a minimum e Only define landmarks on clearly comesponding spots Protein variants should definitely not be used as landmarks e Landmarks should be placed on small sharp spots rather than on large diffuse ones to reduce the enorin the position e Landmarks essentially corect global deformations of gels Therefore it isrecommended not to put landmarks on spots in locally distorted regions because this can worsen the matching results Rather place landmarks around such regions Landmark Landmarks can be defined using the dedicated tool asdescnbed below To define landmarks 1 Click the Landmark icon in the toolbar 2 Place the mouse cursorovera known well defined spot in the first reference
141. mage so that Melanie can export the aligned X and Y coordinates of the selected spots Please bearin mind that properalignment is crucial at this tage especially for images of old gelsthat underwent significant shape change since they were onginally imaged and analyzed and forgel images that were acquired ona high resolution system To exporta spot coordinate file to the ProPic spot picker 1 Inspect your analytical gel image in Melanie and select the spots you want to export to the spot picker Annotate these spotsorsave them in a spot set so that you can easily re select them ata later stage 2 Place yourgelon the ProPic robot bed produce the ProPic image and open it in Melanie Use the gelimage asis Do not resize crop flip orrotate the image 3 Align your analytical gel image to the ProPic image using a sufficient number of landmarks 4 Selectallspotsto be cut with the spot excision robot from the analytical image Make sure only the analytical gel image isselected Choose Fle gt Export gt Spots to Picker gt Genomic Solutions ProPic Entera file name and destination folder The file isautomatically given a tdsextension TwoDSpotlist 8 Melanie exportsthe aligned Xand Y coordinatesforeach selected spot In other words the coordinate system of the ProPic image will be used 166 Melanie User Manual Edition AC Undo redo and history 11 11 Undo redo and history 11 1 Undo redo The software allows you
142. match vectors in blue Vectors link the spotsin a gel with the comesponding spotsin the sheet reference Thissheet reference hasa darkergreen gel name and should not be mistaken forthe match reference which has a red component The sheet reference can be changed by choosing View gt Sheet gt Set Reference A blue upside down triangle on a spot indicates that the spot was matched to one or more spots in other gels but not to a spot in the sheet reference Figure 7 5 A spot with a trangle meansthat the conesponding postion in the sheet reference lies outside the visible area Figure 7 5 To hide the match vectors oron the contrary to display them when they are not visible choose View gt Matches gt Show Vectors Melanie User Manual Edition AC 107 7 Matches 7 6 Display matches File Edit View Select Reports Tools Help OP IRs oi LAOD OARRA S x fz mi g Gels 12 Spots 15 Matches 2 Annotations 0 Figure 7 5 Different representations of matches The selected spots in match set AT2 with a blue upside down triangle are matched but not with a spot in the sheet reference Spots with blue triangles asthe one indicated with a green arrow are matched but the conesponding position in the sheet reference lies outside the visible area To minimize the match vectors after having moved orzoomed an image select the Move tool and double click in the image so that it is synchronized with the otherimagesand the sh
143. me forthe new classand click OK Altematively create an empty class by nght clicking on the Classes folderand selecting Create Class in the contextual menu Drag images ormatch sets into the new class 4 5 Handle project tems Several operationsare available for most itemsin a project mostly by nght clicking in contextual menus 4 5 1 Display To display one or more images match sets or classes in a new sheet nght click ona selected item and choose Display Please note that only complete match sets or classes are displayed This means that if you select one image ina match set all imagesin the match set are shown In the resulting sheet only the highest level and lowest level items match sets orclasses are specifically visualized in the sheet and panes respectively To select intermediary levels you can use the corresponding icon in the pane tab of the match reference forthat level Once the imagesare displayed in a sheet you can start working with them You can change the layout settings to focuson certain images and hide others 4 5 2 Remove To permanently remove an item from the project select Remove in its contextual menu This will delete the item from your hard disk 4 5 3 Properties When choosing Propertiesfor an item certain of itsattnbutesare shown such as itsname creator file path etc Here iswhere you can change the name of an item orentera comment to describe the item Melanie UserManual Edition AC 5
144. ms Raw spot value Relative Spot value Central tendency Thisnomma lization sets the centraltendency valuesto 0 and if the Adaptive Gradations option see above is deactivated you have a good overview of the dispersion and therefore of the homogeneity of the matches This normalization is sensitive to high spot values Melanie User Manual Edition AC Data analysis 8 Spot value Central tendency This normalization divides all values by the central tendency and thusgivesa ratio forall data If you deactivate the Adaptive Gradations option see above all histograms have the same scale and thus it becomes easier to detect matchesthat do not have homogenousvalues This no malization ismore sensitive to low spot values Nomalized Spot value Central tendency Dispersion This nomailization isa compromise between the two nomalizations described above and is sensitive to all values CAUTION The Relative and Nomalized optionsare not available for DIGE expenments when Vol Ratio isselected asthe quantification measure 8 3 4 Factoranalysis The visual task of comparing gelsis rather difficult when dealing with a large number of gels that consist of thousands of spots It can be hard to assesswhetherdifferent sample populationsexistand to charactenze their different protein expression profiles Factor analysis helps here by condensing the information contained in such huge data sets into a smaller number of fac
145. n Thiscan be an open gel orstep tablet image that wasalready calibrated oryou can select a file calor mel from the hard disk All pixel and spot values subsequently calculated and displayed in the software correspond to the calibrated values 3 5 4 Remove a calibration You can remove a Calibration from a gel Note that thiscan also be done for gels that were already calibrated when you imported them into the software To remove a calibration from a gel 1 Display and select the gels from which you want to remove the calibration Choose Edit gt Gels gt Reset Calibration Confim your choice 3 5 5 Control a calibration The Controlcalibration mode allowsyou to venfy whetheryou are using the conectcalibration It requires a different specially calibrated step tablet e g Kodak Step Tablet no 3 which you compare to your previous Calibration results So you have a calibration step tablet for everyday use and a specially calibrated control step tablet to verify your Calibration penodically To contol a calibration l Scan yourcontrol step tablet e g Kodak Step Tablet no 3 and open the image in Melanie If nec essary rotate the image such that the light steps are displayed at the top Choose Tools gt Calibration Tablet gt Contol You are asked to load the calibration to be controlled This Calibration could have previously been saved using the Save icon in the Create Calibration window cal orcan simply
146. n the display align images show dual color orspot ovenap in the current sheet or change the way gels spots annotations or matchesare visualized Select specific spots spot sets annotations or matches Reports Display tabularorgraphical information on about selected gels spots annotations or matches and compute differencesand similanties between gel images The data analysis based on robust statistics factor analysis and sta tistic al tests Tools Change display quantification and other options at the software level and customize the user interface custom toolbars keyboard shortcuts Create and controla calibration tablet while working in the Image Pool Help Accessdocumentation and obtain license product version or system information Most menu commandsalso have toolbar icons and or keyboard shortc uts 2 2 1 Keyboard shortcuts Keyboard shortcuts when available are designated on the nght hand side of the comesponding menu command A list of all shortcuts is given in the Appendix Please note the logic behind the key combinations Ctrl is used to maneuver gels Shift is used to maneuver spots Altis used to maneuver annotations Chi Shift is used to maneuver matches Melanie User Manual Edition AC Graphicaluserinteface 2 You can create yourown keyboard shortcutsformenu commands CAUTION A new keyboard shortcut must be unique Be carefulnotto duplicate a keyboard shortcutthat isalready ass
147. nd orupper limits of your search interval F create spot set Create a spot set to annotate spots of interest for use ata laterstage All currently selected spots are automatically included in the newly created spot set which appearsasa column in the table You see a checked box forspotsthat belong to the set oran empty box forspots that do not belong to the set CH Statistic s Set the statisticsto be used forcalculating the Centerand Dispersion value of each class and consequently the Gap Ratio and Noma lized 138 Melanie UserManual Edition AC Data analysis 8 values These settingsare common to the ClassAnalysis Table and Class Analysis Histograms CAUTION The Center and Dispersion values define the interval that charactenzesthe protein sample of each classin a match To characterze a classonly by the central value set the Dispersion percentage sliderto 0 This is useful if you want to calculate the difference orratio between the central values of your classes With the Mean 100 selected as Central Tendency and the Dispersion slide r set to 0 the Gap and Ratio valuesthat are calculated in the Class Analysis Table correspond to the classical way to calculate the difference orratio between populations Altematively you can use the Fold and versus Class X options see 8 4 3 Fold change Settings Some of the above mentioned columns may not be displayed by default Click the Settings icon to show orhide columns from the t
148. nsthat the software divides the gel width height or the visible screen area by the number of subdivisions The Coordinate Units can be Centimeters Inches Pixels or pl MW units provided data isavailable in the annotation category pl MW The pl MW grid can also be displayed over gelsthat do not Melanie User Manual Edition AC 79 5 Gels 5 6 Gel reports 5 6 Gel reports 80 contain this information but that were matched to a gelhaving pland MW values M 0 map a Wi Grid Settings Attached to Gel Screen Number of subdivisions Horizontal Vertical Centimeters kb Inches Pixelz Figure 5 12 Grid linesin cm attached to the gel image 5 6 1 Gel table Choose Reports gt Gel Table to display a table Figure 5 13 with summarized information about the imagesin the current sheet File name ID path size modification and creation dates Image height and width expressed in pixels Rows and Columns pixel size PixSize Minimum and maximum gray levels before MinGray and MaxGray and after calibration MinValue and MaxValue Calibration information Calibration Formula Unit Name Creator and Date Melanie User Manual Edition AC Gel Table FileName A TL Gell IA Ti Gel A Ti Gel3 lA T2 Geli Gels 5 e Numberofspotsand annotations e Staining method and user defined gel descriptions e MatchSetto which the image belongsand the Classto which it wasassigned Mat
149. ny out several analyses using different matching schemes You can copy match sets to use them in another configuration In Figure 4 10 for example the match setsA Tl A_T2 B Tland B T2 were copied to be used in populations pertreatment T1 T2 ratherthan growing substrate A B Because existing matchesare conserved when copying match sets thiscan save a lot of work Workspace a EF 7 Image Pool Sige UM Project Geh AB E E Match Elei A Sipe AT1 A Il Gel A Il Gelz A Il Gel g _T1_Giel3 Deh ET1_1 Geh T2 Geib ATZ 1 Bei ET2 1 E Classes Figure 4 10 Match sets ATL AT2 BTL and BT2 were copied to be used in the match hierarchy T1T2 Melanie User Manual Edition AC 57 4 Projects 4 3 Create match hierarchy n uh n cb ROD VEZ rer A Tl Gel3 B TL Gel3 A_T2_Gel3 B T2 Gel3 A Tl Gel2 B TL Gel2 A T2 Gel2 B T2 Gel2 A Tl Gell B TL Gell A T2 Gell B T2 Gell Figure 4 11 A tree view of the match hierarchy in Figure 4 10 The match sets A_TI A_T2 B Tl and B T2 have been reused in a different configuration compared to the example in Figure 4 3 You can simply drag one or more match setsto a new destination or copy right click on the match set and paste them in the new destination right click on the destination project or match set The destination can be a match set oropen project Copies ofthe match sets including the existing matches are created A numberis appended to the orginal name You
150. o be uniquely recognized The software can therefore detect forexample if you delete a geland replace it with anotherone with the same name CAUTION You must be very careful when manipulating project match set gel and otherMelanie filesoutside the software in order not to comuptthe data 10 4 Exportto spotexcision robots Please note that in addition to exporting spotsto an excision robot it may be useful to save them aspartofa spotset orannotate them This allowsyou to easily select them lateron to add expenmental data such asmassspectrometry information 10 4 1 Bruker Proteineer SP spot pic ker Melanie can export spot coordinates of selected spots directly to the Bruker Proteineer SP spot picker by choosing File gt Export gt Spots to Picker gt Bruker Proteineer SP For more details about this functionality please see the documentation provided by Bruker Daltonics Melanie User Manual Edition AC 163 10 Data integration 10 4 Export to spot excision robots 164 10 4 2 GE Healthcare Ettan spot picker To use the Ettan Spot Picker two adhesive markersshould be placed on the gel before scanning These markers are used for the calibration of the coordinates that is fordetermining the cormespondence between the X and Y positionsof the analyzed gel image and the coordinatesof the actual gel located on the spot picker Figure 10 2 Once the gel hasbeen digitized and analyzed with Melanie the software can generate
151. o data sets differ Significantly It is used to test whether or not two samples may reasonably be assumed to come from the same distnbution It has the advantage of making no assumption about the distnbution of the data and isfrequently prefered overthe Mann Whitney rank sum test where there are many ties repeated values Please note that this generally comesata price Other tests e g the t test may be more sensitive if the data meet the requirements needed forthat test The assumptions of the Kolmogorov Smimov test are e The probability distributions are continuous e The measurement scale isat least ordinal e The two samplesare mutually independent In the Kolmogorov Smimov test the data points in each sample spot values fora particular match in a class are sorted in ascending order Melanie User Manual Edition AC Data analysis 8 and converted into anempincaldistibution function EDF Thisfunction gives the fraction of data pointsto the left of a given value z Forthe second example in Figure 8 15 the ordered data points from class X are 0 034 0 045 0 056 0 064 0 069 and 0 078 The fraction of data pointsto the left of each of these zvaluescan easily be calculated and plotted full line in an Empincal Distnbution Plot Figure 8 16 Itisclearthat no data lie strictly below 0 034 17 0 17 1 6of the data is strictly sma ller tha n 0 045 33 0 33 2 6 of the data is stric tly smaller than 0 056 50 0 50 3 6 of the
152. o flip rotate crop orinvert yourimages Instead use the software that came with yourscannerorthe dedicated tools in Melanie 3 2 5 File format The supported input formats are GEL Molecular Dynamics MEL Melanie lmageMaster TIFF Tag Image File Format IMG Fuji GSC and 1SC Bio Rad Please note that the default TIFF format doesnot include calibration information although some imaging toolsdo export tif files that contain calibration tags 3 2 6 DIGE file naming convention To facilitate the import of DIGE images it isrecommended that the file names forthe group of two orthree imagescontain a common stnng and their respective dye names Cy2 Cy3 Cy5 3 3 Preview images 34 3 3 1 Open images Youcanopengelimagesthatare in any ofthe above mentioned input formats mel gel tif mg gsc lsc DIGE imagescan also be imported and automatically grouped by opening ds files To open gel images Melanie User Manual Edition AC Image Pool 3 1 Do one of the following e Choose File gt Open e Inthe Workspace window click the Projecticon In the Add Files window search for Ales of Type All Image Files mel tf gel img ds e Inthe Navigatorof the Workspace window nght click on the Image Pool folder and select Add 2 Inthe Open FilesorAdd Files window browse the directory where the image file is located select its name and click Open Use the Shift or Ctr keys to make multiple selections
153. o scana Calibration step tablet orcalibration strip along with your gels These step tablets have known intensity values expressed in optical density OD or diffuse density DD published by the manufacturer of the step tablet Please note that forthe purpose of 2D gel analysis it is only useful to calibrate the image capture device when working in transparent mode No calibration needsto be done when you do reflective scanning When both a transparentand a reflective calibration strip are provided be sure to use the appropnate calibration step tablet Calibraton tablet file The OD values forthe step tablet have to be specified in a Calibration Tablet File together with other information such asthe height and width of the tablet and the number of steps An example of sucha Calibration Tablet File Kodak2 tab can be found in the Template Tablet folder of the Melanie installation directory ThisCalibration Tablet File ismade foruse with the Kodak Step Tablet no 2 If you do not use thisspecific step tablet you can copy the file and edit the data to make yourown Calibration Tablet File You can edit the file with tools such as Windows Notepad Asthe intensity values supplied with yourstep tablet are generally expressed in diffuse density DD you have to convert them to OD values Forthis purpose the manufacturer of the step tablet should provide the appropnate relationship Forthe Kodak Step Tablets no 2 and 3 forexample thisisOD
154. or more selected gels To edit gel descriptions 1 Select the imagesforwhich you wantto add oredit descriptions 2 Choose Edit gt Gels gt Edit Description 3 Inthe Add Gel Description window select an existing gel description category orcreate one Click OK 4 Enterthe geldescnption that appliesto the selected images Click OK 5 The new gel descriptions are displayed in the Gel Table Altematively you can define gel descriptions by clicking on the Add Description icon in the Gel Table toolbar It s behaviour is identical to that of the Edit gt Gels gt Edit Description menu You can delete all gel descnptions of a certain category forthe selected gels by choosing Edit gt Gels gt Delete Description 3 6 4 Permanentgel description categones When geldescnption categonesare created asdescnbed above they are only used forthe gelsthat were selected dunng the creation of the categones To make gel descnption categones permanent in the software alwaysavailabe from the category list you mustdefine them in the Options To create permanent gel description categones 1 Choose Tools gt Optons 2 Click the Add in the Gel Descriptions tab 3 Entera category name in the Add Category box and click OK 4 The category isdisplayed in the permanent category list 46 Melanie User Manual Edition AC Image Pool 3 To remove a pemanentcategory select it from the list and click Delete Melanie User Manual Edition AC 47 3 Im
155. ormation about annotations including the label Table content foreach category the annotation coordinatesand Spot ID if the annotation is linked to a spot Melanie UserManual Edition AC 25 2 Graphical user interface 2 Reports 26 2 7 1 Dynamic content Report content is continuously updated A report selection is synchronized to reflect the most current data from the coresponding sheet that contains the gel images Notice that reports are attached to their comesponding sheet If the sheet is closed the reports will be closed aswell 2 7 2 Content based on enabled spots By default allsootsare enabled and therefore represented in the reports But the content of reportscan be limited to a subset of spots by disabling spots that are not of interest See 6 4 3 to leam more about enabling disabling spots 2 3 Toolbars Most reportshave a toolbar with the following iconsin addition to some report specific iconsand functionalities that are descnbed in later chapters of the User Manual a Suspend synchronization By default the selection in the report is continuously synchronized with the coresponding sheet that contains the images and with other reports Click the Suspend Synchronization icon to stop the synchronization and render the selection in the report independent of the selection on the gel and vice versa H Save Entera name and select the desired format for the file to be saved Tablescan be saved in t
156. ot Asthe region moves with the anchor and vice versa you can easily crop a similar part of each gel by corectly positioning the crop areas of the same size in the gels Figure 3 4 38 Melanie User Manual Edition AC Image Pool 3 To define a crop area 1 FO E m Click the Region tool and place the cursoratthe top left position of the area you want to crop Hold down the left mouse button and move the cursorto the bottom nght position a dashed box is displayed Release the mouse button at the end point Move a crop area by clicking inside the box and dragging tt Change the size of the area by dragging a comeroredge To remove an area double click on the image If you want to attach an anchorto the crop area hold the Alt key while clicking on an easily recognizable protein spot A dark blue circle will be centered on the spot Note that thisanchor may be located inside or outside the crop area To change the position of the anchor hold the Alt key and click another spot To remove the anchor hold the Alt key and click on the anchor Propagate the crop area to the other imagesin your sheet by holding the Shift key while clicking in the crop area Adjust the position of the crop area in each gelby moving itso that the anchor iscentered on the appropriate spot File Edit View Reports Tools Help PES PACCA 3 dm h Sh A 24 TI Add Files to Project 3 Image Pool on D m e H 4a Crop area e
157. ots to select them on the gelsand any other open reports DIGE Histogram bk Id page 4 8 AJ Number of Spots Gel 02 Cy Control 1 1 5 Log Volume EE EE Figure 8 13 DIGE Histogram Two report specific toolsare available y Slider Move the sliderin the toolbarofthe DIGEHistogram window to view the histograms forthe other images in the sheet Measure Select one of the following options from the Measure icon e Max Slope Largest gradient associated with the co detected sp ots e Area Number of pixels within the spot boundary e Max Intensity Largest pixel value associated with the co detected sp ots e Max Volume Volume of the largest co detected spot Melanie User Manual Edition AC Data analysis 8 8 4 Analyze classes 8 4 1 Specify classes To identify protein expression vanations between populations of gels you need to specify what gels belong to which population by creating classes Classesare created in the Workspace Oftentimes you already know the populations for your set of gels This is the case when you are comparing gelsfrom healthy tissue extracts with those from disease associated samples When you have no preliminary knowledge of the populations in the set of gels you can draw possible conclusions from factor analysis 8 4 2 Overlapping measures Spot valuesfora given match within each classcan be summanzed by the central tendency and dispersion In addition to these descriptors t
158. ou want to display information Melanie User Manual Edition AC 15 5 Gels 5 4 Visually compare images 5 4 Visually compare images 76 When working in MatchSet or Classes sheets the software provides different tools to visually compare images to the sheet reference 5 4 1 a Sheet reference You can set the sheet reference by choosing View gt Sheet gt Set Reference Allotherimagesin the sheet are compared to thisreference image forthe optionsin the View gt Sheet menu Align Images Show Dual Color Spot Ovenap 5 4 2 Align images The Align Images feature facilitates the visual comparison of images that demonstrate large vanations in protein migration Choose View gt Sheet gt Align Images to activate it It is especially helpful when the images are in Stacked mode It warps Figure 5 9 the imagesin the sheet so that they are better supenmposed with the sheet reference and therefore with each other This allows easy identification of cormesponding spots To align images the software needsto know which positions in the different images corespond to each other that is represent the same protein fom This isdone by defining landmarks The alignment algonthm then deforms the imagesto supenmpose the landmarks CAUTION Asopposed to matching landmarks do not need to be linked with spots to cany out image alignment Therefore no spots need to be detected To align images in a sheet 1 Setthe desired image asthe
159. pots with a volume higherthan the given threshold are selected together with their matched spots which may have volumes that are smaller than the threshold These are the spots that are kept Choose Select gt Spots gt Inverse Selection 6 The curent selection includes all spots for which the maximum volume in any of the two orthree DIGE images is lowerthan the given threshold 7 Choose Edit gt Spots gt Delete to permanently delete the selected spots from your DIGE gels select By Value gt 30000 Figure 6 3 Select by Value can be used to filter soots based on their volume Please note that instead of deleting spots you can exclude them from the analysis by disabling them Melanie UserManual Edition AC 89 6 Spots 6 4 Select spots 6 4 Selectspots 90 6 4 1 R Select Spots can be selected with the Selecttool Once selected they are highlighted in green Figure 6 3 unlessthe default spot colorswere modified Note that all matched spotsare selected aswell If an annotation is attached to a spot the annotation is also selected Similarly if you select an annotation or label with the Selecttool the linked spot is also selected To select more than one spot select the first one and then hold down the Shift key while clicking on additional spots To select allspotsina region place the cursorat the top left position of the desired region hold down the left mouse button and then drag the cursorto t
160. ption prints one image per page a Sheet Print the current sheet Whatever your choice the image is printed as it is displayed on the screen retaining objectsand properties such asspots annotations contrast mapping and pseudo colors alignment zoom grid etc CAUTION With a zoom factor of 1 the printed gel image takesthe full paper width You can adapt the zoom factor to decrease the size of the pnnted image Page Setup You can change pnnt parameters such aspnntername papersize paperonentation etc Choose Ale gt Page Setup Thiscommand opens the standard pnnt window where pnnterrelated settingscan be modified Melanie User Manual Edition AC Spots 6 6 Spots 6 1 Introduction Once gelshave been added to a projectand you have taken a good look at them you are ready to detect spots A spot delineatesa small region in the gel where protein is present This shape is automatically differentiated bya spotdetection algonthm and quantified itsintensity area and volume are computed There are two different spot detection algonthms implemented e Non DIGEimagesare detected with the Melanie algorithm e DIGE imagesare co detected with GE Healthcare s DeC yder 2D algonthm 6 2 Detectspots in non DIGE gels 6 2 1 Procedure The Melanie spot detection algorithm is optimized to give relevant biological results with minimum user interaction You can preview spot detection and fine tune just a few parameters before
161. r where the license server isto be installed insert the Melanie CD ROM Click Install GeneBio License Server In the installation window click Next 4 Acceptthe default installation path and start the installation By changing the default installation path the file pathsin LMTOOLS will need to be updated accordingly 5 Answer Yesto any question about Windows Firewall this may or may not appear in order forthe license server to work propery 6 LMTOOLS automatically opens once the installation is suc cessfully completed Melanie UserManual Edition AC 9 1 G etting sta rted 1 4 Licensing Click the Config Servicestab and ensure thatalloptionsare setas in Figure 1 1 The file paths shown i e C Program Files GeneBio License Serven are valid if the default installation wasdone Click Save Service even if no changes were made Leave LMTOOLS open and proceed to the next section LMTOOLS by Macrovision Corporation http iwww macrovision com m EI File Edt Mode Help Service License File System Settings Utilities Start Stop Reread Server Status Server Diags Config Services Borrowing Configure Service Service Name Path to the Imgrd exe file Path to the license file Path to the debug log file Save Service GeneBio Remove Service Program Files GeneBio License serverilmgrd exe Browse Program Files GeneBio License server Licenses Browse Program Files GeneBio License
162. rast 67 annotation 152 cursorinformation 75 landmark 103 match gels 105 measure 66 move 63 profile 74 region 65 select 90 106 152 Shortcuts 172 show dualcolor 78 zoom 63 translation 73 transparency settings 74 transparent mode 74 tialperiod 8 timmed mean 114 midrange 114 triple detection 87 t test 132 133 Twain compatible scanners 161 U undo 167 unit 70 un pinned 29 update gels 159 usermanual 13 UUID 163 V value 113 120 122 vectors 107 view 15 Melanie UserManual Edition AC 193 194 signalintensity 67 visibility 74 vol 85 88 ratio 88 W whole image 71 Wilcoxon 134 workspace 20 classfolder 59 classes 59 match folder 54 match hierarchy 54 navigator 23 projects 52 toolbar 23 X XMLformat 162 Z zoom 63 73 altematives 64 apply to allgels 64 overview option 64 Melanie User Manual Edition AC www genebio com Geneva Bioinformatics GeneBio SA c o Swiss Institute of Bioinformatics CMU 1 rue Michel Servet 1211 Geneva Switzerland CENEBIO Geneva Bioinformatics SA Swiss Institute of Bioinformatics This version of Melanie hasbeen developed by the Swiss Institute of Bioinformatics in collaboration with Geneva Bioinformatics GeneBio SA and GE Healthcare Melanie is powering the Image Master 2D Platinum gel analysis software sold by GE Healthcare All intellectual property rights on this User Manual aswell ason Melanie belong to the Swiss Institut
163. ray levels that are present in the selected region This option is practical in combination with the unit and a relatively small region In this case you enter an adaptive mode that allows you to adjust to the local gray levels The effect isa regional increase in contrast that is useful for viewing very faint spots Colors The software offers varous color palettes to visualize the intensities in your image The Gray Saturation palette is helpful when you want to visualize saturated spotsorthe background It corespondsto the default Gray option except forthe maximum gray value saturation that is displayed in red and the minimum value background that appearsin blue To decrease the stringency on what is considered saturation orbackground you can modify the minimum and maximum gray levels Invert You can inverse the gray levels by checking the Invert box 5 3 2 3Dview Another way to examine the intensity vanations in gelimagesisby looking at the three dimensional view of gel regions Figure 5 6 In this type of view the Xand Y axesrepresent the pland MW values whereas the pixel intensity is plotted along the third dimension Zaxis The resulting image showsa peak foreach protein spot with a peak height that is proportional to the spot intensity 3D viewscan be rotated in any direction to look at the interesting spots from all sides thus facilitating spot editing ormatching decisions The 3D View window can be displayed by c
164. re in fact numbers that summanze spot values from a match The central tendency allows you to localize a center for the data whereas the dispersion indicates how closely the data points fall around the center CAUTION Absent spots with zero values are also considered in the calculation of statistics for both central tendencies and dispersion Specify and display the statistics by clicking the Statistics icon ina report toolbar Figure 8 1 Gra x Choose the statistics The sliders restrict the statistics to the central values Central tendency 8 Mean lt Midrange O Reference Dispersion Mean squared deviation O Mean absolute deviation Half range size Third section Figure 8 1 Statistics common to Gelor Class Analysis Tables and Histograms The third section is specific to different report typesand isdescribed in the corresponding sections of the User Manual Cental tendency The central tendency gives the general location of a variable This is commonly calculated by the anthmetic mean also known asthe average orcenter of gravity of a distnbution the median the middle value which dividesthe sample in two equal parts orthe midrange middle location between the two sample extremes Mean and midrange valuesare very sensitive to extreme values outliers and can be seriously affected by a single observation Onthe other hand the median is highly resistant to outliers A compromise is given
165. re likely to be and the more factors are calculated Since the first factors are generally the best ones for charactenzing gelsand matchesthat behave similarly than any subsequent ones the factors are ranked in order of importance Figure 8 11 showsthe Factor Projection Plot obtained when the first two axes in Figure 8 9 were selected In the example only the 20 most significant matches are displayed on the projection plot If all matches were shown one would find that many of them clusteraround the ongin of the graph This illustrates that the majonty of matches i e protein spots are not meaningful in classifying the gels The furtheraway a spot is from the origin the more important it is likely to be in terms of charactenzing the gels Melanie User Manual Edition AC Data analysis 8 Factor Projection Report Mean 100 and M 5 D GA AA Match ID Contrib Axel Contrib Axe Quality Figure 8 11 Factor Projection Report The ten matches with highest contribution to the second axis are selected To find a possible meaning fora given factor one should first identify the matchesthat largely contnbute to this factor The Factor Projection Table is used forthispurpose When sorting the matches according to their contribution to the first axis one discovers that the matchesat the top of the table are those with the highest relative volumes as found in the Gel Analysis Table In fact the first axis is generally co
166. rrelated with protein abundance Please note that the Factor Projection Table also contains the Quality foreach match Thisnumber tells you how close the distance of the projection isto reality Matches with very similar behavior similar expression profiles across gels are close in space However when projected onto a two dimensional subspace matchesthatare actually farapart may appearclose together It istherefore important to lookat the Quality to judge whethermatchesare effectively close Ifthe values are high for both matches the chance is great that they are indeed Melanie User Manual Edition AC 127 8 Data analysis 8 3 Analyze gels nearby and have a similarbehavior Ifone ofthe matcheshasa low value any interpretation becomes tentative Gels that are adjacent on the graph are likely to be similarto each other They may correspond to the same population This is cleary the case inthe example above The gelsfrom T1 clustertogetherbelow the honzontal axis whereas the gels cormesponding to 2 lie above The closera match isto a given set of gels the more charactenstic it is likely to be of those gels That is the more important the match is in detemining why those gelsare different from other gels In Figure 8 11 one can observe that the matches 48 123 180 181 and 248 are close to the gelsbelonging to T1 The histogramsin Figure 8 12 show that these matches have high spot valuesforthe Tl gelsand low values forthe T2 gels Mat
167. rrent selection can also be used asinput Specify the operator And Or Not Equal or Exclude 4 Entera new name forthe resulting spot set Altematively the output can be immediately reflected in the current selection Click OK Select To select spot sets choose Select gt Spot Sets 6 4 3 Enabled spots By default allsootsare enabled and therefore represented in the reports To exclude a specific subset of spots you can disable protein spots that are not of interest or specifically define a set of spots to be enabled Only enabled spots appear in reports The optionsrelated to the creation saving orloading of setsof enabled spots can be chosen from the Edit gt Enabled Spots menu W set Select the spots you want to focusyouranalysison and choose this option to enable the selected spots Afterdeselection excluded spots are disabled and appearin yellow Add Use thisoption to add selected spotsto the curent set of enabled spots Remove Use thisoption to remove selected spotsfrom the current set of enabled sp ots Save Use this option to save the currently enabled spotsasa new spot set Entera name and click OK Load Use this option to enable spots belonging to an existing spot set Select To select the enabled spots choose Select gt All Enabled Spots 6 5 Display spots 92 65 1 spotshape Once spotsare detected you can choose how to display theirshapes outlined crossed filled outline
168. rs The scrollbars on the right and bottom edgesof each image can be used to change the zoom factor of an image by dragging one of the endsof the scrollbar You can also move the visible area of the gel horizontally or vertically by clicking in the middle of the scrollbarand dragging left nght or up down Click on the gray square atthe intersection of two scrollbarsto reset the image to its full image size If you want to view a specific area of yourgel image choose View gt Global gt Scrollbars gt Adjust In the Adjust Visible Area window set the exact honzontal and vertical start and end coordinates forthe area to be displayed You can do thisin termsof different units Pixel coordinate Percentage and Real coordinate pland MW Please note that pl MW annotations must be defined in orderto use this function To hide the scrollbars choose View gt Global gt Scrollbars gt Show In that case You can still move and zoom images by using the Move and Zoom tools in the toolbar 2 5 4 Switch order You can change the orderin which imagesare displayed by dragging the gel name onto another image It isthen inserted before thisimage Simila dy you can re order panes by dragging theirtabsto a new position Swap panesorimagesby choosing View gt Sheet gt Navigation gt Switch orthe Ctr F shortcut This reverses the last re ordering operation applied to a pane orimage Melanie User Manual Edition AC Graphicaluserinteface 2 2
169. rsion the projects are automatically inserted in the workspace Batch File Conversion EH Name Path Creation Date Modification Date Comment U Software Name Version DIGE C Program Files GeneBio Melanie 6 Tu 18 01 2008 02 09 25 01 2008 11 11 5 Melanie Figure 10 1 Select projectsfrom previous Melanie orlmageMasterversionsfor Batch File Conversion 10 2 Acquire images from Twain compatible scanners Melanie can also acquire images directly from TWAIN compatible scanners 10 2 1 oy Select source You must indicate the scanning source by choosing File gt Import gt Twain gt Select Source All WAIN compatible scannersattached to your PC are automatically recognized by Melanie This selection only needs to be done once unless you want to change to a new image capture device Melanie UserManual Edition AC 161 10 Data integration 10 3 Export data 10 2 2 amp Acquire Launch the scan with File gt Import gt Twain gt Acquire The scanner software opens giving you the opportunity to change the necessary settings and subsequently initiate the scanning process Once this is done the image is saved in Melanie file format and added to the Image Pool 10 3 Export data 162 10 3 1 Reports Data obtained in the analysiscan be exported for use in other applications by saving the vanoustabularreports Tablescan be saved in text format txt asa Microsoft Excel Workbook xls or in XML xml format
170. s 63 analysis histograms 121 analysistable 119 description options 30 descriptions 45 export 81 ID 163 print 81 reports 80 save 81 shortcuts 172 table 24 45 80 generalsettings 113 Genetix GelPix spot picker 165 Genomic Solutions ProPic spot picker 165 good images 33 graphical userinterface 15 gray levelmapping 68 gray saturation 71 grid lines display 79 edit 79 show 79 grow spots 93 GUI 15 H half range size 115 help 12 15 hide annotations 156 ID 108 labels 156 histograms classanalysis 139 DIGE 129 gelanalysis 121 history 168 clear 169 copy 169 insert marker 169 print 169 refresh 169 save 169 http link 150 queries 147 Melanie UserManual Edition AC I image pool 20 35 display 35 hide 35 properties 35 remove 35 sheet 35 images 63 calibrate 40 Calibration 33 compare 76 depth 33 describe 45 editing 33 export 81 file format 33 file names 33 good 33 manipulate 63 open 34 preview 34 print 81 82 process 37 resolution 33 save 81 selection 21 to clipboard 82 to file 82 import 159 162 annotations 159 croparea 37 labels 159 match set 58 insta II license server 9 software 8 installation cd rom 8 installer 8 intensity 70 85 interface 15 interpreta factoranalysis 126 invert 71 gray levels 37 ipconfig 9 isunique 146 K keyboard shortcuts 16 Kolmogorov Smimov 136 L labels 159 add 159 copy 158 Melanie UserManual Edition AC 185 186 create 143 create categores 145 delete 15
171. s activated the Edit Label window is displayed Change the text in this box to modify your label content The Selecttool should also be used to change the position of an annotation In this case simply select an annotation and drag itsbasis to the new location 9 8 2 Edit menu Various options for editing labels or annotations are available from the Edit gt Annotations menu All of these featurescan be used to edit several labelsorannotationsata time hee Ta Delete annotations and labels Two possibilities are available fordeleting selected labelsor annotations e Edit gt Annotations gt Delete deletes the selected annotations Edit gt Annotations gt Labels gt Delete deletes the selected labels To delete labelsfrom a specific category only first select all labels from the desired category with Select gt Annotations gt By Category K4 Edit labels You can change the content of selected labels belonging to a single category by choosing Edit gt Annotations gt Labels gt Delete and entering the desired modifications in the Edit Labels window Copy paste annotations Youcanselectannotationsin a given gelimage and copy them to the comesponding locationsin other gels Thisisa simple meansof creating a set of similar annotations in a seres of gels Subsequently you may need to adjust the annotation positions Select annotations on one image and choose Edit gt Annotations gt Labels gt Copy Then select the
172. sare descnbed in detail elsewhere in the User Manual This toolbar contains the basic tools of the software More importantly their functions Move Zoom Region Selection Measure and Landmark are not available in any menu Although they do have corresponding keyboard shortcuts Display PROD Thistoolbarcontainsthe options Undo Zoom Move Redo Zoom Move Adjust Contrast and Cursor Infomation Window Image Ad amp ty dh k Di addFies to Project The image toolbar Rotate Flip Invert Gray Levels Crop and Add Files to Project iscontextual itisonly available when working with imagesin the Image Pool Detect And Match Spots OG di Thistoolbarisoniy displayed when working with gelsin a Match orClass sheet i e once gelshave been imported into a project You can Detect Enable Edit Match Gels Add Match Delete Match and Show Vectors Edit Spots GQ SB OC This toolbar is only available when spot edition is enabled Choose Edit gt Spots gt Edit Enabled to get into this mode 2 3 2 Customize toolbars Toolbarscan be configured according to your individual specifications Melanie User Manual Edition AC Graphicaluserinteface 2 Toolbar position To change the position of a toolbar click the leftedge and drag the toolbarto the position you want You can drag a toolbarto any of the edgesofthe GUI When a toolbarisdragged outside of the frame of the Melanie window it becomesa floating window Toolbar format
173. scamed outon a gel Menu Command Ctr Shift Z Edit gt Redo Melanie UserManual Edition AC 171 A Shortcuts A 2 Tool shortcuts A 2 Tool shortcuts mea Je A 3 Gel shortcuts mea Mecom Guo ct ca ce cb i i EE 172 Melanie User Manual Edition AC Shortcuts A AA Spotshortcuts mea peu A 5 Annotation shortcuts mew Momma a a mu ES ms EG m A 6 Match shortcuts mea pemco Ctn Shift A Select gt Matches gt All Ctn Shift G Edit gt Matches gt Add Match Melanie UserManual Edition AC 173 A Shortcuts A 6 Match shortcuts es mp Ctr ShiftH View gt Matches gt Show ID Ctr ShiftHK View gt Matches gt Hide All ID Ctr ShiftHN Select gt Matches gt Inverse Selection Ctn Shift U Edit gt Matches gt Delete Match Ctn Shift Y View gt Matches gt Show Vectors 174 Melanie User Manual Edition AC References B AppendixB References Bl Software Appel RD Hochstrasser DF Roch C Funk M Muller AF and Pellegrini C 1988 Automatic classification of two dimensional gel electrophoresis pictures by heunstic clustering analysis A step toward machine leaming Electrophoresis 9 136 142 Appel RD Hochstrasser DF Funk M Vargas R Pellegrini C Muller AF and Scherer jJ R 1991 The MELANIE project from a biopsy to automatic protein map interpretation by computer Electrophoresis 12 722 735 Appel RD Palagi PM Walther D Vargas R Sanchez C
174. se HTIP addressand query engine are entered asconstraintsto the annotation category see Figure 9 3 Type them in asone string in the Extemal Engine field forexample http www expasy org swiss 2dpage Create Category Define constraints for category MEW Category attributes Url External Engine pas ong swiss 2dpage Display properties Color C By default Hidden Figure 9 3 Setting the Extemal Engine asa category constraint Federated 2 D PAGE databases A list of federated 2 D PAGE databases with the required database query formats can be found at http www expasy org ch2d 2d access html or by clicking on the Ud button to the nght of the Extemal Engine field Copy the desired database addressand query engine from this site Other databases It is possible to find the required query format fordatabasesthatare not federated orthat do not contain 2 DE data Directly query the database until you find a specific protein orother entry Then copy the addressof the corresponding Web page in yourbrowserto the Extemal Engine field of the Create Category window without including the accession or identification number of the curent entry Generally this address consists of the database HTIP addressand query engine followed by a question mark or equal sign Melanie User Manual Edition AC Annotations 9 Forexample you might display the entry forthe protein structure 1BMT in the well known Protein Data Bank
175. se the mouse scroll wheel to zoom in or out e Use the View gt Gels gt Navigation gt Zoom menu e Use the shortcut keys forthe above mentioned menu commands e Use the scrollbars e To temporanly enlarge an area ina gel image hold down the Ctr key while the Zoom tool is activated The area under the cursor is enlarged asif you were looking through a magnifying glass Ld Overview option When you zoom in ona gel itcan be helpful to have an overview of where the region islocalized on the full image Figure 5 1 This overview enablesyou to easily locate and move to any region you want on your gel Choose View gt Global gt Show Overview to show or hide the overview of each image in its lower right comer The green rectangle in the overview correspondsto the curent view of the gel You can drag the green rectangle to another position to display a new region Melanie User Manual Edition AC File app Gels 5 Edit View Select Reports Tools Help MU ATi AT2 BT1 B12 PER eoig soo Gels 12 Spots0 Matches 0 Annotations 0 Figure 5 1 Overview option activated In the lower right comer of all images in the Display Zone isa small overview of the entire gel with a green rectangle comesponding to the visible gel area 5 2 3 i Region A region isa rectangular area in an image that is of interest for displaying a 3D View for previewing spot detection parameters or adjust contrast settings for cropp
176. sed ina single gel Spots to be picked are selected highlighted in green 10 4 3 Genetx GelPix spot picker To export spot coordinates of selected spots directly to the Genetix GelPix spot picker choose File gt Export gt Spots to Picker gt Genetix GelPix 10 4 4 Genomic Solutions ProPic spot picker The ProPic spot picker producesa TIFF file of the entire gel holderarea with a resolution of 1035 x 1317 pixels each pixel representing approximately 330 x 330 microns of the image area This ProPic image can be analyzed with Melanie and spot coordinatesin the image can Melanie UserManual Edition AC 165 10 Data integration 10 4 Export to spot excision robots be exported back to the ProPic spot pickerin a predetermined file format The ProPic software subsequently translates the image X Y coordinates into ProPic robot X Y coordinates by using a robot map Since the robot mapping assumes that each X Y coordinate in the image always cormespondsto the same position on the robot bed the ProPic image must remain in its orginal fom It can never be resized cropped orrotated The ProPic image isa picture of the curent state of the gel from which the spots are to be picked It isnot necessarily the image used to determine the spotsto pick One can select spots from any analytical image ofthe same gel obtained from a different resolution system and analyzed with Melanie However the analytical image needsto be aligned to the ProPic i
177. sed to flag items with their specific characteristics protein name database accession numberand so on orto mark spots with common character stics They also offerthe possibility of linking and associating gel objectsto extemal query engines ordata sourcesof any format text html soreadsheet multimedia database located locally oron the Intemet his annotation is attached oa pixel cross basis E Thi annotation is attached o a spot square basis Figure 9 1 Annotations are composed of an annotation basis square or cross an annotation flagpole and a set of labels flag An annotation isdefined by its position on the gel Xand Y coordinates and itssetof labels Each label belongsto a predefined or user defined category As shown in Figure 9 1 each annotation iscomposed ofa basis which can be a square ora crossdepending on whetherthe annotation is attached to a spotora pixel a flagpole and a flag that consists of a set of colored labels Spot and pixel annotations You can create two typesof annotations Figure 9 1 s Annotations linked to pixels are visualized with a cross basis They are simply connected to a pixeland have the same coordinates asthat pixel e Annotations linked to spots are visualized with a square basis They are linked to a spot and have the same coordinates as that spot that is asthe spot s center of gravity These annotations are automatically selected when the linke
178. send the physical addressofthe computerwhere Melanie isto be installed If purchasing a floating license send the physical address of the computer where the license server isto be installed To find the physical address while running the software 1 Launch the software 2 Choose Help gt License Info 3 Inthe License Information window click Copy to clipboard 4 Paste the clipboard content into an email and send this to melanie genebio com so that your license can be created To find the physical address of the license server or when the software is not yet installed 1 Onthe computer where the license file isto be placed for example the license server fora floating license choose All Programs gt Accessones gt Command Promptin the Windows Start menu 2 Inthe Command Prompt window enter ipconfig all There must be a space between ipconfig and all 3 The response should contain several parameters for the network Look forthe line Ethemetadapterlocal Area Connection Then look forthe Physical Address 4 Note down the PhysicalAddressamong the displayed information and send it to melanie genebio com so that your license can be created The physical address must be correctly recorded forthe license file to work 1 4 2 Install the license server This section only pertainsto floating licenses If you have a node locked license then skip to the next section To install the G eneBio license server 1 Onthe compute
179. sequently labels and categories e Name ofthe image on which the annotation was created Melanie User Manual Edition AC 159 9 Annotations 9 9 Annotation table e SpotlD ifthe annotation is attached to a spot e Coordinates ofthe annotation e Calculated pland MW values ifpl MW annotations were defined on the image ora matched image e Acolumn foreach label category with the label content in the corresponding cells Annotation Table GRA AIS FileNa i Divel X Pie B_T2_Gel3 94 IB_T2_Gel3 230 B_T2_Gel3 254 B_T2_Gel3 B_T2_Gel3 651 E E rd si sl Figure 9 9 Annotation Table 160 Melanie UserManual Edition AC Data integration 10 10 Data integration 10 1 Convert projects from earlier software versions Melanie can Convert Projects created with versions 4 5 and 6 of Melanie and ImageMaster 2D Platinum Images analyzed with older versions must be added to a project in order to be imported into this new version and forspots annotations and match data to be recovered You can do this batch conversion for many projectsata time Choose File gt Import gt ImageMaster 2D Platinum or Melanie Data and indicate the folder where some orall of your projects pn files are saved The software searchesand displays all project files found in this folder and allows you to select projects for conversion Figure 10 1 Then you must indicate the location where the converted projects Should be saved After conve
180. several classes in the Workspace To cany out advanced expression analysis you must work in this type of sheet When you move the mouse cursorovera sheet tab the screentip specifies the type Melanie User Manual Edition AC Graphicaluserinteface 2 Selection The current sheet isalwaysin frontand itsname isin bold Selecta sheet by clicking on itstab Close Click the Close icon in the uppernght comerof the curent sheet when you are finished working with it Layout The Layoutic on to the left of the Close icon can be used to choose the arrangement of the panesin a sheet Sta cked Panes in a sheet are one on top of another Tiled Panesina sheet are side by side One Row Panesin a sheet are ina single honzontal line One Column Panesin a sheetare in a single vertical line Free You specify the numberof panes laid out honzontally and vertically in a sheet 2 5 2 Panes There can be one ormore panesin a sheet see Figure 2 3 Each pane hasa tab with its name On the left side of the tab one or more icons describe the match orclass hierarchy Selection Selecta pane by clicking on itstab Use the Shift or Ctrl keys to make multiple selections Click the sheet tab to selectall panesin a sheet Selected panes have green tabs By default panesare laid outin Tiled mode When working ina different mode like Stacked bring a hidden pane to the front by clicking on its tab Layout Click the Layouticon on the nght side of
181. sheet reference with View gt Sheet gt Set Reference 2 Select the Landmark tool and define a few landmarks bearing in mind the rules listed below Choose View gt Sheet gt Align Images 4 If partsof yourimagesare still not sufficiently aligned you can add extra landmarks The alignment is automatically updated 5 To view the original images choose View gt Sheet gt Align Images The original images replace the aligned ones Melanie User Manual Edition AC Gels 5 es aqa dy m a D aja Sk hn i ta NEON SE e gao Bel am e A 1 mm m h zgi r gt Si dn A i r 3 ogo b ka a Ze My o SA ua p 4 THM AN KE W ex un wf eege e Sy Sekt Lh Wee ee wa ET a ae i N H al BA ac wa 33 74 L KE Soe D So gen H Bon a a KC SR g dh 3 en Fot o n t A gr Tay OE AZ R SIP SA 3 a aa a ani ae Porri v q ZE apts Z VM Da em PZ gei o o gt eh w a aw 7 T a SIL lk s a P da Se e u d A a WEE re de Ae 4 a bit H E KA Ki o Z nrz ii d 2 KR S i de L 4 po a Pp aur WT en Pr r d a pa ei f HBr d d i As WW A CIS d rd Ke UP e ly ee ca S F toi l fr Pe eae l a s a 4 b Figure 5 9 Image a before and b after alignment The match vectors and landmarks are displayed Note that the addition of suspect or badly positioned landmarks can worsen the alignment results The following rules mus be considered when defining landmarks e he numberof lan
182. sses isto find the protein expression vanations between different biological states The classes created in the Classes folder of a given match hierarchy can contain any of the imagesin that hierarchy One image can be in different classes In Figure 4 4 forexample the same 12 imagescan be compared as part of the 4 classes AT1 AT2 BTL and BIZ oras part of the two classes Tl and T2 You can define classes at any time even in the very beginning of your gel analysis study when no spots are detected To cany out statistical analysis however your gels must be detected and matched Workspace Ow I 7 Image Pool Sige UM Project E nfh AB E Match Gi A Ge ATI Geh AT Elek B E DI Geh BT2 el Classes g a ATI A Ti_Gell 4 T1_Gel2 A T1_GelS eH AT fe BT eH BT eS T1 Figure 4 4 The Classes folder 4 2 Create a project 52 4 2 1 The firsttme the software is launched Aslong asno projects have been created oradded to the Workspace e g the first time you open the software you are prompted to create Melanie User Manual Edition AC Projects 4 a project see Figure 4 5 Entera project name browse to where the project foldershould be saved and possibly add a comment New Project Project Name Location CA Documents and settings Melanie UsernMy Documents M elan Comment Figure 4 5 New Project window 4 2 2 Atany time You can create a new project at any time by selecting New from th
183. stematically biased Stain intensity va nations differences in protein loading orimage acquisition problems are some typical causes In the Scatter Plots window you Can visualize a scatter plot foreach gel in the sheet versus the sheet reference together with the best fit line corelation coefficient and the number of matches displayed Scatter plots are interactive You can click on the points representing the matched spots This in tum selects the spots in the gelsand other reports J Glider Move the slider in the toolbar of the Scatter Plots window to view the scatter plots forthe other images in the sheet H Scatter table Click the Scatter Table icon in the toolbarto show orhide the table below the plot The Scatter Table displays foreach pairof matched spots in the scatter plot the comesponding spot values in the gels and the erorin relation to the regression line Ba Copy fomula to clipboard Select the Copy Fomula to Clipboard option from the Save icon in the drop down list to copy the regression formula and comelaton coefficient to the clipboard for use in other applications Melanie User Manual Edition AC Data analysis 8 8 3 2 Gel analysis table The GelAnalysis Table and Histogramsprovide valuable toolsforlooking at unusual matches within a set of gel images Analyzing protein expression changes checking spot detection orvenfying matching operations are just a few of the potential uses Choose Reports gt
184. tandards the software can compute approximate pland MW valuesforall the spots pixels in this image as well as any other images matched to tt The principle israthersimple You just have to define pl MW annotations fora certain number of spoty pixels in the gel The calculated pl and MW values forall spots in this geland any matched gelsare 96 Melanie User Manual Edition AC Spots 6 automatically available in the Spot Report or Cursor Information window see below In addition pland MW grid linescan be displayed over the images To define pL MW annotations on an image 1 Select the image for which you know the pl and MW values for several protein spots Spots may or may not have already been detected in this image Click Selectin the toolbar Double click ona spot pixel forwhich you know the pland orMW values 4 Select the pl MWcategory in the Create Annotation by Click window 5 Enterthe known pland MW values respectively separated bya space Replacing one of the values with 1 meansthat no value is set 6 Do thisfora sufficient number of protein spots that are distributed overthe whole image area Obviously the more spots and annotations the betterthe approximated pland MW valueswillbe Melanie doesthe following to calculate approximate pland MW values In the case of pl it looks up the two closest annotations to the left and to the nght of the spot for which the pl will be determined and then interpolates b
185. tensity Region Whole Image Colors Grap Invert Apply Figure 5 5 Adjustmentsto the gray level mapping forthe selected image are immediately reflected in the preview region In this example the maximum gray level was set to 13300 by moving the right side of the slider to the left All pixels with a gray level higherthan 13300 appearasblack By setting the Bending sliderto 3 the image becomes darker e You can additionally use a non linearmapping function The Bending parameter i e sliderto the left of the histogram expands orcompresses the contrast at the dark or light ends of the range When the bending parameter is positive the image is lighter When the bending parameter is negative the image is darker Contrast Select the image for which you want to see the current minimum and maximum gray level settings When you make changes to these settings the list will disolay Modified to reflect this fact Unit Two different unitscan be used fordisplaying the gray level minimum and maximum Melanie User Manual Edition AC Gels 5 e Intensity uses the raw pixel values as displayed in all the reports When a calibration isdone these correspond to the calibrated pixel values e Choosesthe scale asa percentage of the total gray level range in the histogram Region You can display the histogram forthe gray levels in the e Whole Image This isthe default option e Selected Region he software only considers the g
186. the position you want Apply to all gels To move all gelsin the current sheet by the same displacement hold down the Shift key while changing the position in one of the gels Double click To move allgelsin the current sheet to the same position with the same zoom factor double click on one of the gels The cormesponding position in the different gels is estimated by interpolating between the surrounding matches or if no matches exist between the two nearest landmarks Finally when no landmarks exist the gelsare aligned atthe same location using the X and Y coordinates Altematives There are other waysto change positions in gels e Use the View gt Gels gt Navigation gt Move menu e Use the shortcut keys forthe above mentioned menu commands e Use the scrollbars 5 2 2 ES Zoom Select this tool click repeatedly in the area of the gel where you want to zoom in Right click repeatedly on the gel to zoom out You can also define a zoom area place the cursorat the top left comer of the area hold down the left mouse button and move to the bottom right position a red box isdisplayed Release the mouse button atthe end point Melanie UserManual Edition AC 63 5 Gels 5 2 Manipulate images 64 Apply all gels To move allgelsin the current sheet to the same position with the same zoom factor hold down the Shift key while zooming in orout on one of the gels Altematives There are other waysto zoom gels e U
187. the tab to change the arrangement of the imagesina pane The optionsare the same asfor panesina sheet 2 5 3 Images By default the gel name is displayed in the upper left comerof an image The colorof the name indicates whether the image isselected green ornot gray If an image name hasa red comer thismeans that it isthe reference image forthe matching If an image name isa darker green ordarkergray than the other images then it isthe current Melanie User Manual Edition AC 21 2 Graphical user interface 2 5 Display zone 22 sheet reference All other images in the sheet are compared to this sheet reference when using the menu commands in View gt Sheet To hide the image names choose View gt Global gt Show Gel Names The image name isreplaced by three dots Move the mouse overan image name to view a screentip specifying the match hierarchy In a Class sheet the classand path to which it belongsare also given Ifthe image isthe sheet reference then thisfact is included in the screentip Selection Select an image by clicking on itsname Use the Shift or Ctrl keys to make multiple selections Click the pane tab to selectallimagesina pane When imagesare hidden like in Stacked mode bring an image to the front by clicking on itstab atthe bottom ofthe pane Quickly siftthrough images using the Page Up and Page Down keys on your keyboard or click on the navigation tnangles in the lowernght comer of a pane Sc roliba
188. tion The Cursor Information window is available from the menu View gt Global gt Cursor Information Window or by clicking the corresponding icon in the Display toolbar It can be used at any time to display information on pixels and spots located at the position of your mouse Cursor Figure 6 9 Information on the pixel under the cursor 98 Melanie User Manual Edition AC Spots 6 e Name ofthe image e Calibrated pixel intensity e Xand Y coordinates expressed in pixels in pland MW units if available and in cm orinches Information on the spot under the cursor s Spot ID e Quantification values Intensity Area Vol and Gool Depending on the spot detection algorithm used the Saliency Vol Ratio and Slope are also provided Cursor Information Properties Values File ame E_T1_Gell Fisel Intensity fede Pizel 1059 Pixel 699 10 59 6 99 Spotl 390 Area n59 Intensity 1848 Vol 423352 aval 0 0583 Salency 469 Vol Ratio Slope Figure 6 9 Cursor Information window Melanie UserManual Edition AC 99 6 Spots 6 8 Spot reports 100 Melanie UserManual Edition AC Matches 7 7 Matches 7 1 Intoducion You know how to create an efficient match hierarchy possibly with different levelsof match sets and to display thishierarchy in a sheet You can also detect spotsin allthe gelsin sucha hierarchy You are now ready to initiate the matching process itself Matching isa key operation in 2 DE image ana
189. tors or dimensions that explain most of the vanance observed The factor ana lyss tool is used to examine the inte relationships between large numbers of vanables i e spot values fora senesof gels and to explain these relationships e g gel populations in tems of common undenying factors or associations with specific soot pattems Factor analysis isa complex statistical technique whose comprehensive description is beyond the scope of this manual For more information references are given in the Appendix Perfom a factor analysis A factoranalysis is ca med out on all orselected matches in the Gel Analysis Table You must judge which of the optionsdescnbed below is most applicable to your analysis To cany outa factor analysis 1 Click the Factor Analysis Table icon in the Gel Analysis Table toolbar Melanie User Manual Edition AC 123 8 Data analysis 8 3 Analyze gels 124 2 Ifanymatchesare selected the software askswhetheryou wantto use all oronly the selected rows 3 The Factor Analysis Table Figure 8 9 is displayed with the lines corresponding to the axes that can be drawn in the Factor Projection Plot Select two axesto be displayed in this plot generally the first two ones 4 Click the Factor Projection Reporticon in the Factor Analysis Table toolbar Factor Analysis Table Mean 100 and M 5 0 M D Axe Variance A_T1 Geli A_T1 Gel2 A_T1i Gel3 AT Geli A T2 Gel A_T2_Gel3 3 101623
190. ts size 3 3 2 Image pool sheet The gelimagesappearin the Image Poolfolderin the Workspace and are automatically opened in the Image Pool sheet Figure 3 2 Non DIGEimagesallappearina single pane with the name Files A separate Melanie UserManual Edition AC 35 3 Image Pool 3 3 Preview images pane isdisplayed foreach DIGEgel containing the imagesbelonging to that gel Remove Gels remain in the Image Pool until they are added to a Project When you are finished working with an image nght click on itand select Remove o remove all imagesin the Image Pool nght click on the Image Pool folderand select Remove All Please note that this action neveraffectsthe onginal image files which alwaysremain unchanged in their original location Hide display If you do not want to display all gels in the Image Pool sheet you can nght click on certain itemsin the Image Pool folderand select Hide To show a hidden image nght click on it and select Display To open the Image Poolsheet atany time nght c lick on the Image Pool folder in the Workspace and select Display Properties To rename a gelorimage orto see the file path nght clickona geland select Properties File Edit View Reports Tools Help ly bt PR DO id 4h Sh Ae kW AddFies to Project Workspace a 3 Image Pool a ES z FP Image Pool Pick EI Gel 01 Gel 01 Cy Standard Gel 01 Cy3 Control Gel 01 Cy5 Treated El p Gel 02 Gel 02 Cy Standard Gel 02 Cyd Treated Gel 02
191. u can also choose whether you want to force the calibration curve to go through the ongin see Reports icon below 9 Once you are satisfied with the calibration close the Create Calibration window The software asks whether you want to apply this new calibration If you answer Yes Melanie applies the calibration to the image The iconsin the toolbarof the Create Calibration window and their use are EG Open anotherstep tablet definition H Save the calibration with the extension cal ta Printthe calibration graph Ba Copy the calibration graph to the clipboard EE Display related reports e The Fitting Table displays the calibration formula e The Calibration Table displays foreach step the step number the measured average gray level the theoretical intensity value and the fitting eror difference between the curve and the point The Force Curve Through Ongin option allowsto force the Calibration curve to go through zero 3 5 3 Apply a calibration Once you have created a calibration using a step tablet and saved thiscalibration in the step tablet image file ora calibration file cal you can apply the calibration to newly scanned image files Melanie UserManual Edition AC 43 3 Image Pool 3 5 Calibrate images 44 To apply a calibration to a gel 1 Display and select the gelsthe calibration should be applied to 2 Choose Edit gt Gels gt Apply Calibration 3 Select the source of the calibration informatio
192. usly updated to stay synchronized with the coresponding sheet that contains the gel images Simplified import and visualization of images Improved population matching Management of multiple matches and composite spots Reorganized menu structure with icons Custom and context related toolbars One click to choose desired layout of sheets and panes Option to suround spot selections by boxes for easier identification Single tool to select edit sootsand annotations Dedicated landmark tool Measure tool to compute pixel pl MW or real world centimeter orinch distances between spots Reviewed contrast adjustment feature New 3D View Customized report templates Adaptive display of histograms And much more Melanie User Manual Edition AC Graphicaluserinteface 2 2 Graphicaluserinterface 2 1 Aboutthe interface The graphical user interface GUI isdivided into fourmain parts shown in Figure 2 1 They are the Menu Bar the Toolbars the StatusBar and the Display Zone The Display Zone isthe center of the interface Thisis where gel images are arranged in sheetsand panes The Workspace and any reportsare dockable windows found along the edgesof the Display Zone File Edit View Select Reports Tools Help PERLO PB OB 0 GPARs rts 7 Image Pool 1 aoeds HOR GH A AV FileName SpotID Match ID Pixel X Pixel Y pI MW Intensity Area S vol Comment Landmark A_T2_Gel2 608 219 972 633 98384 663 223855
193. ust to outliersthan the statistics above Figure 8 2 b e The Midrange 100 and Half range 100 define an interval that includes all sample values Figure 8 2 c e The Midrange 50 and Half range 50 are known asorder statistics and interquartile ranges Figure 8 3 0 8 0 8 0 8 0 6 0 6 0 6 0 4 0 4 0 4 0 2 0 2 0 2 0 0 0 Figure 8 2 Histograms showing the sensitivities of central and dispersion values a Mean 100 and M S D 100 b Median and M A D c Midrange 100 and Half range 100 Figure 8 3 Histograms showing the effect of suppressing outliers Midrange and half range values are given for a 100 b 80 c 50 and d 33 8 3 Analyze gels 116 8 3 1 Scatter plots To analyze gel similantiesorexpenmental vanations such asdisparitiesin stain intensity orsample loading you can produce Scatter Plots for matched spots Figure 8 4 by choosing Reports gt Analyse Gels gt Scatter Plots Melanie User Manual Edition AC Data analysis 8 Scatter Plots 0 951 x 0 0125 Corr 0 9222 Count 419 5 B_T2 Gell Match ID SpotlD Ref Gool A _T1 Gell B_T2 Geli Fitting Error 560 563 0 379515 0 138808 0 174151 559 of 4 0 0943084 0 0999504 0 00817633 0 224839 0 04354845 Figure 8 4 Scatter plot for matched spots Scatter plots give an idea of the relationship between the spot values from two gels by searching forthe lineardependence between the spot valu
194. ut enabled spots e Name ofthe image on which they were detected e SpotID see below and Match ID if the spot was matched e Coordinates ofthe spot scenter of gravity Xand Y e Quantification values Intensity Area Vol and Gool Depending on the spot detection algonthm used the Saliency Vol Ratio and Slope will also be given e Calculated pland MW values if p MW annotations were defined on the image ora matched image e Alllinked labels and spot sets Spot Table GA LV VW E FileName SpotlD Match ID Pixel X PixelY pI MW Intensity Area Vol byl Saliency Anovap lt 0 001 A 2198 A T1 Gel 136 392 857 275 1590 250 163584 0 0309635 408 192 0 2199 A_T1_Gel3 135 645 Op ZPH 3466 258 s264 2 0 099585 2552 47 be 2200 A_T1_Gel3 134 ESO SAD ZU 1220 2539 143642 00 0271722 409 032 ke 2201 4 T1_Gel3 133 sda SSS 1922 383 287018 0 0542941 727 33 LI 2202 A_T1_Gel3 132 gd DGL 207 4558 473676 0 0896035 2020 34 w Figure 6 8 Spot Table Spot ID Each spot in a gelhasa unique identifier called the Spot ID Spot IDs of deleted spotsare not reused Melanie attibutesa new ID to each new spot When a spot is split the child spot for which the coordinates arme closest to the parent spot keepsthe existing spot ID the otherchild spot getsa new ID When two spotsare merged the resulting spot is attributed the ID of the initial soot that wasclosest to the new center of gravity 6 8 2 OO Cursor informa
195. vels are not included in the History Melanie User Manual Edition AC Undo redo and history 11 Insert marker You can place a markerin the History by choosing Edit gt History gt Insert Marker Clear Clearthe list of actions in the History by choosing Edit gt History gt Clear Refresh Please note thatthe content of the History isnot automatically updated when you workon the imageswhile the History window isstillopen Click the Refresh icon to update the list Save print copy By using the options in the Save icon drop down list in the History window the content of the History can be e Saved inan XMLtype file with the extension hst e Printed It will first display in your Web browser using the XSL stylesheet located in the Template Script folder of the Melanie installation directory Use the print option in your browserto geta papercopy e Copied to the clipboard Melanie UserManual Edition AC 169 11 Undo redo and history 11 2 History 170 Melanie UserManual Edition AC Shortcuts A AppendixA Shortcuts A 1 Shortcut keys Several menu commandscan be activated by keyboard shortcuts They are indicated to the right of the cormesponding command in the menus Please note the logic behind the key combinations Ct for gels Shift for sp ots Altfor annotations Chi Shift for matches Some exceptions do exist The most important onesare the following two shortcuts used for undoing and redoing action
196. view e Next Previous Move to the next or previous image in the stack e Animate Switch automatically between the different imagesin the stack This option is useful for visualizing the expression vanations in a set of images e Transparent Mode Display one of the images Reference witha transparent surface This option is useful for visualizing expression vanationsin a single static view To move one view with respect to the other choose Translation in the Tools icon Hold down the Ctr key gray orthe Shift key green forthe Reference while dragging the view e Set Reference Select the image to be used asthe Reference for the Transparent Mode e Transparency Settings Choose the color forthe display of the Reference and increase ordecrease the transparency BB Display options Visibility Select the imagesto be displayed in the 3D View window e Spot Shape Set the way the spotsare displayed in the 3D View Crossed Outlined Filled None e Display Options Display the X pink Y purple and Z blue axes the coordinates of the point on which these axesare centered and change the way the surfaces of the 3D Views are visualized Gnd Wireframe Smooth e Color Palette Combine the 3D View with one of the color palettes available It issometimes diffic ult to judge whether spots should be split are saturated ornot or have other problems such asso called donut structures low intensities in the center compare
197. visibility hidden orshown of the columns In some cases Histogramsand Match Statistics Table you can define other display options Y Select by value Thisfeature allowsyou to selectitemsin the report based ona numerical search cntenon Clickthe Select by Value icon then select the measure Le column you want to use for refinement and finally set the lower and orupper limits of your search interval 2 7 4 Customize reports Sorting data in columns Data intabularreportscan be sorted by the column content If you click once on the header ofa specific column a triangle is displayed indicating that the column s numencal or textual data issorted in ascending order When you clickonce more onthe header the triangle inverts indicating that the data issorted in the opposite order Note that ascending order means that numbers are sorted from Oto 9 and text is sorted from A to Z Column visibility Load a predefined template orsave your own template indicating what columns should be hidden or shown using the Settings icon in a reporttoolbar Thisisparticulany useful when you only want the essential information to appear for clanty orforprnting purposes To apply an existing report template 1 Click the Settings icon in a report toolbar 2 Inthe template window Figure 2 5 select a template name with the Load icon By default only the Gel Table has more than one predefined template Properties Files Decnptions Calibration 3 Clic
198. w Edit Show Select Analyze Reports Tools Window Help Haf elei Ld 4 TestCalibration2 33EdS 10 FileName TestCalibration2 SU Vale 034863 Detection parameters S Oe e Area 106 Parameters 3 Intensity 1509 e Smooth 8 gt 2 Min Area H EJ D 5 Saliency faoo000 0 IV Auto Preview Cancel Gels D Spots 0 Annotations o Figure 6 1 Adjusting spot detection parametersin real time 6 2 2 Spotdetection parameters Spot detection parametersare best adjusted in the following order e Smooth Set this pa ra meter first It fixes the numberof times Melanie smooths the image before detecting spots using a smooth by diffusion algorithm The Smooth parameter should be optimized to detect all real spotsand split as many overlapping spotsas possible without being concemed about noise spots these can be filtered out with the Saliency and Min Area parameters s Saliency Thisparameterisa measure based on the spot curvature It indicates how fara spot stands out with respect to its environment Real spots generally have high saliency values whereasartifactsand background noise have small saliencies Although the Saliency isan efficient quantity for filtering spots it is also highly dependent on the images e g image resolution and 84 Melanie User Manual Edition AC Spots 6 depth Some gels need a saliency value of 10 for comect filtering Othersmay necessitate a value of 50
199. wapped fourtimes in order to separate the samples The software displays the smaller of the two calculated U values in the Class Analysis Table The lowerthe number the higherthe probability is that the means of the two samples are different Knowing this value and the sample size you can easily look up the probabilities in a Mann Whitney table Caution should be used when analyzing the results of a Mann Whitney test First the assumptionsare often violated Thisisthe case when spots are completely absent in one of the classes in this case you have repeated values of 0 Moreover if you have small samples the Mann Whitney test is meaningless If the total sample size is seven or less the test alwaysgivesa probability of finding different means in the case of identical populations greater than 0 05 no matter how little the samples differ Melanie User Manual Edition AC 135 8 Data analysis 8 4 Analyze classes 136 0 15 0 10 0 05 0 bedacghfikj jikghfedcba 598 613 Spot values Ranks Spotvalues Ranks X Y 1 5 1 5 3 4 5 Figure 8 15 Two examplesto illustrate the Mann Whitney and Kolmogorov Smimov tests In a the two classesdo notoverap atall whereasin b 4 shifts are needed to completely separate the spot valuesofthe two classes The bold valuescorespond to the spot valuesand ranksfrom classY the others belong to classX Kolmogorov Smimov test The Kolmogorov Smimov test tiesto determine if tw
200. xport import Crop areascan be exported and imported to ensure thatthe final size of all yourcropped gelsis identical even between work sessions To exporta crop area 1 With the Region tool define a crop area 2 Selectthe gel in which you defined the crop area Melanie UserManual Edition AC 39 3 Image Pool 3 5 Calibrate images 3 Choose Edit gt Gels gt Crop Area gt Exportto save the crop area toa file with the extension cpt Crop Tool 4 Browse to locate a folderand entera file name Click Save To importa crop area 1 Select the gelsto be cropped to a previously defined crop area 2 Choose Edit gt Gels gt Crop Area gt Importand select the previously saved file Click Open The crop area appearson the selected images 4 Byclicking inside the area with the Regiontooland dragging it you can move the crop area to superimpose the anchorona characteristic spot 3 4 4 k Invert gray levels You can invert the gray levels of selected gels Ihismeansthat if your image showswhite spotson a black background the inversion displays black spotson a white background the required mode for analysis in Melanie CAUTION When your images open in the software with white spots ona black background this may indicate that you used incorrect scanner settings orthat yourfiles are not properly imported Please verify your image acquisition parameters If you think the software doesnot corectly support your files i e saved
201. y Max Match Count A_T1i_Geli a 170 0 678044 0 145679 0 214853 2 16773 0 0862424 0 874034 12 0 508486 115 0 730117 0 106354 0 145668 1 6817 0 065628 0 895849 12 0 700301 0 458706 0 235954 0 51439 0 179034 0 866777 11 0 124425 0 177178 0 260686 1 47133 0 526836 0 738571 5 0 7478 w i gt Figure 8 5 Gel Analysis Table In addition to the standard functionalities for saving printing copying content to the clipboard and navigating in the report the following tools in the Gel Analysis Report are particularly useful Melanie UserManual Edition AC 119 8 Data analysis 8 3 Analyze gels 120 Y Select by value Select itemsin the table based ona numencalsearch criterion Choose the measure i e column to be used forrefinement and setthe lower and orupper limits of your search interval F create spot set Create a spot set to annotate spots of interest for use ata laterstage All currently selected spots are automatically included in the newly created spot set which appearsasa column in the table You see a checked boxforspotsthat belong to the set oran empty box forspots that do not belong to the set H H Factor analysis table Cary outa factor analysis CH Statistics Set the statistics to be used in the report These settings are common to the Gel Analysis Table and Gel Analysis Histograms Settings Some of the above mentioned columns may not be displayed by default Click the Settings icon to show or

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