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Melanie 3 User Manual - Bio-Rad

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1. 45 Images 18 68 Open 22 ieu ee Re 17 Page Setup 19 Print GOS AWAY 19 Gels Window 19 Raw Image in teo 20 Select Gels with Loaded Image 20 nde EE 20 Unload saa 20 Revert from File 18 18 Save 18 file format opening foreign image formats 18 opening Melanie images 17 filtering gels 121 fitting report 45 flipping gels 124 G Gaussian spots 44 gels Leuten otis ee Guten ence 7 aligning eee 31 closing 2 nme 20 e lee WEE 121 definition mee 7 deleting inas nia 121 display order 24 displaying 2 43 eth tn ata 22 displaying grid lines 31 displaying information 47 duplicating eese 121 eraslnig incre eie 121 ele lte WEE 18 filtering rns 121 flipping neon A 124 inverting grey levels 124 Ee ET 22 loading se of WE 19 matching 79 MOVING EPEE rte irme erc 27 opening ass nepotes 17 prititlng sa oz irr rene 19 reference gel 31 79 85 reporting ese andy a cedes 37 reverting Save
2. 18 rotating ee eee 124 SAVING uii dE ki 18 Scaling 124 selectlhig 21 SHOWING 2 5 teet 23 STACKING sets 25 UNAlIGNING cot eti 32 unloading eene 19 ZOOMING ertt ee 28 gels report to classes 104 gray offset 45 gray slope ies 45 green objects 10 grid lines over gels 31 e leie EE 8 85 coefficient of dispersion 96 coefficient of variation 96 correspondence analysis 97 creating groups by matching 85 e le WEE 8 deleting 1 1 ELA 86 emeret 86 PROTO 85 in matched gels 79 Elle SC EE EE 96 reporting 94 96 restriction uice eto 87 selecting seeseessss 87 separability 97 SHOWING Sees ce eet idee 86 histograms 93 spots report 98 GEET 90 ungrouping enn 86 with multiple pairs 85 groups report for classes 104 H hand symbol See manual hand tool eee i 27 hardware requirements 2 Help Index itc 6 User Manual _ 6 help on line 5 6 131 hidden gels zonea
3. 6 histograms eee 69 e EE 104 108 classes groups 105 exporting ssa 71 OUPS unice c ote dert e 93 PINGING i ee tette 71 SAVING Sa 71 selecting groups 72 images sanal ege Ee 18 importing annotations 68 foreign image formats 18 labels 2 ye 68 installation treten 2 interface uwa emere 4 Internet accessing remote databases 113 114 querying the database 115 setting the browser 114 setting the database 114 inverting grey levels 124 Melanie 3 User Manual 157 158 K kolmogorov test data analysis 109 L ADOSER 53 LEE 53 accessing remote databases 53 113 adding uten 55 Comment gee Ege 54 creating intei 55 deletirig eite 59 rro Lalo ZO ACP 59 IMPOMING aa 68 Landmark 53 modifying esse oe eerte tne 59 E iei d et 54 reporting weds eh ees 67 Selecting e 60 SES uc eee 54 SNOWING i thee 64 licen oe eiae ade pe 3 loading gels 19 loading protein name 116 M magnify glass 29 magnify tool 28
4. RE 143 Annotations handling eA 144 Pairs handling sese eee 144 KNOWN BUGS 1332 a seiko o ENEE A Soaks 145 REFERENCES NN dE NR Eed WG ENER 147 The Melanie project RESEND EES TG eee 147 Two dimensional electrophoresis 2 DE 148 2 DE databases creme aS 148 Proteome and ExPASy 150 TECHNICAL SUPPORT Wie ERO Ee to Vee EA 153 INDEX M e EMSC OER RE RS 155 CREDITS Melanie 3 User Manual BASICS Welcome to Melanie 3 the third generation of the well known Melanie two dimen sional electrophoresis analysis software Melanie is a complete state of the art soft ware application designed to analyze images of two dimensional electrophoresis 2 DE It was conceived in close collaboration with biologists and proteomics scien tists from Hochstrasser s group in Geneva to insure fast and reliable comparisons of spots analysis of gels and identification of proteins Its design and functions address both experts and beginners needs Melanie has been designed to be easy to use very intuitive user interface never theless we encourage you to read this manual in order to understand all the exten sive power of this software This manual shows you how to make the best of Melanie and its advantages while explaining all its functions in detail It includes exhaustive explanations and useful tips which will help you to analyze
5. Short cuts See page Gels Only Selected Ctrl Z 23 Hide Selected Ctrl H 23 Show All Ctrl Y 23 Sorted by Name 23 Sorted by Classes 23 Spots Shape Crossed Shift 6 45 Outlined Shift 7 45 Filled Shift 8 45 Show All Shift Y 45 Hide All Shift H 45 In Region 45 Only Selected Shift Z 45 Hide Selected 45 Show ID 45 Hide All ID 45 Set Color 46 Annotations Show All Alt Y 64 Hide All Alt H 64 In Region 64 For Spots 64 Only Selected Alt Z 64 Hide Selected 64 Visible Categories 64 Hidden Categories 64 Pack Categories 64 Unpack Categories 64 Linked Data 64 Set Color 66 Groups Show ID 86 Hide All ID 86 Melanie 3 User Manual Table 10 10 Select menu Short cuts See page Gels By Name 21 Visible Ctrl A 21 Aligned 21 Stacked 21 All 21 Inverse Selection 21 For Groups 21 For Classes 21 Spots By ID 46 By Color 46 Only Gaussians 46 In Region 46 From Report File 46 All Shift A 46 Restrict by Value 47 Inverse Selection 46 Annota tions By Content 60 By Category 60 By MS 62 Combine 63 Intersection 63 Union 63 Difference 63 Exclusion 63 By Color 60 Common Labels 63 In Region 63 From Report File 63 All Alt A 63 Inverse Selection 60 Pairs For Spots Ctrl Shift M 82 M
6. 122 when detecting spots 40 software requirements 2 sort by similarity histograms on groups 95 sorted values histograms on classes 105 histograms on classes groups 106 histograms on groups 95 sorting data eports o Ree Ret 74 Spot tool ueniet 46 SPOS AE as 39 adding sise 42 adding from gel 43 adjusting detection parameters in real time 42 calibrating ss et esas eed 44 i ice 46 definition sR 8 detecting eese 39 detecting in region 41 detection parameters 40 editing i recens 39 Melanie 3 User Manual 44 Die SE 8 39 modifying assesses 42 QUANTIFYING 44 unie ber 49 for groups 98 templates 50 selecting i 46 SNOWING aad Mia pete 45 stacked gels selecting eem 26 stacking gels 25 Statistical tests 108 statistics central tendency 90 dispersion eese 90 ej goi ORAN DER 90 half range size 92 url cc 91 mean absolute deviation 92 mean squared deviation
7. 60 adjusting contrast in real time 34 Link with Spot 55 correlation coefficient 88 Modify rca ecco 59 correspondence analysis Paste Annotations 59 groUps iR 97 corresponding location 27 59 Paste Labels DEM 60 creating Unlink with Spot 55 annotations 55 Classes categories 56 Add to Class 101 classes este 101 Remove from Class Met EN 102 groups as starting pairs 81 Gels Sets ATE Z 56 Calibrate Intensity 45 starting pairs 80 Reset Calibration 45 synthetic gels 119 Se ZARY PISZAC cropping gels 121 roups cursor information 47 Group Spots 86 Set Reference Gel 86 D Ungroup Spots 86 data analysis Pairs kolmogorov test conecte 109 tee 81 student t test 108 Auto Match Gels 80 wilcoxon test TOO database sa eec cee teeta 113 Delete 81 2 DE databases 113 Match Annotations 81 accessing remote databases 114 Preferences accession number AC 53 Cursor Information
8. 81 coloring se nr eere 66 ee rulle WEE 59 de NES dee ed 55 definition 8 deleting 59 e le Le WE 59 importing 68 modifying EEN 59 MOVING BREET 59 moving tags 59 PASUNG cinere pes 59 reporting 67 Selecting aub ctm 60 showing 222 64 c calibrating SPOS id CEE 44 calibration plot 45 calibration report 45 categories ORE EEEE 53 addihg uiuere 56 Comment essen 54 terc rn gro Ep E 56 Landmarks 53 beet 54 reporting 67 e 54 56 145566 see aie cerei ecrit exe qs 9 101 definition 9 gels report 94 104 histograms eee 104 108 overlapping measures 102 OBO oec 104 reporting aa 107 classes groups histograms eee 105 closing gels Rene 20 coefficient of dispersion 96 coefficient of variation 96 coloring Melanie 3 User Manual 155 annotations 66 CODY olivate 59 60 1i TEE T 46 Copy Paired Labels 60 contextual Delete 59 dies EE 5 Delete by Category 522 59 adjusting contrast 33 Duplicate Labels
9. 21 For Groups 21 Inverse Selection 21 Stacked 21 Current xD 49 Visible siet 21 Groups All Lites Rott 87 ia 87 For Spots cca ek 87 Inverse Selection 87 Restrict Selection 87 Pairs All teste tm det 82 Bad Pairs iie 82 For Spots 82 Multiple Pairs 82 Spots ALLS 46 By Color Rass 46 By ID tinh OS 46 From Report File 46 In Region asia 46 Inverse Selection 47 Only Gaussians 46 Restrict by Value 47 selecting annotations 60 from histograms groups ccc tpe 72 from report annotations 62 ite ec 21 groUpS 87 labels dz oai AM 60 72 masal ea aasa 82 SA 47 gels ie tentent et 21 JIOUDSN onte be ie 87 labels iet rco 60 BAIS odia canit el 82 SPOS sccm e es n 46 stacked gels 26 separability for groups 97 serial numbers 116 Sets Creating SS dS e ea 56 setting preferences 36 Shortcuts Sess 5 Show Annotations EOF Spots anh ee 64 Hidden Categories 65 Hide IR 64 Hide Sel
10. 26 Hide Overlapped 27 Hide Pails i tice etti 27 81 is aie eee Ee SN 26 PrEVIOUS noe trees 26 Selected Gels 25 Set Stack Reference 26 Show Overlapped 27 Show Pairs 27 81 Unstack Selected Gels 26 LOOM siada E 29 wilcoxon test data analysis 109 Window Information 47 Volet 29 window Printing WATA 19 World Wide Web See Internet See database 2 zone hidden gels 6 reference gel 6 ZOOMING gels sees 28 with magnifying glass on line 29 Melanie 3 User Manual 161 162 Melanie 3 User Manual The Melanie project originated in 1985 at the Geneva University Hospital and the Computer Science Department of Geneva University Switzerland by Ron Appel Matthieu Funk Denis Hochstrasser and Christian Pellegrini Melanie 3 its third version has been developed at the Swiss Institute of Bioinformatics in collaboration with other groups of the Geneva Proteomics Network The following key persons have contributed to Melanie 3 Software design and developments in Geneva Daniel Walther G rard Bouchet Patricia M Palagi Software developments in Cochabamba Bolivia J Reynaldo Vargas Gonzalo Argote Oscar Antezana Soraya Ordonez 2 D PAGE applic
11. Uniform Exponential e Rayleigh Cube Root Logarithmic 3 Remap Grey Levels Creates copies of the selected gels with modified grey level mapping see Gray levels on page 33 This is especially useful if you analyze images with a very low grey level range For example the Melanie spot detection algorithm may not work properly on an image with only 25 grey levels You can therefore remap it to an image with 4096 shades of grey so that spots may be correctly detected 4 Contrast Enhancement Applies a contrast enhancing algorithm to the images in order to highlight small spots Fig 10 3 The following three contrast enhancement algorithms are available e Quadratic Sigmoid 122 Melanie 3 User Manual FILTERING GELS e Wallis Figure 10 3 The original image left and the same image after contrast enhancement right 5 Filter Background Applies one of the two following background filtering algo rithms to the selected gels in order to eliminate gel background Fig 10 4 e Subtraction of global image minimum e Background subtraction by polynomial transformation Figure 10 4 The original image left and the same image after background subtraction right 6 Morphological Filter Applies a morphological filtering algorithm rank filter to the selected gels Fig 10 5 according to 2 parameters the window size which can be 3x3 5x5 or 7x7 pixels wide e and the value index 0
12. 37 48 ona RSA E a Database 37 57 114 abels Tor loading labels 116 Grid Preferences 37 private database 113 Raw Image 36 querying the database 115 Spots remote database 113 Add From Gel 43 setting the browser 114 Auto Detect 39 setting the database 114 BOCA 43 SWISS 2DPAGE master gels 115 Edit 43 SWISS 2DPAGE serial numbers 116 FE deleting Steg 59 annotations 59 Mg A E 86 gels eerte ne tdg 121 ARE R DEAD PORADE bes 86 80 EI amg 59 a a i oS 39 PASS E Here 81 d cing Up t E 121 SPOS feet deceret 43 exiting Melde c o m NS 20 detecting WAS SPOTS oic ask 39 D m 71 Pe e E E 41 Melanie WAGES oen ies e c 18 isplay order lend 24 TOPOS zad za 71 displaying F gels aer RE ue rat 22 SE 31 oe 2 DE database 114 pseudo colors 35 Cis 58 EE A iut PI EE 20 Export E Edit Gel Region to Clipboard 19 Annotations Gel to File 19 Add Label 5 2 ue 55 56 Gels Window to Clipboard 19 156 Melanie 3 User Manual Gels Window to File 19 Import Annotations 68 Calibration
13. Central tendency gives the general location of a variable This is commonly calculated by the arithmetic mean also known as average or center of gravity of a distribution the median the middle value which divides the sample in two equal parts or the midrange By selecting Mean the arithmetic mean is calculated i e the sum of all the sample values divided by the sample size e selecting Midrange the midpoint of the sample value is calculated i e the middle location between the two sample extremes By selecting Reference gel values the central tendency is the value from the spot in the reference gel Mean or midrange values are sensitive to extreme values outliers and can be seriously contaminated by just one observation On the other hand the median is highly resistant to outliers A compromise between mean or midrange and median is given by the trimmed mean or midrange where a predefined number of outliers is removed from the sample The trimmed measures are more robust than the mean or midrange but are more sensitive than the median The percentage sliders available in the dialog box allow you to remove outliers 100 value means that all the spot values available in a group will be used to calcu late the statistics i e no suppression of outliers 0 corresponds to the Median measure For example 8096 value means that the 1096 minimum values and the 1096 maximum values will be discarded from the sample and
14. Figure 4 8 Report on annotations Menu Files Importing labels You may import annotations and labels into open gels To do so select a gel and choose Labels In the displayed window type the file name and select the file type You may import labels from a file containing a Report on Labels for example or from any Labels file type In the last case it must contain the columns SpotlD X Y Category and Label If the spot ID is not known use 1 in this field and Melanie will position the label in the corresponding X and Y positions of the gel If X and Y posi tions are not known use 1 in this field and Melanie will position the label in the cor responding spot ID Columns in the file must be separated by tabulators In the same way you may import annotations from a file containing a Report on Annotations To do so select Annotations and type the file name in the displayed window 68 Melanie 3 User Manual Introduction The reports available in Melanie provide not only powerful options to display infor mation on selected objects but also a useful tool to navigate through data Reports may be accessible through the Reports menu the Analysis menu or through dis played reports In the Reports menu Report on Gels Displays summarized information about the selected gels such as intensity calibration number of detected spots etc Report on Spots Displays specific information about selected
15. Figure 8 9 Example of two populations Wilcoxon test indicates that the last two values from A must be swapped four times to the left so that both populations no longer overlap The report values are the normalized number of shifts needed to sort the two popu lations You may then use this value to look for the probability of confidence in a Wil coxon table Kolmogorov The Kolmogorov Smirnov test is used to test whether or not two samples come from the same distribution The Kolmogorov Smirnov test does not require the assump tion that the population is normally distributed nor does Wilcoxon The statistics measure the number of values in percent from a population which is not covered by values from the other population For the example given in Fig 8 9 the Kolmogorov test shows that 5 7 values from A are not covered by the values from B Melanie 3 User Manual 109 8 KEN CLASSES The sample percentage not covered by the other population is displayed on the Report on Statistical Test You may then use this value to look for the probability of confidence in a Kolmogorov table We 2 Note that all statistical values are only useful when dealing with more than 2 gels 2 7 per class Sort the columns in the report by clicking in their headers and re select the groups lis which correspond to your own criterion of statistical value 110 Melanie 3 User Manual PART IHN TOOLS 2 DE databases A
16. Matches at least one occurrence of the pre P0 1 matches PO1 ceding character P001 P0001 Melanie 3 User Manual 61 62 EEEE ANNOTATIONS Table 4 1 Regular expressions to search patterns in labels Description Example Matches zero or one occurrences of the pre P0 1 matches P1 and ceding character PO1 Matches zero or more occurrences of the P0 1 matches P1 P01 preceding character P001 P0001 Groups a subexpression P 24 matches P24 P2424 P242424 Matches any of the characters inside the P a d matches Pa Pb a z is the range of characters between a Pc Pd and z and 0 9 is any numeral from 0 to 9 Matches any character not in the set of char P bd matches Pa Pc acters following the Pe but not Pb ou Pd Matches the expression before or after the P ab cd 1 matches Pab1 Mostly used within a subexpression and Pcd1 n Matches exactly n times the preceding char PO 3 1 matches P0001 acter but not PO1 nor P001 n Matches at least n times the preceding char PO 2 1 matches P001 acter and P0001 but not P01 Matches at least n times and at most m PO 1 3 1 matches P01 times the preceding character P001 and P0001 but not P1 or P00001 For example to select all labels which contain the string POO you may either type the expression P00 and choose the Regular Expression field in the dialog
17. A DIFFERENCES BETWEEN MELANIE II AND MELANIE OR WHERE TO 128 Landmarks and labels Landmarks and labels were generalized with the new object Annotation The object Landmark is now replaced by the pre defined label category Landmark Landmarks are also used as reference starting points to align gels Labels were also replaced by label categories The pre defined label category Ac replaces the Accession Number field from Labels in earlier versions of Melanie and it also keeps the main property of being the link to Melanie s remote database query engine The short ID information is held by a category designated ProteinName Aligning gels To be able to perform operations easily and faster in many gels simultaneously such as magnifying moving etc an implicit alignment is automatically done when gels have landmarks with the same names This alignment calculates corresponding pix els in gels based on the simple interpolation of the two closest landmarks with same names in gels This implicit alignment does not replace gel warping needed to com pensate deformations on gels and to improve matching We still encourage users to align gels before stacking and matching For this purpose select gels and choose View Align Gels Align Stacking many gels Earlier versions of Melanie were able to stack two gels to better compare given gel areas and to create and visualize pairs Selecting one gel on the stack immediately s
18. Next PageDown View Stack Previous Shift Up View Zoom In Shift Down View Zoom Out Melanie 3 User Manual Spots handling Table 10 15 Short Cut Menu Command Shift A Select Spots All Shift G Edit Groups Group Spots Shift H Show Spots Hide All Shift M Select Groups For Spots Shift O File Open Shift S File Save Spots Shift U Edit gt Groups Ungroup Spots Shift X Edit Spots Delete Shift Y Show Spots Show All Shift Z Show Spots Only Selected Shift 1 Show Spots Shape Crossed Shift 2 Show Spots Shape Outlined Shift 3 Show Spots Shape Filled Shift 4 Show Spots Set Color Purple Shift 5 Show Spots Set Color Orange Shift 6 Show Spots Set Color Brown Shift 7 Show Spots Set Color Default Melanie 3 User Manual 143 EEEE SHORT CUTS AND MENU STRUCTURE Annotations handling Table 10 16 Short Cut Menu Command Alt A Select Annotations All Alt C Edit Annotations Copy Alt E Edit Annotations Modify Alt H Show Annotations Hide All Alt L Edit gt Annotations Link with Spot Alt S File Save Annotations Alt U Edit Annotations Unlink from Spot Alt V Edit Annotations Paste Labels Alt X Edit Annotatio
19. Melanie 3 User Manual RN CONTENTS Displaying gridlines cleri 31 Aligning GAE sre ER err NEE EP ei a RE cx 31 Cc VH I II A a AO E 33 Calibrating gels o sss ne oot e REN 36 Setting preferences _ 36 Reportingongels eee eee eee eee nnn nnn nic 37 3 SPOTS eee OPRZE zed KO 39 Editing Spots iuo y rer e NETA rk xxx PA WWR e E E SE 39 Preview detection results 42 Adding modifying spots _ 42 Spots quantification _ 44 Calibrating spots saci toe oes et IIIA SUITES 44 SHOWING SPOtS xime Ee eee ee eh exem 45 Selecting SPOTS lt meta ate tea ere e Mechela ale 46 Cursor information satre RN 47 Report ON Spots sa owa eee naue as aaa ee 49 d ANNOTATIONS EE E AEN E 53 Pre defined label categories 53 Creating annotations 55 Creating specific links _ 56 Modifying and deleting annotations 59 Editing annotations 59 Selecting annotations and labels 60 Showing annotations and labels 64 Reports On Annotations 67 Importing lab
20. Morphological Filters 121 Scale By 124 Rotate 124 Invert Gray Levels 124 Create 119 Synthetic Gels Database Settings 114 Load Protein Name from 115 Swiss 2D Load Swiss 2D Serial Num 115 ber Toolbar Hand Ctrl 1 Magnify Ctrl 2 Region Ctrl 3 Spot Ctrl 4 Annotation Ctrl 5 140 Melanie 3 User Manual Table 10 13 Window and Help menus Short Menu cuts See page Window Zoom 29 Information 47 Help User Manual Index Tutorial About Melanie Melanie 3 User Manual 141 SHORT CUTS AND MENU STRUCTURE 142 Gels handling Table 10 14 Short Cut Menu Command Ctrl A Select Gels Visible Ctrl F View Stack Front to Back Ctrl G View Stack Selected Gels Ctrl H Show Gels Hide Selected Ctrl O File Open Ctrl S File Save Ctrl U View Stack Unstack Selected Gels Ctrl W File Close Ctrl Y Show Gels gt All Ctrl Z Show Gels Only Selected Ctrl Up View Move Gels Up Ctrl Down View Move Gels Down Ctrl Left View Move Gels Left Cirl Right View Move Gels Right Ctrl 1 Tools Toolbar Hand Ctrl 2 Tools Toolbar Magnify Cirl 3 Tools Toolbar Region Ctrl 4 Tools Toolbar Spot Ctrl 5 Tools Toolbar Annotation PageUp View Stack
21. check box in the bottom of the histograms window Fig 8 4 Figure 8 4 Histograms on Classes with unsorted central values and with sorted in ascending order central values To have the same gradations for all histograms unselect the Adaptive gradations check box in the bottom of the histograms window Fig 8 5 Figure 8 5 Histograms on Classes with adaptive gradations set individually and set according to the minimum and maximum values Use the Report on Classes to change the order or to refine your histograms selec tion Histograms on Classes Groups From the Report and Histograms on Classes windows you may request more detailed and complete histograms on classes for selected groups They display for each group the extracted classes intervals and their corresponding spot values Select gels and groups of spots Choose Classes Histograms or Classes Report Select one of the spot value types Od Vol Area Od Vol In the dialog box Melanie 3 User Manual 105 8 KEN CLASSES 106 select central and dispersion statistics In the toolbar of the displayed window select the item Groups Histograms in the Histograms menu The histograms are displayed for each group of spots and for each class The avail able information from the classes are the central value and the dispersion interval A vertical gray line separates the spots values for each class Fig 8 6 The information displayed in these specif
22. is estimated by interpolation between the two nearest common landmarks i e landmarks with the same name 5 Adjust the annotation positions using the Annotation tool Melanie 3 User Manual 59 EEEE ANNOTATIONS Edit L gt Annotations This option copies annotations from one gel to their corresponding locations in the other gels Copy pasting labels You may also copy some labels from one gel to selected spots or annotations in a set of gels Perform the following steps 1 Select one or more labels 2 Choose Copy The selected labels will be copied into memory 3 Select one or more spots or existing annotations you want to copy the labels to 4 Choose Paste Labels n the dialog box confirm the action Copy paired labels When gels have been matched and pairs have been created labels existing in one gel may be copied to their corresponding spot pairs in the other gels 1 Select annotations in the source gel 2 Select the gels you want to copy the labels to and the source gel 3 Choose Copy Paired Labels In the dialog box confirm the action Duplicate labels You may copy selected labels to another category Select labels then choose the Duplicate Labels command In the dialog box choose the receiving category Note that the selected labels may also belong to different categories You can use this menu to merge several categories or to rename one category Menu Select Selecting annotations an
23. manual 1 on line help 5 6 131 mass spectrometry 62 master gels loading labels 116 SWISS 2DPAGE masters 115 match ms data 62 Matching issa eterne 79 aligned gels 80 automatic matching 80 creating groups 79 85 ms spectrum 62 starting pairs 80 using annotations for matching 81 melanie format 18 Inienus o oeste eee a 4 Analyz8 nue bp iun 4 131 contextual asas 5 Edit 2 5 Saa 4 131 File iui tein 4 131 Help i tn eee 5 131 REDO es 4 131 Select ohn nian 4 131 ShOW ZERA 4 131 ee 5 131 VIEW sisi ree hist 4 131 WindOw 5 131 modifying ANNOTATIONS u tet at 59 groups Ags ese hse aa 86 labels uin tei 59 SPOS ase ee Ee gd 39 42 moving annotations 59 annotations tags 59 Melanie 3 User Manual gels stacked gels multiple pairs O OD ects 2 deese tui annotations SPOS ieget wi eek opening gels foreign image form
24. see Heu ristic clustering on page 99 Powerful normalization techniques see below Queries Melanie s versatile search engine allows to easily formulate complex queries The query power allows to answer your biological question based on e Sophisticated textual request see Selecting annotations and labels on page 60 Qualitative and quantitative information see Restriction of groups selection on page 87 and Restrict spots selection on page 47 Protein expression change for example by ratio or similarity see His tograms on groups on page 93 and Heports on groups on page 96 MS spectrum see Mass spectrometry on page 62 Analysis results and data subsets see Combining sets of annotations on page 63 Normalization The reproducibility of the 2D separation process can be perturbed by a number of factors including differences in sample preparation and loading staining and image acquisition To accurately compare the quantity of any spot across gels it is essen tial to compensate these variations You can analyze these variations with the scatter analysis and iron out them with several powerful methods e Removal of the image scanning variation by Intensities calibration see Calibrating spots on page 44 Removal of the varying stain intensities and sample loading by rela tive spots quantification see Spots quantification on page 44 Melanie 3 User Manual 13 14 1 EE INTRODUCTION R
25. you can normalize the spot values as described for the Histograms on Groups In the Report on Groups window choose in the Displayed value list Fig 7 11 the type of normalization used to display the spots values Reports Displayed values 113 1 rrr nz e E ea a RE e Tm E c SEE vmm ve n ZOE ram TEE Te TEE Figure 7 11 Report on Groups Clicking Histograms in the toolbar Fig 7 11 will display the histograms on selected groups from the report You may immediately analyze the selected groups and directly iterate on the screen through your data see Histograms on groups on page 93 If the Correspondence Analysis check box has been selected in the dialog Fig 7 10 a Correspondence Analysis is done and the C A column displays the amount by which each spot group contributes to the constitution of the axis in the factorial space the inertia value This method creates a projection onto a factorial space of small dimensionality in which gels and their significant spots appear as points Con sequently we can determine which gels are similar and which are the characteristic spots of these gels In fact the natural formation of classes among the gels is high lighted with this method and can be seen in a graphical representation of gels and spots that are projected into the 2 D factorial space Choose the Plot C A button in the toolbar Fig 7 11 A dialog box lets you choose to disp
26. 2DPAGE master gels contain annotations for all proteins corresponding to entries in the SWISS 2DPAGE 2 DE database Each annotation holds the acces sion number in the Ac label category of the database entry as well as the entry s short name in the ProteinName label category For example the Human Plasma protein map possesses the Ac label P02768 and the ProteinName label ALBU HUMAN for each spot that corresponds to albumin SWISS 2DPAGE entries contain detailed data on the related proteins such as the protein s full name bibliographical references annotations protein function patho logical variations etc and the p and Mw of the related spot on the 2 DE maps They also hold cross references to other databases such as the SWISS PROT pro tein sequence database that in turn has cross references to numerous other data bases such as nucleotide sequence databases disease databases etc or the Melanie 3 User Manual 115 MNES DATABASES Tools Database 116 L gt Load Protein YEPD Yeast 2 DE database and the ECO2DBASE E Coli 2 DE database Fig 9 1 Gasse TWISTIDEACH Hemma 11 Pets 1735 m lari rakir z Erin 10 amr 19559 rin Timor APOLIFOFACTEIK B APSE Fim Viana ageet Hori Rkary z k a Teda Vere eae Rhee Frosi iocur Carum Ionic Horas HAPPEE OH GEL Mu THOME ICE jene inl ARCOEERSCH HL ANDERSON H Tigh ore a
27. 91 midrange 91 reference gel values 91 significance levels 108 Status Dar own 5 student t test data analysis 108 SWISS 2DPAGE master gels 115 serial numbers 116 SWISS PROT 115 synthetic gels 119 T templates Spots Teport se css eee 50 Tools Create Synthetic Gel 119 Database Load Protein Name from Database 116 Load Swiss 2D Serial Number 117 Settings e es 114 Gels etes 119 Crop ko ated 121 Duplicate 121 Lone 121 Filter Image 122 FID ss eesti tie 124 Invert Gray Levels 124 ROBIE eee 124 Scale By esis 124 oz EE 6 U unaligning gels 32 ungroup Spots 86 unloading gels 19 V View Align Gels AGM aint 32 Other Alignment 32 Unalign 32 Gray Levels Adjust Contrast 34 35 Pseudo Colors 35 Grid Lines Hide ccm 31 CJA 31 Layout Settings 24 Move Gels nee 28 Same Location 28 Stack Front To Back
28. An index on menu items is given in Appendix Short cuts and Menu structure on page 131 Contextual menu When you click the right mouse button in Melanie s display a Contextual menu appears containing the main functions to edit show and select objects like gels spots and annotations They reproduce the same commands found in the Menu bar but they are quicker to access at any time Fig 1 3 Figure 1 3 Contextual menu Shortcuts Several menu options may be activated by keyboard shortcuts These are indicated at the right hand side of the corresponding menu option A list of shortcuts is given in Appendix Short cuts and Menu structure on page 131 Status bar The status bar describes at a given moment the total number of gels spots and annotations that are selected Fig 1 2 item 4 Note the importance of the status bar as it allows you to know what objects are selected even if not all of them are visible for example when gels are zoomed Melanie 3 User Manual 5 1 ES INTRODUCTION Reference gel zone and hidden gels zone These two zones enable you to quickly change the gels visibility or choose a refer ence gel The hidden gels zone enables you to hide or show your displayed gels with just a drag and drop operation Fig 1 2 item 6 In the same way the reference gel zone enables you to choose the groups reference gel Fig 1 2 item 5 For more details on reference gel see Pairs and groups on page 8 and
29. F Melaniell a third generation software package for analysis of two dimensional electrophoresis images l Features and user interface Electrophoresis 1997 18 2724 2734 4 Appel R D Vargas J R Palagi P M Walther D Hochstrasser D F Mela niell a third generation software package for analysis of two dimensional electro phoresis images Il Algorithms Electrophoresis 1997 18 2735 2748 5 Vargas R J Two dimensional gel electrophoresis computer analysis systems from image acquisition to protein identification Ph D thesis Faculty of science Geneva University 1996 6 Wilkins M C Hochstrasser D F Sanchez J C Bairoch A Appel R D Integrat ing two dimensional gel databases using the Melanie Il software Trends in Bio chemical Sciences TiBS 1996 December 252 21 12 pp 496 497 7 Appel R D Bologna G Hochstrasser D F Classification tools for diagnostic rule formation from protein maps Proc MIE 93 11th International Congress of the European Federation for Medical Informatics Jerusalem April 18 22 1993 40 44 8 Appel R D Hochstrasser D F Funk M Vargas R J Pellegrini C Muller A F Scherrer J R The MELANIE project From a Biopsy to Automatic Protein Map Interpretation by Computer Electrophoresis 1991 12 722 735 Melanie 3 User Manual 147 D C 148 9 Appel R D Hochstrasser D F Roch C Funk M Muller A F Pellegrini C Automatic classification of two dimensional gel e
30. Hand tool 8 Magnify tool 9 Region tool 10 Spot tool and 11 Annotation tool Menus Once objects have been selected you may choose actions from the menus to be performed on them Move the cursor to the desired menu on the menu bar Fig 1 2 item 1 hold down the mouse button Certain menu options have sub options you may choose one of them by pointing over the small arrow on the right hand side of the option The following menus are provided in the melanie program from left to right in the menu bar File to handle whole gels and data on disk open and close gel images save data print images import and export data View to modify the window display fixed or variable number of cells and gels display stacked aligned zoomed grid lines Edit to edit add modify delete specific objects Show to show or hide specific objects Select to select specific objects Analyze to compute differences and similarities among gels statistical and artificial intelligence data analysis Reports to display information on selected objects and to navigate in gels by means of the data Melanie 3 User Manual INTERFACE Tools to create new gel images from selected ones duplicate crop filter gels create synthetic gels flip and scale gels as well as erase gels Window to display information and zoom windows and to list open report windows Help to obtain on line help
31. S Gasteiger E Binz P A Bienvenut W Hoogland C Sanchez J C Bairoch A Hochstrasser D F Appel R D Improving protein identification from peptide mass fingerprinting through a parameterized multi level scoring algorithm and optimized peak detection Electrophoresis 1999 in press 39 Gay S Binz P A Hochstrasser D F Appel R D Modeling peptide mass fin gerprinting data using the atomic composition of peptides Electrophoresis 1999 in press 40 Binz PA Wilkins M R Gasteiger E Bairoch A Appel R D Hochstrasser D F Internet Resources for Protein Identification and Characterization In Microcharac terization of Proteins R Kellner F Lottspeich H E Meyer Eds Wiley VCH 2nd ed 1999 277 300 41 Wilkins M R Gasteiger E Bairoch A Sanchez J C Williams K L Appel R D Hochstrasser D F Protein Identification and Analysis Tools in the ExPASy Server In Methods in Molecular Biology Vol 112 2 D Protocols for Proteome Anal ysis A J Link Eds Humana Press Inc Totowa NJ 1998 pp 531 552 42 Wilkins M R Gasteiger E Tonella L Ou K Tyler M Sanchez J C Gooley A A Walsh B J Bairoch A Appel R D Williams K L Hochstrasser D F Protein identification with N and C terminal sequence tags in proteome projects Journal of Molecular Biology 1998 278 3 599 608 43 Wilkins M R Gasteiger E Wheeler C Lindskog l Sanchez J C Bairoch A Appel R D Dunn M D Hochst
32. also be created and modified manually see Pairs on page 79 The reference gel is the central node of spot groups All spots in selected gels that are paired with the same spot in the reference gel form so called groups Groups may change when the reference gel has changed If you have matched your gels with multiple pairs you may then obtain very large groups because a given spot may be paired to several spots in the reference gel You should therefore use multiple pairs with care Melanie 3 User Manual 85 EE GROUPS Edit L gt Groups Show L gt Groups Menu Edit Editing groups A convenient way to add pairs between a set of gels and a reference gel is to create groups You first have to set the reference gel Select the reference gel and drag to the Reference Gel area or choose Set Reference Gel Select one spot in the refer ence gel and spots in the other selected gels By choosing Group Spots pairs will be added between each of the selected spots and the selected spot in the reference gel To remove some spots that are part of a given group select a number of such spots Choose Ungroup Spots This removes the pairs between the selected spots and the reference gel To completely remove groups select the spots in the reference gel do Shift M to select the corresponding groups and take Ungroup Spots Menu Show Showing groups You may display the group IDs of the reference gel on all visible gels which
33. an Excel file containing the amino acid composition of a protein Fig 4 6 Du eh LOU Scene ere bfa jee rege kapa and LE A 4 Hr frag a Figure 4 6 Composition label containing a link to an Excel file Text link you may have a long piece of text inside any label without over loading the display To create a text link add an annotation to a gel and type the string which must contain the word text A double click on this type of label displays a dialog box containing the entire text For example you may have text containing the sample preparation procedure the detection method or the bibliography for a spot attached to your gels and only part of this information is displayed over the gel In Fig 4 7 the label Sample prepara tion has the protocol used for this purpose ge es mE pon FER Figure 4 7 Sample preparation label containing a link to a text field describing the procedure of the ECOLI sample preparation Melanie 3 User Manual Edit L gt Annotations Edit L gt Annotations MODIFYING AND DELETING ANNOTATIONS Modifying and deleting annotations You can modify and delete annotations and labels By double clicking in one label an edition window is displayed where you may change its text contents On the other hand once one annotation is selected it may be modified by taking Modify In the edition windo
34. and proteomics scientists If you have any sug gestions problems or questions on the use of the features please contact your local Bio Rad office or in the U S call Bio Rad Technical Service at 1 800 4BIORAD 1 800 424 6723 Please report any reproducible software problems to Bio Rad Laboratories Melanie 3 User Manual 153 E EEEE TECHNICAL SUPPORT 154 Melanie 3 User Manual A accession number AC 53 adaptive gradations histogram on groups 95 histograms on classes 105 histograms on classes groups 107 adding annotations 55 labels oet 55 E CN EN SDOLS euer eet tees 42 adjusting contrast 33 in real time 34 aligning OelS aine ee e 31 special alignments 32 Analyze Classes Histograms 104 105 Classes Report 105 107 Groups Histograms 93 Groups Report 96 Heuristic Clustering Do Clustering 99 Hl meses 100 Report ues 100 Scatters Plot 89 Statistical Tests 108 annotation tool 60 annotations 8 53 adding items 55 as starting pairs
35. banner You may also use the Shift or Ctrl keys to select continuous or dis continuous gels on the stack Moving stacked gels Melanie provides tools to superimpose corresponding spots of stacked gels in a simple way In stack mode you can move one or several gels as usual see Moving gels on page 27 Melanie 3 User Manual View L gt Stack MOVING GELS To superimpose two spots put the cursor on the given spot in the front gel then hold down the mouse button Press Ctrl F to display the other gel Move the cursor to the corresponding spot and release the mouse button The two spots will be superimposed Verify this by pressing Ctrl F again Displaying overlapped spots While two or more gels are stacked visible spots from the reference gel of the stack can be seen in transparency mode on the front gel The spots from the reference gel of the stack are displayed in blue below the spots from the front gel Set the reference gel of the stack as described above see Stacking gels on page 25 Some spots of the reference gel should be displayed Choose Show Over lapped Fig 2 8 To dismiss the overlapped spots choose Hide Overlapped ef SC gt EL Gi e 7 rf de I w E a PI He o Figure 2 8 a Overlapped spots spot shapes displayed b Overlapped spots with pair vectors spots filled When gels are in stack mode you may also display the pairs between the reference gel and the other gels of the s
36. can be defined using the Region tool When a region is defined certain actions may be limited to it Fig 1 5 Melanie 3 User Manual 7 1 ES INTRODUCTION Spots Spots represent the proteins on the gel They can be automatically detected by Mel anie or manually adjusted by the user Fig 1 5 Each spot in a gel has an associ ated spot ID a unique sequential number automatically given when it is created Spots may be quantified i e their optical density area and volume are computed Alternatively spots may be modeled as Gaussian functions Annotations Spots or individual points pixels may be marked by annotations An annotation is defined by its position and its set of labels Fig 1 5 It contains relevant information which is separated into label categories pre defined or user defined categories Melanie provides a number of pre defined categories such as Pi_Mw Landmarks Comments Sets and ACs The label category Pi_Mw contains the known pl and Mw values which are used to propagate this information to any point in a gel The label category Landmarks is used as a reference point for operations such as gel alignment The Set category is used to mark a set of spots sharing the same prop erties or results from analysis User information may be saved in labels of Com ment category or in any other user defined category The category Ac holds the protein s accession number AC taken from a user selected database and may be a
37. create the same reports found in the Histograms on Classes window for the selected corresponding histograms The available reports are the Report on Gels used as a legend for classes and gels as well as the Report on Groups separated by classes In the Histograms menu button you may choose to display specific histograms on classes and on groups Statistical tests Melanie also includes statistical tests to determine differently expressed proteins see reference 11 The main characteristic of statistical tests is to establish the probability that the measured difference between populations can be due to chance In order to express the results as probabilities the protein values must follow the restrictive assumptions about these tests Because protein expression among several gels rarely follow these assumptions Melanie only displays the numerical values for each test and lets the user search in the appropriate tables for the corresponding significance levels Select a given number of groups among the selected gels Choose Statistical Tests You then have to select one of the spot value types for the analysis Od Vol Area Od Vol Choose the statistical test values which you would like to have displayed from Stu dent t test Wilcoxon test and Kolmogorov test The Statistical Tests report displays for each selected group of spots the chosen statistical values The displayed values should be considered as qualitative indica
38. faster and will produce better and more accurate results especially with distorted gel images see Aligning gels on page 31 Melanie has been designed to automatically match gels Nevertheless you may speed up and improve the matching process by specifying a few starting pairs You may define starting pairs by manually adding pairs or by automatically pairing iden tical annotations such as landmarks or ACs for example 80 Melanie 3 User Manual Edit L gt Pairs View gt Stack MATCHING TIPS Using annotations as starting pairs If two or more gels have annotations with identical labels for example landmarks you can use these to create starting pairs Select the gels then Match Annota tions Melanie will ask you to select the category of labels to match and the refer ence gel It will then for each spot in the reference gel that has a label of the selected category defined in it pair it with spots in the other gels that have the same label defined in them These newly created pairs may then be used as starting pairs for matching Note that only annotations that are linked to spots may be matched Adding deleting pairs Select one spot in one gel and one or more spots in the other gel using the Spot tool click on one spot then hold down the shift key and click on the other ones Choose Add or Ctrl Shift L To delete pairs select spots in one gel and select the gels from which the corre sponding pairs will be
39. gap of 0 5 from the range size of the current class to its furthest class Because this value is not symmetrical a value from class A compared to class B is not the same as the value from class B compared to class A Fig 8 2 No overlapping case Interval class A o Interval class B p Overlapping case Interval class A Interval class B QQ i 0 75 0 5 Figure 8 2 Schema of the Normalize value Note that the calculations where the protein is absent in a class are represented in the report and histograms by the number 1000000 Melanie 3 User Manual 103 8 KNEW CLASSES Analyze Classes Histograms Histograms on Classes As for groups of spots you may visually study statistics values of classes by display ing histograms Select groups of spots and choose Classes Histograms Select one of the spot value types Od Vol Area Od Vol In the dialog box choose central and dispersion statistics see Statistics for Groups on page 90 The dis played histograms show for each class the chosen Displayed value and the disper sion interval Fig 8 3 Groups Histograms menu Displayed value list Reports menu Classes Histograms Dispersion Sal EU interval Histograms on Classes Vol The used statistics are Mean 100 and M S D Central value Group ID Sorted values Figure 8 3 Histograms on Classes The
40. gel image see Annotations on page Melanie 3 User Manual 31 2 ES GELS 53 The other gels must contain the same landmarks i e same landmark label inside the same spot as the alignment algorithm will try to deform the images in order to best superimpose the landmarks Fig 2 14 Figure 2 14 Gels before and after alignment Gels are in stack mode and their pair vectors are displayed Alternatively if you align gels that have already been matched you may choose to use existing pairs rather than labels from the category Landmarks as reference points View Select all gels that have to be aligned Choose Align In the displayed window gt Align Gels choose the reference gel The selected gels will be aligned relative to the reference gel so that corresponding landmarks are best superimposed To get back to the original images select aligned gels then choose Unalign The original images will be displayed again in place of the aligned ones By default gels are aligned relative to the X and Y image axes using a second degree alignment function in both the X and Y axes You may decide to align images according to only the X or Y axis or to use another alignment function Choose Other Alignment and select the desired option First second and third degree functions are available that request at least two three and respectively four land marks In the event that gels are not aligned but have common landmarks operations such
41. histogram toolbar 1 Save 2 Print 3 Copy to Clipboard 4 Select on Gel Image 5 Select next 6 Select previous Reports also have particular characteristics in common The lines in the tables are numbered indicating in this way the number of selected objects used to create the report The columns are independent elements which may be displaced resized and sorted in descending and ascending order of their values similar to a spreasheet window Saving printing Reports may be saved or printed Tables are saved using the Excel table format i e with tabulations separating the columns Graphics are saved using TIFF or BMP for mats To save reports click the Save button in the toolbar item 1 Fig 5 2 Enter the file name in the displayed window and choose the file format in the Type field To print a report click the Print button item 2 Fig 5 2 The usual PC system win dow is displayed where you can choose printing options such as the printer name Exporting data You may export your data directly from a table into another program for example a spreadsheet software like Excel Select the desired lines in the table using the Shift or Gtrl keys Copy the selection to the clipboard by choosing the Copy to clipboard button in the toolbar item 3 Fig 5 2 or in the contextual menu by clicking the right mouse button Paste into the desired software Melanie 3 User Manual 71 72 5 EEEE REPORTS AND NAVIGATION Yo
42. is set Label Ldmk1 of pre defined category Landmark Category Name Content his protein is a component of the EUCINE ISOLEUCINE VALINE THREONINE transport system IVJ ECOLI a Annotation Set of labels b i tim H reste O amp alele Information on selected Labels c LOM XD jm _ ar Si ERT Em ECO LI ace PO H 1157 267 508 Gar LM protein is a component of the LEUCINE ISOLEUCINE VALINE transporte system pru EEE Figure 4 1 a Annotation over one spot with 5 labels b The editable window of the annotation showing categories and contents of the displayed labels c Report on labels 4 Comment is an indicatory category to users It exists as an example of a gen eral category where users may store any comments 5 Set is a generic category used to mark spots which have some common char acteristics All labels from a Set category share the same text i e the set name In Fig 4 2 a set called St contains spots belonging to the same result of Student T test analysis et p 3 Figure 4 2 A set of spots marked by the same category of type Set St 54 Melanie 3 User Manual Edit L gt Annotations CREATING ANNOTATIONS To help you visually identify the different categ
43. key aspect of the Melanie program is its ability to link spots on gel images to pro tein data in 2 DE databases 2 DE databases contain data on proteins identified on 2 DE images such as pl and Mw bibliographical references to protein related litera ture information on protein functions etc Several specialized 2 DE databases exist that Melanie can work in conjunction with Melanie is especially well suited to work with data from the SWISS 2DPAGE global 2 DE database that is accessible over the Internet see SWISS 2DPAGE master gels on page 115 Spots on 2 DE gels analyzed with Melanie may be linked to two different types of 2 DE databases 1 Private 2 DE databases This is a database you build yourself by defining your own master gel on which you add protein related data You may then match gels with your master protein map 2 Remote 2 DE databases If your computer is connected to the Internet you may link your gels to remote databases such as SWISS 2DPAGE and remotely retrieve protein data related to spots on your gels The spot annotation with Ac label The annotation with an Ac label is a small tag associated to a spot which identi fies the corresponding protein see Annotations on page 53 The Ac label holds the proteins accession number taken from the 2 DE database The Ac label is the link between the gel and the 2 DE database Melanie 3 User Manual 113 9 ES DATABASES Tools Database Querying a remote d
44. link to Melanie s remote database query engine Labels may also contain direct links to other files and Web sites Pairs and groups The pair is the association between two corresponding spots i e it represents the same protein in two gels Pairs may be manually defined by the user or may be automatically determined using Melanie s powerful gel matching program When two gels are matched the corresponding spots are paired Figure 1 6 One pair A group represents the same protein in several gels When several gels have been matched to a given reference gel the latter provides a unique spot numbering scheme across all gels Each paired spot in a gel image may then be associated to the corresponding spot ID in the reference gel The spots in a set of gels that are paired to one given spot in the reference gel together form a spot group The group is the basic element for analyzing and searching changes in protein expression across gels Melanie 3 User Manual OBJECTS Variation in protein expression may be visualized and analyzed using histograms or through powerful statistical methods Reference gel Group Figure 1 7 One group Classes The assembling of a number of gels which have a common biological meaning or common characteristics establishes a class of gels It corresponds to a set of gels representing the same population For example gels from the same infected tissue but from different patients belonging
45. of Geneva Switzerland Copyright The Swiss Institute of Bioinformatics retains all ownership rights to the Melanie software and documentation No part of this publication may be reproduced or transmitted in any form or by any means electronic or mechanical including photocopy recording or any information storage or retrieval system without permission in writing from Swiss Institute of Bio informatics Copyright 1992 2000 Swiss Institute of Bioinformatics Geneva Swit zerland All rights reserved Copyright 1988 1999 Sam Leffler and 1991 1999 Silicon Graphics Inc for the TIFF library Technical support Bio Rad provides technical support on the features of the Melanie soft ware The staff of Bio Rad is receptive to your suggestions If you have any suggestions problems or questions on the use of the features please contact your local Bio Rad office or in the U S call Bio Rad Tech nical Service at 1 800 4BIORAD 1 800 424 6723 Please report any reproducible software problems to Bio Rad Laboratories rademarks TIFF is a trademark of Aldus Corporation Microsoft Excel Microsoft Word Microsoft Internet Explorer Windows 95 Windows 98 and Win dows NT are trademarks of Microsoft Corporation PostScript is a trade mark of Adobe Systems Inc Netscape Communications Netscape and Netscape Navigator are trademarks of Netscape Communications Corpo ration Rather than put a trademark symbol in every occurrence o
46. other options in the Tools menu you will have to spec ify file names Inverting grey levels The last option in the Tools menu lets you change the grey levels of selected gels by inverting them reverse video Select gels then Invert Gray Levels As for other options in the Tools menu you will have to specify file names 124 Melanie 3 User Manual PART IV APPENDIXES DIFFERENCES BETWEEN MELANIE 1 AND MELANIE or WHERE TO FIND WHAT SEEMS TO HAVE DISAPPEARED Show and select objects Most operations in Melanie may only be applied to selected objects on selected gels For instance a report on spots will only be enabled if there are selected spots on selected gels Likewise most of the main operations in Melanie may now only be carried out when objects are visible on gels i e you can only select the objects that are visible on the display For example to select annotations or spots they must be displayed on gels Nonetheless when a hidden object is selected by the means of a report for exam ple it may be viewed on the screen but it will immediately disappear once it is unse lected again Select gels Selecting gels is much faster and easier To select one gel you just need to click on the gel s selection banner the horizontal stripe located at the top of the gel which contains its name The action of selecting gels may be done at any time with any of Melanie s tools Melanie 3 User Manual 127
47. same directory as the matched gels and pairs will be automatically created against the synthetic gel The resulting synthetic gel will be created and displayed Fig 10 2 See Gja jaa daiis ipn Di iis Hip Figure 10 2 A synthetic gel and its three contributing gels The reference gel is ECOLI 120 Melanie 3 User Manual Tools gt Gels DUPLICATING GELS You may now perform a quantitative study on their spots using spot groups see Groups on page 85 The synthetic gel can be updated by adding new spots with Edit gt Spots Copy from Gel see Adding modifying spots on page 42 The image will be updated according to these new spots Duplicating gels You can create copies of selected gels and include existing objects such as spots or annotations Select the gels you want to duplicate Choose Duplicate and give new file names for them You may also add an extension or replace the extensions of the original image files The gel image files will be copied to disk They will then be opened and displayed in the Melanie window Erasing gels To delete existing gels select them then Erase All selected gels will be removed from the screen as well as from the disk Associated spot pairs are deleted as well Cropping gels Define regions in selected gels using the Region tool Choose Crop You may choose what objects you want to keep with the cropped image such as spots and annotations You may als
48. used statistics Fig 7 4 item a Notice that the standard deviation is the Mean squared deviation multiplied by N N 1 where N is the sample size This dif ference comes from the fact that the standard deviation should be an unbiased estimator Median Mean 0 and Mean absolute deviation 100 are much more robust to outliers than the statistics above Fig 7 4 item b e Midrange 100 and Half range 100 define an interval which includes all sample values Fig 7 4 item c Mean 100 Median Midrange 100 M S D 100 M A D 100 Half range 100 b c Figure 7 4 Histograms showing the sensitivity of central and dispersion values a Mean 100 M S D 100 b Median M A D 100 c Midrange 100 Half range 100 Melanie 3 User Manual Analyze L gt Groups Histograms HISTOGRAMS ON GROUPS e Midrange 50 and Half range 50 are known as order statistics and interquartile ranges Fig 7 5 Figure 7 5 Histograms showing the sensitivity of outliers suppression Midrange and Half range values with a 100 b 80 c 50 and d 33 Notice that absent spots represented by zero values are also considered in the cal culation of statistics Histograms on groups Histograms and Report on Groups provide valuable operations which may be used to look for peculiar groups of spots Analyzing groups or verifying the matching or spot detection operations are some of the endles
49. well as with TIFF files Analyzed gels are saved into the Melanie release 3 file format unless otherwise specified by the user Importing foreign image formats Melanie allows you to import images stored in foreign image file formats such as TIFF 8 or 16 bits GIF Bio Rad scan Choose mages In the Import Image dialog box choose one of the available formats You may also choose the reduction factor to be used when importing images Saving gels Any changes carried out on gels have to be saved in order to be stored in the gel file Otherwise the changes will be lost when exiting the Melanie program or when closing the gels Choose All Changes to save all changes performed on the selected gels or any of the individual saving options For example Save Spots will only save the detected spot shapes but not other changes After performing changes on gels you may revert to the saved status on disk by choosing one of the Hevert from File options in menu File Current changes will be lost The Save As command enables you to create copies of selected gels or parts of them if regions have been defined in gels You may also use this command to save files created in Melanie as earlier Melanie versions see What happens when you open your old Melanie files on page 129 Exporting gels and windows To files Alternatively you may save any gel images in one of the supported foreign file for mats TIFF or BMP In this case gel
50. which the current gel was assigned to The name of the gel to which the current gel was aligned to Melanie 3 User Manual SHOWING GELS The zoom factor of the current gel Gel banner Figure 2 3 Gel banner is the gel selection area For example a gel called 94 0002 with a zoom factor 4 assigned to class CLASS A and aligned to gel ECOLI will show the following information in the yellow stripe 94 0002 CLASS A ECOLI x 4 The gel banner is used to select gels and to drag them Showing gels Once you have opened a given number of gel images you can decide which ones you want to display and which ones you prefer to hide at a given point in time To hide a gel drag it to the hidden gels zone Fig 1 2 number 6 To hide several gels at a time select the gels as described previously and while holding the Shift key drag them to the hidden gels zone Fig 2 4 LIME mmm 3 Bis gt im Ld Sr Kemi Took iz Be ee ro m ECOLE rei Hidden Gels F j D e di e i e D Visible Gels k 7 ei e 7 x z a gt e Gale Spe E keete D Figure 2 4 Hidden and visible gels Melanie 3 User Manual 23 2 ES GELS To make a hidden gel visible on the display area drag the selected gel from the hid den gels zone to an available cell on the screen If you drag the hidden gel to a cell already occupied by another gel b
51. will appear on the screen To select the displayed information click Settings in the Cursor Information window s toolbar and the Settings Cursor Information dia log box will be displayed Fig 3 6 Select the attributes which you would like to have in the Hidden list left column and transfer to the Visible list right column using the gt gt button You may choose many attributes at once by using the Shift or Ctrl keys Click Apply to confirm your choice and the selected attributes will be displayed on Cursor Information window You may also double click on one attribute and it will be transferred from one list to the other On the other hand you can remove attributes from the information window by selecting the items on the Visible list and clicking lt lt The selection order is pre served on the display You may also save the current chosen template of cursor information by clicking Save and typing the file name in the Name field In the same way you may load an existing template by pressing Load and choosing the file name These settings may also be made permanent from the Preferences Cursor Information menu option Melanie 3 User Manual Reports La Spots Report REPORT ON SPOTS Menu Reports Report on Spots Select some spots and choose Current or one of the other report templates that you may have defined previously see below A report will be displayed The Spots Report summarizes all relevant informatio
52. 2 1 962 2000 Genera Switzerland Melanie 3 User Manual MELUMBRO 199 Warranty Bio Rad Laboratories warrants that the Melanie software shall substantially conform in all operational features to Bio Rad s current specifications as pub lished in Bio Rad s user and installation guides and that when properly installed it will be free of material defects which affect system performance The Purchaser must notify Bio Rad in writing within 30 days of delivery of the soft ware not including delivery of any subsequent modifications to the software of any defect If the software is found to be defective by Bio Rad Bio Rad s sole obligation under this warranty is to remedy the defect in a manner consistent with Bio Rad s regular business practices For a defect which adversely affects the performance of the software Bio Rad shall use its best efforts to cure such defect as soon as reasonably practicable after receipt of Purchaser s notice For minor defects Bio Rad shall use its best efforts to correct such minor defects in the next release of its software If however Bio Rad is unable to cure a major defect within 90 days of receipt of Purchaser s notice Purchaser shall have the option to cancel this agreement whereupon Bio Rad shall refund only the software fees paid Liability of Bio Rad to Purchaser if any for breach of warranty or any other claim relatin
53. 67 LEUCINE ISOLEUCINE VALINE THREONINE transporte system penu pomme wig TIT se Figure 4 6 Report on selected labels On the other hand you may display information about all labels belonging to selected categories and selected gels In this case select the gels then choose Categories Report and select the desired categories in the dialog box The infor mation displayed in this report is the same as for the Report on labels Fig 4 7 3 Categories Report Bisi ES Hiel sl z e e Information on selected categories aeg Beane Label Seat X Y moje E S 2 pe po t meets p po Jl ssp ws 19 p poo Jet amp ss Els Ecs zl s e p Ju Jr eis Figure 4 7 Report on categories Melanie 3 User Manual 67 NENNEN ANNOTATIONS Reports Annotations Report File L gt Import L gt Labels File L gt Import L gt Annotations You may also create reports on selected annotations which display information about all visible categories and their labels Fig 4 8 Select annotations and choose Annotations Report 3 Annotations Report of E uai sl zje e Information on selected Annotations seo x v A Comet s Prasitne menu maa uetecmeernni _ ecu iz ms su 12037100 beermat enu m zw snp LLL JL exu ug zw PE Sen 5 a DE p wor un sa aT mr jerez sr sas p EEUU
54. A 729 ZY gt Figure 10 5 The original image left and the same image after a morphological filter with 3x3 and value index 8 right Melanie 3 User Manual 123 REN CREATING IMAGES Tools L gt Gels This algorithm produces an image by replacing each central pixel of a window by the grey value specified by value index in the list of pixel values from the defined window sorted in ascending order If the value index is 0 the filter corresponds to an erosion shrink spots If the value index is the maximum window size the filter corresponds to a dilation grow spots If the value index is the central window size the filter is a median filter Flipping gels If you have scanned images in the wrong direction you may correct this by applying the Flip Gels options Select gels then Flip and choose one of the flipping options Horizontally Vertically or Both Directions As for other options in the Tools menu you will have to specify file names Scaling gels The mage menu lets you create larger or smaller copies of selected gels Select gels then Scale By and type the horizontal and vertical scale factor values As for other options in the Tools menu you will have to specify file names Rotating gels If you have scanned images in the wrong position you can correct this by applying Rotate Gels Select gels then Hotate A dialog box lets you specify the rotation angle with the sliders As for
55. AGE database what has changed during the last year Nucleic Acids Res 1999 27 289 291 19 Appel R D Bairoch A and Hochstrasser D F 2 D Databases on the WWW In Methods in Molecular Biology Vol 112 2 D Protocols for Proteome Analysis A J Link Eds Humana Press Inc Totowa NJ 1998 pp 383 391 20 Appel R D Hoogland C Bairoch A and Hochstrasser D F Construciing a 2 D Database for the WWW In Methods in Molecular Biology Vol 112 2 D Protocols for Proteome Analysis A J Link Eds Humana Press Inc Totowa NJ 1998 pp 411 416 Melanie 3 User Manual 21 Tonella L Ou K Sanchez J C Frutiger S Appel R D Bairoch A Walsh B J Yan J X Tyler M Hoogland C Rouge V Morsch f S Gooley A A Hughes G J Williams K L Hochstrasser D F 97 Escherichia coli SWISS 2DPAGE data base update Electrophoresis 1998 19 1960 71 22 Appel R D Interfacing and Integrating Databases In Proteome research new frontiers in functional genomics M R Wilkins K L Williams R D Appel D F Hoch strasser Eds Springer Verlag Berlin Heidelberg 1997 pp 149 175 23 Hoogland C Baujard V Sanchez J C Hochstrasser D F Appel R D Make2ddb a simple package to set up a 2 DE database on the WWW Electro phoresis 1997 18 2755 2758 24 Peitsch M R Wilkins M R Tonella T Sanchez J C Appel R D Hochstrasser D F Large scale protein modelling and integration with the SWISS PROT and SWISS 2DPAGE datab
56. And immediately at the end of this operation Melanie will implicitly unload the raw images thus freeing the used memory Note that the chosen Display depth that is set in your computer determines the memory used by Melanie to display gels see the Display icon on the Control Panel of your computer to find out more details For example 16 million colors require 3 times more memory than 256 So you may also control Melanie s performance in managing the memory by choosing the display depth according to your needs Closing gels and exiting Melanie Use Close to close all selected gels If any of the gels have not been saved since changes were performed Melanie lets you choose to save them before closing them To exit Melanie select Exit Melanie lets you choose to save any unsaved gel images before exiting Our mascot Barry greets you with a folklore song from Switzerland Melanie has been developed in Geneva at the foot of the Swiss Alps where the Saint Bernard dog is at home and people sing yodel to communicate between moutaintops 1 http www ips k12 in us mskey pods Big_Mac_Pod gwaltney yodel The_Yodel_Course html 20 Melanie 3 User Manual Select La Gels SELECTING GELS Menu Select Selecting gels Most of Melanie s functions can only be performed on selected gels Gels may be selected either by clicking on their banners or by using one of the Gels options in menu Select Regions of gels may also be sele
57. Fig 7 7 defacb 345 Figure 7 7 Histograms on Groups with unsorted values and with sorted in ascending order values Also to highlight spot variations inside a group the histograms gradations are adjusted according to each group Unselect the Adaptive gradations check box to display the same gradation in all histograms Fig 7 8 Figure 7 8 Histograms on Groups with adaptive gradations set individually and set according to the minimum and maximum values You may look for similar histograms easily on Histograms on Groups window Select one histogram and press Sort by Similarity button in the toolbar A new histogram Melanie 3 User Manual 95 EE GROUPS Analyze L gt Groups Report 96 window will be displayed where the selected histogram is first displayed and the other histograms follow a similar criterion This criterion is based on the selected histogram s shape For example the histograms shown in Fig 7 9 can be consid ered similar abcdefghijk abcdefghijk abcdefghijk abcdefghijk 859 699 817 58 Figure 7 9 Histograms displayed according to their shape similarity Reports on groups Similar to the Histograms on Groups the Heport on Groups describes numerical data concerning groups of spots as well as their corresponding statistical values Once you have selected a given number of groups possibly all you may display a report on them Choose Groups Report Melanie will ask you to
58. Groups on page 85 Toolbar Objects on screen may be displayed manipulated or processed using the various options from the Melanie menus or by selecting them with the tools provided in the toolbar The Melanie toolbar contains 5 buttons Fig 1 4 the hand tool 7 the magnify tool 8 the region tool 9 the spot tool 10 and the annotation tool 11 Is eje ep 00066 Figure 1 4 The toolbar enlarged 7 Hand tool 8 Magnify tool 9 Region tool 10 Spot tool and 11 Annotation tool The Hand tool moves gels in order to show other parts The Magnify tool lets you repeatedly zoom in or out the whole gel The Region tool is useful to select a rect angular region within a gel The Spot tool lets you select individual spots and the Annotation tool is used to select annotations and labels On line help The manual is also available on line in the Melanie program To consult it on line Make sure that the Help folder is located in the same folder as the Melanie applica tion Then in the Help menu choose User manual An index is also available To access the on line user manual and index you must have the Netscape Navigator World Wide Web browser installed or Microsoft Internet Explorer You may choose the table of content document from which you can read any of the on line manual chapters or you can request the on line index then click on the desired concept to obtain the related on line chapter Melanie 3 User M
59. I as simultaneous gel moving gels matching etc are done based on a simple inter polation between the two nearest common landmarks i e landmarks with the same name Otherwise when the gels are not aligned and have no landmarks the oper ations are based on the same image location 32 Melanie 3 User Manual GRAY LEVELS Gray levels A 2 DE gel image is traditionally displayed as a gray level image where gray levels represent the gel s optical density You may opt to display images using pseudo col ors You may also change the way gray levels are displayed Adjusting contrast Modern scanners are usually able to scan 2 DE images with 12 or even 16 bits per pixel that is with 4096 or 65536 gray levels respectively Because common screens are only able to display 256 colors or gray levels mapping has to be undertaken between the 4096 or 65536 gray levels respectively and the 256 screen gray levels By default Melanie uses a linear mapping function where the lightest point in the image is mapped to 0 white and the darkest point is mapped to 255 black In Fig 2 15 the minimum and maximum gray levels zero and 2361 respectively are remapped by the default linear mapping Define the display transfer function Display Figure 2 15 1 Gray level distribution of selected images 2 Mapping transformation 3 Minimum and maximum gray levels slider 4 Gray level distribution after the function transformatio
60. Just take View Move Gels then choose the corresponding sub option The equivalent of a double click inside a gel may be found in Melanie s menu Select one gel and take Same Location All gels will be positioned at the same location with the same magnification of the selected gel If you cannot see a gel because it was moved outside the visible parts of the screen while using the hand tool then select the gel and take one of the options from View Move Gels Zooming gels The Magnify tool lets you repeatedly zoom in or out the whole gel To zoom in pick the Magnify tool and click over the gel in the wanted region To zoom out click the right mouse button Fig 2 9 Zoom out times 0 25 Zoom in times 4 Zoom in times 8 Figure 2 9 Zoom in and out 28 Melanie 3 User Manual View Zoom Window L gt Zoom MAGNIFYING WINDOW To zoom in or out of all visible gels simultaneously hold down the Shift key and then click the left or right mouse buttons respectively After the zoom all gels will be moved to the same position Alternatively you may choose to zoom in and out by taking Zoom and selecting the corresponding sub option The minimum possible zoom factor is 1 16 times the original gel image The maxi mum zoom factor is 32 times the original gel image however the resulting images will have a maximum size of 32 000x32 000 pixels Magnify glass The Magnify glass is an alternative way to vie
61. Manual 35 2 ES GELS Edit L gt Gels L gt Calibrate Edit L gt Preferences back to gray levels choose View Gray Levels Pseudo Colors and select Gray Levels among the displayed options Wen Ep ies Jet Dope De Beie Ek Figure 2 18 Gels displayed using the FiveRamps color lookup table Menu Edit Calibrating gels Melanie enables you to calibrate pixel intensities and as a consequence to calibrate gels Rather than displaying gray level values taken from the scanner device you may display image values indicating the real world values for example optical den sity values see Calibrating spots on page 44 Calibrated gels have an asterisk in their names in the reports Setting preferences A few preferences may be set and preserved between Melanie sessions using the options in the menu Edit In Raw Image you may decide that all further open gels will keep their raw data image in memory see Loading unloading gels on page 19 36 Melanie 3 User Manual Reports Gels Report REPORTING ON GELS In Grid Preferences you may set the grid attributes i e the grid units and number of subdivisions see Displaying grid lines on page 31 In Cursor Information you may select spot and pixel attributes to be displayed in the Cursor Information window see Cursor information on page 47 In Database you may set preferences for the automatic queries in the internet see Databases on pag
62. OLI LIYK ECOLI protein is a component of s P04391 E XS E UCINE ISOLEUCINE VALINE OTCL COLI Bt vi THREONINE transport system t t j St SU m zza 6P2 FCOLI P04384 gt e 5 20 43000 E 00 1 e 4816 b og e P13857 his protein is a component of the EUCINE ISOLEUCINE VALINE THREONINE transport system 04391 s 06999 Figure 4 3 a All categories and labels are visible b Only Categories Ac Pi Mw and Comments are visible and only for certain spots When you click on an annotation which has visible and hidden labels all its labels will be displayed on the screen during the time it remains selected Fig 4 4 The hidden labels will immediately disappear when the annotation is unselected wre wre AEN Figure 4 4 Annotation with hidden labels is unselected During the time it is selected the hidden labels become visible Melanie 3 User Manual 65 NENNEN ANNOTATIONS Show L gt Annotations 66 L gt Set Color If several labels hold the same content then by default each spot will be displayed with its own label You may display a single label for all identical labels by making Pack Categories and choosing the desired category name Unpack Categories separates the label tags again Fi
63. Report and Histograms on Classes Displays chosen statistical values for classes of gels calculated on selected groups such as central tendency devia tion and overlapping measures Report on Statistical Test Displays statistical values for classes of gels such as Student t Wilcoxon and Kolmogorov significance levels You may display as many reports and histograms simultaneously as you wish Once a report or histogram is displayed on the screen it is listed in the Window menu and may easily be brought in front of all the other displayed windows Fig 5 1 Eun Help Zoom Information Spots Report Groups Report Groups Histograms Groups Report 1 Figure 5 1 List of open reports Note that report and histogram windows may hide other existing report and histo j gram windows by popping up in front of them Many of the report and histogram properties such as saving printing organizing etc are shared by all of them This chapter is dedicated to these common proper ties 70 Melanie 3 User Manual SAVING PRINTING Toolbar AII Melanie report histogram and plot windows have in common the toolbar shown in Fig 5 2 Reports also have the property of being navigable meaning that from one or more items in a report histogram or plot you may re select corresponding objects on the gel as well as travel through the objects which are listed in the reports PP o PP gel zl ass Figure 5 2 Report and
64. You may also calibrate gels using calibration annotations from the reference gel Set the reference gel The calibration values from the reference gel are propagated to the other gels through the paired spots You may also import a calibration by using the mport Calibration command A dialog box asks you the file name The specified file for example a gels report must be in a text file format and must contain the columns Gray Slope and Gray Offset with the corresponding values Once gels have been calibrated Melanie allows you to display the best fitting line through the calibration values After displaying Calibration Plot similar to the scat ter plot window see Scatter plot on page 88 you may open two other reports by selecting one of the menu item of the report button in the Calibration Plot toolbar These reports are Fitting Report shows the gel name gray slope and offset values the corre lation value and the number of spots Calibration Report displays spot ID optical density calibration and fitting error values as well as X and Y coordinates of spots You may change the calibration at any time by taking Edit Gels Calibrate Intensity and choosing a calibration category or remove it by taking Reset Calibra tion Menu Show Showing spots Once spots have been detected you may display their shapes over the gels in three different ways Fig 3 5 filled or outlined shapes as well as crosses locate
65. also inside the classes In the Histogram on Classes Groups window unselect the Adaptive gradations check box to have the same gradations in all displayed histograms Report on Classes Specific reports on classes may be generated to display their statistical values Once classes have been set select the gels and groups of spots to be included in the report Choose Classes Heport Select one of the spot value types Od Vol Area Od Vol In the dialog box select central and dispersion statistics which are explained in Statistics for Groups on page 90 Histograms menu Displayed value list Reports menu 3 Classes Report Hiel j hur Center Report on Classes Vol The used statistics are Mean 100 and M 5 D Gee Wa Cesk Gost cssc P e Lem re SIS mee mee mee meer 4 E SI Weer OAC LL Gora SIS We oo veer STI SI Heel med eem 0 0899008 0 0855545 0 0893008 0 0829942 8 2 00236457 0 0236457 QO06I8310 0 0210032 Figure 8 8 Report on Classes The Report on Classes displays the chosen Displayed value Fig 8 8 for each class and the maximum value among all the classes The values available in the Melanie 3 User Manual 107 8 MN CLASSES Analyze L gt Statistical Tests Report on Classes allow you to differentiate one class from the other classes and this according to each group of spots From the Report on Classes window you may also
66. anual OBJECTS Objects A Melanie object is an entity that may be displayed and manipulated The objects are gels as well as gel components such as spots regions annotations and labels Pairs groups and classes are objects which allow for spots comparison across sev eral gels Gels A gel is a two dimensional electrophoresis image that has been digitized and stored on disk Several image formats may be opened as gels but they have to be saved into the Melanie file format in order to keep objects such as detected spot shapes and values pairs etc If a gel is saved with its objects into a foreign format then the objects will simply be part of the saved image and will have lost any structure or value Annotations 4 uet 7400 lt gt Label category Ac UCC ECOLIT v Fak a Label category ProteinName Label category Landmark Label category Pi_Mw Figure 1 5 Spot shapes annotations labels in a region The gel image is the raw input data to the Melanie program from which proteins may be detected and quantified Gels may also be duplicated erased cropped fil tered flipped or scaled Gels may be displayed in tile mode one gel next to the other or in stack mode gels on top of each other Gels may be aligned that is dis torted gels will be stretched pixel wise in order to make a set of images completely superimposable Regions A region is a rectangular subpart of a gel that
67. ases The example of Escherichia coli Electrophoresis 1997 18 498 501 25 Appel R D Bairoch A Sanchez J C Vargas J R Golaz O Pasquali C Hochstrasser D H Federated 2 DE database a simple means of publishing 2 DE data Electrophoresis 1996 17 540 546 26 Appel R D Sanchez J C Bairoch A Golaz O Ravier F Pasquali C Hughes G J Hochstrasser D The SWISS 2DPAGE database of two dimensional polyacry lamide gel electrophoresis Nucleic Acids Research 1996 22 17 3581 3582 27 Pasquali C VanBogelen R A Wilkins M Frutiger S Appel R D Vargas R Sanchez J C Hochstrasser D F The ESCHERICHIA COLI SWISS 2DPAGE data base Electrophoresis 1996 17 547 555 28 Sanchez J C Golaz O Frutiger S Schaller D Appel R D Bairoch A Hughes G J Hochstrasser D F The YEAST SWISS 2DPAGE database Electro phoresis 1996 17 556 565 29 Gravel P Sanchez J C Walzer C Golaz O Hochstrasser D F Balant L P Hughes G J Garcia Sevilla J Guimon J Human blood platelet protein map estab lished by two dimensional polyacrylamide gel electrophoresis Electrophoresis 1995 16 1152 1159 30 Sanchez J C Appel R D Golaz O Pasquali C Ravier F Bairoch A Hoch strasser D F Inside SWISS 2DPAGE database Electrophoresis 1995 16 1131 1151 31 Appel R D Sanchez J C Bairoch A Golaz O Miu M Pasquali C Vargas R J Hughes G Hochstrasser D F SWISS 2DPAGE a database of two dimen s
68. at well adapted to 16 bit images In this case you have to tune them Detect spots in a region You may also only detect spots in a given region If a region has been defined in a gel Melanie enquires whether the detection must be done in the selected region only or in the whole gel This allows you to refine the detection in a gel s area with other parameters Spots outside the region are not affected Melanie 3 User Manual 41 42 3 EE 5007 Preview detection results If a region has been defined each change of a parameter value may immediately display the result from the spots detection in the selected region Fig 3 3 Figure 3 3 Adjusting detection parameters in real time Note that if the small rectangle temporarily disappears from the display change one of the detection parameters slightly to make it visible again Adding modifying spots The usual way to create spots is to detect them using Melanie s powerful spot detec tion algorithm Nevertheless you may manually create modify or delete selected spots To add a new spot on the gel choose the Spot tool and double click in the desired location In the dialog box select Disc radius to draw a circumference and use the slider to choose its radius units in number of pixels Click OK and make the desired modifications of the recently created spot in the Edit spots window To modify a spot double click on the spot shape and the edit window will be dis
69. atabase through the Internet In Melanie you may work with your own master images or you can link your gels to any SWISS 2DPAGE master gels An interesting possibility to link your images to 2 DE databases is offered if your computer is connected to the Internet You may retrieve data from remote 2 DE databases for any label containing a valid accession number AC The http query is formed by two parts e the fixed part which contains the http address and the query engine e the variable part which contains the Ac label Querying one annotation means that the fixed and variable parts are concatenated Setting the browser To retrieve data from remote databases you must have the Netscape Navigator World Wide Web or Microsoft Internet Explorer browser installed Choose Settings and select your favorite browser Setting the database You have to set the http site of the remote federated 2 DE database see reference 25 i e a 2 DE database that may be remotely queried from within 2 DE analysis software Choose Settings and enter the whole http site For example you may decide to query on the ExPASy server the SWISS 2DPAGE 2 DE database using its query engine Nice2dpage or the SWISS PROT protein sequence database using the query engine Niceprot In the first case type http www expasy ch cgi bin nice2dpage pl in Database URL field In the second case type http www expasy ch cgi bin niceprot pl Click the World 2D button in
70. ation tool Click on any label to select it The selected label will be highlighted in green To select more than one label use the Shift key You may also select labels and annotations by using the menu items Select a region on a gel using the Region tool then In Region to select all annotations in the selected region or All to select all annotations in all selected gels If colors where assigned to annotations they may be selected by taking By Color and choosing the corresponding color in this menu option You may select a group of labels which have a sequence of letters in common in their contents For example LAT designates all labels from the chosen categories whose names contain the sequence of letters LAT For this purpose take By Con tent Choose one or many categories and enter a label content to select the corre sponding labels Expressions in this field are taken literally when the Regular Expression field in the dialog box is not selected By selecting the Regular Expression field in the dialog box you may use regular expressions that can be inserted into the search field A regular expression is a con cise and flexible notation for finding patterns of text A list of regular expressions which may be used in search criteria is given in Table 4 1 Table 4 1 Regular expressions to search patterns in labels Syntax Description Example Matches any single character e oli matches eaoli aboli ecoli
71. ations Jean Charles Sanchez V ronique Rouge Luisa Tonella Catherine Zimmermann The following persons have also contributed through code development sup port or the development of earlier versions of Melanie Melanie 3 User Manual e Peter Burke Horia Ciobanu e Ottavio Cremona e Anca Dima e Roberto Fabbretti e Olivier Golaz e Andrew Goodman e Christine Hoogland e Romica lancu e Elena Manoila e Marina Miu e Markus M ller e Christian Pasquali e Gheorghe Popescu e Florence Ravier e Jan Snydr Michael Melanie 3 User Manual
72. ats Melanie format organizing aeos eise Mes re overlapping measures for classes P paS AC AG RIAA AGE definition dieses e ees KEPORUIN ww ene eed tette selecting NEE Showing ette pixels information preferences Ee cursor information database 5 o ere grid lines tees raw image easiest printing gels eap histograms ree regions nece Lees tees Velo ON pseudo colors Q quantifying spots R range ratio reference gels reference gels zone region tool E e EE printing oerte Reports All Matches Annotations Categories Gels ede i edt TOPOS AS Ea 69 all matches 83 exporting EE 71 on annotations 67 on categories 67 On classes esiste ntn ther 104 107 on gels 37 on groups 94 96 ON labels em 67 on matches 83 on ms spectru
73. ay also change the rectangle s size by dragging its corners or borders To dese Melanie 3 User Manual 21 22 2 ES GELS lect the drawn region select the Region tool and click outside the rectangle region or double click inside one region Figure 2 2 Selected region in gel To select the same region on all visible gels hold the Shift key and select the rectan gular region on one of the gels In this case the selected regions are also calculated proportionally to corresponding pixels Menu Show Displaying gels Melanie provides numerous ways of displaying gels and related information on the screen each one with specific and helpful functions You may display gels in tile mode one gel next to the other or in stack mode piles of gels where only the first one is displayed You may arrange gels as you wish fixed or variable number of display cells and whenever you wish show and hide gels You may display numer ical gel information Cursor window or complementary information Zoom window grid lines Each displayed and visible gel has two special areas that indicate its name and the operations that have been performed on them Fig 2 3 The gel banner horizontal stripe located at the top of the gel which contains its name indicates whether or not the gel is selected The gel legend located in the top left corner of a gel yellow background besides indicating its name also provides The name of the class to
74. box or type POO and unselect the Regular Expression field You may select all labels from specific categories Choose By Category and pick out the category names If you have already saved a Heport on Labels or a Report on Annotations you may reselect all labels by choosing From Report File A dialog box lets you choose the name of the file which contains the report Finally if some labels have already been selected you have the possibility to do Inverse Selection This will unselect the selected labels and select all unselected ones inverting the criteria used to select them You may also select annotations directly from a displayed Report on Annotations Labels or Categories see Reports and navigation on page 69 Note that by selecting spots their linked annotations are also selected Mass spectrometry You may match spots according to their mass spectrometry data using a correlation algorithm For this purpose the mass spectrometry data must be available in labels through one of the following formats Melanie 3 User Manual Select L gt Annotations gt By MS Select L gt Annotations L gt Common Labels Select L gt Annotations SELECTING ANNOTATIONS AND LABELS e as a file link see Creating specific links on page 56 i e using the file key word followed by the name of the file which contains the ms data This file must be either a ms file type PerSeptive GRAMS 386 or a text file containing a sequen
75. ce of mass values separated by carriage return e asa text link see Creating specific links on page 56 i e using the text keyword followed by a sequence of mass values To match spots according to their MS data select one label then choose By MS In the dialog box choose the label category with which the selected label will be matched A Mass Spectrum Match report is displayed showing the match results and a score is given for each match from 0 to 1 i e from bad to good match The selected label is also in the report with a score of 1 match with itself Common labels You may easily retrieve common labels among many gels If two or more gels are selected you may choose Common Labels to select common labels in all gels In the dialog box choose the categories of labels to which the selection will be applied If only one gel is selected this special kind of selection will correspond to selecting packed labels i e selecting labels from the specified categories containing the same content Combining sets of annotations You may combine the selection of categories and annotations For this purpose decide for a first criteria and select annotations using one of the above commands Take the menu option Combine and choose one of the four operations from Inter section Union Difference and Exclusion In the dialog box choose the label cate gory corresponding to your second criteria Click Ok The result of the logical opera
76. cell will be inserted at the dragged location Stacking gels Very often users prefer to visually compare gel regions simultaneously arranging them side by side in the display i e in tile mode which is Melanie s default mode But in the case where many gels are open at a time their visible parts may be too small Stacking gels i e displaying one gel on top of the others thus creating a pile of gels is another way to display and compare gels You may compare each gel of a stack one by one for example against the reference gel of the stack Fig 2 6 Besides the stack mode allows you to quickly permutate gels letting you visu ally discern changes in protein intensity You may stack many gels at a time and you may also have as many stacks as you want Dh Bu Le Eee Jiri dem Wie st Figure 2 6 Gels in tile mode and stack mode To stack two gels select one gel and drag it onto the other gel To stack several gels select the gels and drag them onto one of the cells Alternatively select gels and take Selected Gels Certain operations that can be performed on stacks let you compare the front gel with one of the other gel in the stack called the Stack Refer ence Gel To set the reference gel of a stack select the stacking cell and choose Melanie 3 User Manual 25 26 2 ES GELS Set Stack Reference The name of the reference gel of the pile is displayed with a blue background over the gel and th
77. cted using the Region tool To select one gel click on the gel s selection banner The gel will be surrounded by a green line indicating that is has been selected To select more than one gel select the first one as described above then hold Shift or Ctrl keys and select additional gels Alternatively you may select all visible gels in the Melanie window by choosing Visi ble or only the aligned gels by choosing Aligned see Aligning gels on page 31 or just the stacked gels by choosing Stacked see Stacking gels on page 25 You may select specific gels by choosing their names in the list given by the By Name command You may inverse an existing selection of gels by choosing Inverse Selection or select all gels by taking All Gels which have spots matched with the reference gel may be selected by taking For Groups In the same way Gels belonging to specific classes may be selected by taking For Classes You may also select gels directly from a displayed Report on Gels see Reports and navigation on page 69 The region tool The Region tool is useful to select a rectangular region within a gel Position the cur sor at the top left position of the desired region hold down the mouse button then move the cursor to the bottom right position A rectangle will be drawn around the region Fig 2 2 Release the mouse button at the desired point You may move the region by clicking inside the rectangle s area and dragging it You m
78. d groups The group concept is essential to analyze spots in a set of gels Elaborate reports histograms statistics and analyses may be produced from them see Groups on page 85 Melanie 3 User Manual 79 o EE Pairs Edit L gt Pairs In Melanie you can match gels with single pairs or multiple pairs Matching with single pairs means that one spot in a gel will be paired with exactly one spot in the other gel Matching with multiple pairs means allowing that one spot in one gel be paired with several spots in the other gel Fig 6 1 Figure 6 1 One spot paired to two spots in the other gel Menu Edit Automatic matching To match two or more gels select them by clicking on their banners while holding Shift or Control keys or by using the Select Gels menu options Choose Auto Match Gels Melanie will first request that you designate the reference gel from the selected gels You will then be asked whether you wish to allow multiple pairs All gels will then be matched with the reference gel Depending on the computer you are running Melanie on this might take time Saving pairs creates a pairs file that resides in the same directory as the matched gels Therefore only gels that reside in the same directory may be matched Matching tips The only prerequisite for matching two gels is that spots have already been detected Aligning gels prior to matching is optional but in many cases the matching process will be
79. d in the center of the spots Select gels then choose one of the options in the Shape menu w Ww e w 5 p Figure 3 5 New spots display outlined shape filled shape and crosses Spots may be displayed with the Show All menu option and hidden with the Hide All option Melanie 3 User Manual 45 3 IN SPOTS Show L gt Spots L gt Set Color Select gt Spots Select a region on a gel using the Region tool then In Region to show all spots in the selected region You may show only selected spots with Only Selected Only the spot shapes of the initially selected spots are kept on the screen It is very help ful for example to mark some spots on the screen when you are performing a visual inspection of gels or a visual inspection of the analysis results You may hide only selected spots with Hide Selected You may also show respectively hide the IDs of selected spots Select some spots then Show ID respectively Hide All ID Hiding spots also has the additional effect of displaying images faster Coloring spots Spots may be displayed on the screen using 3 different colors other than the default red To do so select spots then choose Set Color and one of the proposed colors The Default command colors spots in red the default color in Melanie The colors remain displayed until you select Reset All to color all spots in red as the default The colors are not saved Menu Select Selecting spots T
80. d labels The Annotation tool is useful to select annotations and labels To select one annota tion pick the Annotation tool and click annotation basis To select one label just click on it Annotations and labels may only be selected when they are visible on the gels Annotations may be selected with the Annotation tool Click at the basis of any annotation to select it or at one of its labels The selected annotation will be high lighted in green and will be displayed in front of all the other displayed annotations 60 Melanie 3 User Manual Select L gt Annotations SELECTING ANNOTATIONS AND LABELS Fig 4 9 If the annotation is attached to one spot the spot will also be selected To e OLI Lone Hout pi 32168 SYS ECGLPD ECt p32168 HSLU ECOLI HSLU ECOLI t PO i t PO R AM L aes SR b Figure 4 9 a Annotation hidden by the other annotations b Same annotation displayed in front of the other annotations after selection select more than one annotation select the first one then hold the Shift key and select additional annotations To select all annotations in a given region position the cursor at the top left position of the desired region hold down the mouse button then drag the cursor to the bottom right position All annotations in the selected region will be selected and highlighted in green Labels may also be selected with the Annot
81. deleted Choose Delete or Ctrl Shift U Note a very convenient way to add pairs between a set of gels and a reference gel is to create groups see Groups on page 85 Corresponding spots may also be easily discerned when gels are in stack mode and spots are displayed in overlapped mode In this case use also the flip facility Ctrl F to locate spots Menu Show Showing pairs You can look at the matching results in stack mode Stack gels then choose Show Pairs The pairs between the reference gel of the stack and the other stacked gels are displayed in the form of blue vectors linking the locations of paired spots Fig 6 2 To hide vectors choose Hide Pairs Figure 6 2 Matched gels blue vectors indicate paired spots Melanie 3 User Manual 81 o EEE Pairs a In tile mode groups of spots are easily visualized by displaying the group IDs see Showing groups on page 86 Menu Select Selecting pairs To select all pairs among two matched gels select them then choose All Pairs All Select paired spots will be selected L gt Pairs You can highlight only pairs for a given set of spots Using the Spot tool select a number of spots in one of the gels Choose For Spots Spots in selected gels that have been paired to the selected spots will be highlighted Fig 6 3 Figure 6 3 Selected paired spots across gels and the corresponding report To select only multiple pairs from two selected ge
82. dly classify similar gels into two or more classes and to determine the characteristic spots of each class that is the proteins that are potentially differently expressed in the various classes Select gels and groups of spots Choose Do Clustering Select one of the spot value types Od Vol Area Od Vol to be considered by this classification algo rithm In the following window define the minimum and maximum number of classes to be formed by Melanie respectively Minimum number of classes and Maximum number of classes parameter For example if you choose 2 and 4 Melanie will successively split the gels in 2 3 and 4 classes then compare the resulting classifications and according to a simi larity function show the best one Select the sensitivity parameter to be considered between the highest and the low est values in groups Sensitivity parameter The smaller this parameter the smaller Melanie 3 User Manual 99 EE GROUPS Analyze L gt Heuristic Clustering the accepted difference among groups will be and more spots will be present in the results This parameter may be seen in a schematic way in Figure 7 13 Sensitivity Spot Interval Class 1 Interval Class 2 min max min max Figure 7 13 Sensitivity parameter Depending on the computer you are running Melanie on this might take time espe cially when the number of maximum classes is high The resulting classification may be visualized in a den
83. dogram which plots the classes and their gels It may also be analyzed through its Report where the signifi cant spots are also displayed The Classification Level parameter in the Heuristic Clustering window establishes the depth of classification which may be seen in the plot and report as well Fig 7 14 After the analysis has been performed you may display a report which shows the names of the selected gels and their classes as well as information on each selected group such as group ID and their intervals of minimum and maximum val ues in each class In this case take Report Another way of visualizing the classification made by Heuristic Clustering is to dis play a dendogram which shows the classes and their gels Choose Plot Premrn CEREA Figure 7 14 Two classes and one classification level 100 Melanie 3 User Manual Edit L gt Classes A number of gels having common biological meaning or common characteristics establish a class of gels It corresponds to a set of gels representing the same pop ulation One of the main goals of Melanie is to help you find significant protein expression changes among several populations of gels For this purpose two com plementary techniques are available e Overlapping measures Summarizes each class by an interval and mea sures the overlapping across them e Statistical Test Measures the difference among populations and estimates if this difference is significant i e is
84. double click on their sep aration The column at the left of the cursor will regain its default size You may also enlarge or reduce the row size In this case drag the separation between two rows to the top to reduce the row or to the bottom to increase its size To enlarge or reduce all rows at once hold the Shift key and drag the line s separa tion To resize rows so that their whole content is displayed double click on their separation Melanie 3 User Manual DATA ANALYSIS Introduction Gel matching is a key operation in 2 DE gel image analysis The basic gel match ing algorithm consists of comparing two gel images and finding pairs of related spots i e spots depicting the same protein in both gels A pair is represented by a doublet f f where f is a spot ID in the first gel and f a spot ID in the other gel One generally says that gels have been matched and that individual spots have been paired Melanie can match two or more gels Matching two gels means finding all the pairs between spots of the two gels Matching several gels means picking out a refer ence gel then successively matching each gel with the reference gel In this way spots in all gels may be compared with spots in the reference gel For consistency when matching only two gels Melanie will also ask you to specify which one is the reference gel All spots in selected gels that are paired with the same spot in the reference gel form so calle
85. e 113 Menu Reports Reporting on gels Select gels as described previously and choose Reports Gels Report Gels Report display summarized information about the selected gels such as image height and width in pixels Fows and Columns minimum and maximum gray levels number of detected spots number of existing annotations pixel dimensions of the scanned image PixWidth and PixHeight The class to which the gel was assigned to and the intensities calibration is displayed as well The Group information also available in the Report on Gels indicates the reference gel Ref and whether the gels are matched against it Value 0 indicates no match ing and 1 indicates matching between the gel and the reference gel Fig 2 19 4 Gels Report Bisi x Mal Si e Information on selected Gels TT ations T Gas Amoi D s Mary Sage SE Ji zm 2 ZC zemes Stam D IS SI Wen ma i seo Tr zer zast Gato DIR Figure 2 19 Report on gels Gels ECOLI and 94 0002 are calibrated Melanie 3 User Manual 37 2 ES GELS 38 Melanie 3 User Manual _ SPOTS The elementary component of a gel is the spot Spots are spot shapes automati cally detected by Melanie s spot detection algorithm or manually adjusted by the user Each spot in a gel has an associated spot ID a unique sequential number automatically assigned to it when it is created Spots may be quantified i e
86. e click on an http link label will launch your favorite browser software Netscape or Explorer on your screen and it will automatically retrieve the corre sponding Web page To set your favorite browser choose Edit Preferences Database For example you may have a direct link to EXPASy Fig 4 5 Pm e es ss Dee TRE x 0 m ud rr m i Ta T CH his is a link to ExPASy me fei beis bech bei rr rese LL Can abel sedi ceger i Uoseeg gt ExPASy Molecular Biology Server The pake FaF y It ons CH Tia s e res ESP rea roba bob agp UTE arr x Ser La D sors SAL This yw d d mid di ama eode aoro lish THT Figure 4 5 Label This is a link to ExPASy has a link to a http page File link you may link spots or regions of gels to software files To create a file link add an annotation to a gel and in the text field enter the string which must contain the word followed by the file name Click OK A double click on this label will launch the specified file with the default system application associated to the file extension The linked files must be located in a specific directory which may be defined by choosing Edit Preferences Data base and by filling the Annotations folder field see Setting preferences on page 36 Melanie 3 User Manual 57 58 NENNEN ANNOTATIONS For example you may have
87. e name of the first gel of the pile the front gel is displayed as usual Fig 2 7 Reference gel of the stack Figure 2 7 Stacked gels Cell on the left has 3 stacked gels The front gel is 94 0005 the gel in the top of the pile 94 0002 is the second gel the gel in the middle of the pile and Ecoli is the bottom gel located in the bottom of the pile Cell on the right has other 3 gels stacked together To put a gel in front of the stack you may either press the Page Up or Page Down keys You may also click on a gel s banner or take Next or Previous To switch from the front gel to the reference gel of the stack choose Front To Back or simply Ctrl F By pressing Ctrl F quickly several times or the Page Up or Page Down keys the differences between the stacked gels can be observed visually To unstack the gels select the gels and choose Unstack Selected Gels When many gels are stacked or hidden their names are not entirely displayed Click on their banners and their names will be displayed in the front gel respectively in the stack or hidden pile Selecting stacked gels You can select each gel from a stack individually You may also choose to select two or several gels in the stack and operations will be performed only on these selected gels To select one gel click on its banner To select all the gels in the pile double click in the front gel s banner which corresponds on the stack to the left most name on the
88. e of displaying gels allows the user to instantly see how the proteins are expressed through a series of gels see Displaying gels on page 22 Stack mode of displaying gels gives you the possibility to track protein varia tions flicking gels see Stacking gels on page 25 Transparency mode allows you to visually link spots from the front gel with spots from the gels behind see Displaying overlapped spots on page 27 Reporting results More than 20 reports tables and graphics are available which contain over 50 data types Intuitive quantitative filter possibilities see Sorting data in tables on page 74 Fully customizable reports see Heports and navigation on page 69 and Report on Spots on page 49 e Simultaneous navigation in the reports and on the gels see Navigating through data on page 73 Database and Internet ready Seamless exchange between your own database and Melanie see mport ing labels on page 68 Selecting annotations and labels on page 60 and Saving printing on page 71 Direct database querying through Internet such as Swiss Prot see Data bases on page 113 Comprehensive and unlimited annotation capabilities You may add any type of information in default or user definable categories see Pre defined label categories on page 53 and Creating annotations on page 55 Fast landmarking allows to easily define tie points in multiple gel studies see Pre defined label categories
89. e selection methods described above Then take Pairs Report You will get the list of all paired spot IDs identifications among gels Fig 6 3 When many gels are selected this report corresponds to a report on group IDs dis playing the list of all paired spot IDs among all gels Another way to display the matching result is to select two or more matched gels then Matches Heport This will display the number of pairs between all the selected gels and any open gels Finally you may choose All Matches Report This displays information on match ing between all gels in a given directory Melanie 3 User Manual 83 6 EEE Pairs 84 Melanie 3 User Manual Introduction The spot group is an essential concept in the Melanie software When several gels have been matched to a given reference gel the latter provides a unique numbering scheme for spots across all gels Each paired spot in a gel image may then be asso ciated to the corresponding spot ID in the reference gel The set of spots that are paired with one given spot in the reference gel forms a spot group see Figure 1 7 on page 1 9 The group ID is the ID of the corresponding spot in the reference gel The group is the basic element for analyzing spot variations across gels and for pro ducing histograms and reports as well as statistical and artificial intelligence analy sis Groups are automatically created when matching a set of gels with a reference gel but they may
90. e tables by the values of the columns Click once on the header of the desired column and a triangle will be displayed indicating that the col 74 Melanie 3 User Manual SORTING DATA IN TABLES umns data is sorted in ascending order Click once more on the header and the tri angle will be inverted indicating that the data is sorted in descending order Ascending order means that numbers are sorted from 1 to 9 and text is sorted from A to Z 3 Spots Report oj x Mel alle Information on selected Spots rise seb A spe ui ee 08 UT ECCE REI EE 2 330018ew 178 171 1087 86 0 260573 2 23531 0 318753 703 001 ser en el reet zem 4 33006vew 103 171 404 539 0 178323 1 81944 0 261873 327 000 Slam Ter leed zz veer eg omest TGA wis ELI 0 728783 6 54998 0 248587 658 001 mr 1 00213 5 04245 0 503736 629 000 3 330026c w 1 23687 5 82221 4 Sa R SpotlD 171 rt 171 171 184 184 184 184 74 b Figure 5 5 Items sorted by two criteria group ID R SpotID and volume Spots were first sorted according to volume ascending order then according to spot ID a Sort ascending b Sort descending 171 You may sort the columns elements using multiple criteria Rows that contain equal items in a sorted column appear together When using two or more criteria the rows wit
91. easures 102 Histograms on Classes 104 Histograms on Classes Groups 105 Report Classes 107 Statistical tests 2 hit ho eis oe ores 108 PART III TOOLS 9 DATABASES ceeds sene CE ERO VE ECHOS EE A Rien 113 2 DEdatabases eeeeeeee enr 113 Querying a remote database through the Internet 114 SWISS 2DPAGE master gels 115 10 CREATING IMAGES 119 Creating synthetic gels 119 Duplicating gels 121 Erasing Gels maszt seperti eae cee te ete ee ee 121 Gropping gels a cia tac steele ee Geta aa 121 Filtering rico oba aes Fae be ee yo corn eee oe 121 FlIPPING GEIS udo Saree ER D ee o a teas 124 Scaling gels Sanus western deals Se esce eS we eee ae ie 124 Rotating Gels inlet I tae MOR Ee a eee 124 Inverting grey levels 124 Melanie 3 User Manual i RN CONTENTS PART IV APPENDIXES iv A DIFFERENCES BETWEEN MELANIE II AND MELANIE 3 OR WHERE TO FIND WHAT SEEMS TO HAVE DISAPPEARED 127 SHORT CUTS AND MENU STRUCTURE 131 Men structuren uere wi oki a a 132 Gels handling Seed Seege ren NEE in 142 Spots handling
92. ected 64 In Region 64 Linked Data 66 115 Only Selected 64 Pack Categories 66 Set Color Default 66 Melanie 3 User Manual 159 160 Reset All 66 Set Colors 66 Show All sss 64 Unpack Categories 66 Visible Categories 64 Gels Hide Selected 24 Only Selected 24 Show 24 Sorted by Classes 24 Sorted by Name 24 Groups Hide All ID 86 Show ID 86 Spots Hide All Sita 45 Hide All ID 46 Hide Selected 46 In Region 46 Only Selected 46 Set Golor uo tures 46 Default 46 Reset All 46 EEE 45 Show All 45 Show ID esses 46 showing annotations 64 circularly e drittes 49 gels acies pee tense 23 groups nt sieja etude 86 labels 64 Eo ee ee 81 ele 45 single pairs 80 smoothing smooth filters
93. elanie 3 User Manual 137 ENIE SHORT CUTS AND MENU STRUCTURE 138 Table 10 10 Select menu Short cuts See page All Ctrl Shift A 82 Bad Pairs 82 Multiple Pairs 82 Groups By ID 87 For Spots 87 All Groups Shift M 87 Restrict Selection 87 Inverse Selection 87 Select All Unselect All Melanie 3 User Manual Table 10 11 Analyze and Reports menus Menu Short cuts See page Analyze Groups Report 96 Groups Histograms 93 Scatters Plot 88 Classes Report 107 Classes Histograms 104 Statistical Tests 108 Heuristic Clustering 99 Do Clustering 99 Report 99 Plot 99 Reports Gels Report 37 Spots Report 49 Current 49 Labels Report 67 Annotations Report 67 Categories Report 67 Pairs Report 83 Matches Report 83 All Matches Report 83 Melanie 3 User Manual 139 ENEI SHORT CUTS AND MENU STRUCTURE Table 10 12 Tools menu Short See cuts page Gels Crop 121 Duplicate 121 Erase 121 Flip 124 Horizontally 124 Vertically 124 Both Directions 124 Filter Image 121 Smooth 121 Histogram Equaliza 121 tion Remap Gray Levels 121 Contrast Enhance 121 ment Filter Background 121
94. elected the other one You may now stack many gels at a time i e display one gel on top of the others thus creating a pile of gels By switching from one gel to the other many new oper ations may be done on the gels of the stack such as displaying overlapped spots for example You may also choose to select one two or many gels in the stack and operations will be carried out only in these selected gels Analyzing data In Melanie 3 not only the display has changed but also the way to reason about groups of spots and classes of gels Anyhow some operations used to analyze gels Melanie 3 User Manual SHOW AND SELECT OBJECTS in Melanie 2 3 or earlier may still be found in Melanie 3 with the difference that zeros absent proteins are taken into calculations Differential analysis with classes set take Analyze Classes Report In the Classes Report dialog choose Mean 100 and dispersion 0 In the displayed report choose Ratio in the Display value list The results will be displayed in the report and by sorting the values in this column you may select the most important group values according to your own criteria Differential analysis without classes set take Analyze Groups Report and select the Range ratio item Sort the resulting column to enable the selection of the most important values Correspondence analysis without classes set take Analyze gt Groups Report and select the Correspondence Analys
95. els waa OOS eee 68 5 REPORTS AND NAVIGATION 69 IntrOd CcllOn case ai see ONG a beets eI Re M ERE Bee x rz 69 Bee TEE 71 Savitig printlng wo eee eet eee aid O 71 Exporting data ze RIES IR En oh 71 Selecting objects 72 Navigating through data _ 73 Sorting data intables ees 74 Organizing table display 76 PART II DATA ANALYSIS 6 PAIRS nace sevice oai oz to ee Dente Radna 79 Introduction illia e Iac ewe Sees ede eee eee CN 79 Automatic matching esen esereke eee 80 ii Melanie 3 User Manual CONTENTS MA NAGTE RT Crave ete POW D eaten 80 Showing BANS ssec met ma MR rmx athe ace ree Ee 81 Selecting pairs eebe ee ee lee DA 82 Reports On pals ex 83 T GROUPS eects VEA ERES hee Seka VERRE 85 IntroOcUctloni wasze test ree cat z Moa St 85 Editing groupS ueste nd eek o een chanel ete tek 86 Showing groups NN REESEN Rx ex EN ETE Rr Mere 86 Selecting I hh 87 Scatter PIOSENEK d uomo 88 Statistics for Groups nnn 90 Histograms On groups usas yeu iii OWEN Lick wee ey 93 Reports on groups ee eee eee cee eee Eha 96 Heuristic clustering e 99 S8 CLASSES Gaerne eee este GC OSEE 101 Specifying classes wk eee eee 101 Overlapping m
96. emoval of interference and outliers by using robust statistics see Statistics for Groups on page 90 Varying staining absorption across proteins by normalizing protein expression change see Comparing protein expression changes on page 95 Merge of several gels from the same sample in a composite gel with fine control over the chose proteins see Creating synthetic gels on page 119 Compensate the gel distortion introduced by variation in the protein migration by gels alignment see Aligning gels on page 31 Quality control Margins of error and validation tools allow to reach a high quality standard The pur pose of the numerous validation tools is to ensure that you don t miss any relevant data Analysis and control are closely related both focus on peculiar spots These pecu liar spots can result from a protein expression change or from an error induced by the detection and matching process Margins of error Scatter analysis see Scatter plot on page 88 Matching score bad pairs see Selecting pairs on page 82 All statistical measures see Statistics for Groups on page 90 and Statistical tests on page 108 Validation and verification tools Melanie 3 User Manual Contrast adjustment see Adjusting contrast on page 33 Report navigator and selector see Navigating through data on page 73 Matching vectors display see Displaying overlapped spots on page 27 Protein identificator overlay see Showing
97. f other trade marked names we state that we are using the names only in an editorial fashion and to the benefit of the trademark owner with no intention of infringement of the trademark PART I BASICS CONTENTS INTRODUCTION 5353x939 22305352 anos 1 Hardware and software requirements 2 Installation ne prc cec er SERB OK neuen 2 lat Le 4 ODBJSCIS a Eed yesh ath Seabee tase 7 Selecting and showing objects 10 Features Overview 11 Important Notes oue I RO a a ELT Lecce REA 15 GELS bier bine bik wrod eb Saeed Gu wie eee WA 17 Opening gels as ee EPIRI Se 17 Saving gels use siara kage un ee wis E eee ee ee ee 18 Exporting gels and windows _ 18 Paninggal EET e Re mE a ei 19 Loading unloading gelS leeren 19 Closing gels and exiting Melanie 20 Selecting 21 Displaying gels Ie ume ERR 22 Showing gels 2 5 RR REI ERI t RR ER aS 23 Changing the display gels order 24 Stacking gels lt a ma ele lI eee I EUR exe XM E ES 25 Movirig gels d Rex HIE Gu RE eet AW 27 Zoominggels eee Peete 28 Magnifying window sa e eee aan anna anna RI III II III 29
98. g 4 5 PEDA Lk 004 50 50 P ea Figure 4 5 and unpacked labels for protein 00450 in the Plasma master gel Swiss 2DPAGE database The label belongs to the pre defined Ac category Coloring annotations Annotation tags may be displayed on the screen using 3 different colors other than the default cyan To do so select annotations then choose Set Colors and one of the proposed colors The Default command colors annotations in blue the default color in Melanie The colors remain displayed until you select Reset All to color all annotations in cyan as the default The colors are not saved You may open a label link by double clicking on it Alternatively to open any label link select the label and choose Linked Data Melanie 3 User Manual Reports L gt Labels Report Reports gt Categories Report REPORTS ON ANNOTATIONS Menu Reports Reports On Annotations Melanie provides reports for annotations categories and labels Select labels and choose Labels Report he Labels Report displays information about selected labels such as content category spots ID and X and Y positions on the gel Fig 4 6 Labels Report Bisi X sl zlelo Information on selected Labels Get ll Sed X Y 1 andmar mi mm LEES nw a ej a m 3 ECOLI be o 1157 267 267 508 LI LM This DEE is a component of the 157 2
99. g to this agreement shall be limited to the total amount of software fees paid by purchaser to Bio Rad In no event shall Bio Rad be liable for incidental or consequential damages loss of business or profits special or indirect damages of any nature whatsoever No amendment waiver or other alteration of the warranties in this agreement may be made except by mutual agreement in writing Purchaser agrees that Bio Rad s liability arising out of contract negligence strict liability in tort or warranty shall not exceed the amount of software license fees paid by Purchaser Under the copyright laws this manual and the software program contained herein may not be copied in whole or in part without the prior written consent of the Swiss Institute of Bioinformatics except in the nor mal use of the software or to make a backup copy This exception does not allow copies to be made for others whether or not sold but all of the materials purchased with all backup copies may be sold given or loaned to another per son Under the law copying includes translating into another language or for mat A multi use license may be purchased to allow the software to be used on more than one computer owned by the purchaser including a shared disk sys tem This warranty as well as the contract of purchase of the Melanie software is governed by and construed in accordance with Swiss law and is submitted to the exclusive jurisdiction of the Court of the Canton
100. groups on page 86 Circularity measure see Report on Spots on page 49 Wide variety of graphical and comparison table IMPORTANT NOTE Important Note In the following chapters all features of Melanie will be described in detail For each operation the corresponding menu option is indicated in the left margin and the text describes how to carry out the operation after having chosen that menu option Sub options for the selected menu option are indi cated in bold face in the text Melanie 3 User Manual 15 1 MEE INTRODUCTION 16 Melanie 3 User Manual This chapter presents basic Melanie procedures such as opening saving and clos ing gels displaying gel images such as arranging and stacking gels changing con trast and zoom and printing them Menu File Opening gels To open a gel image click in the File menu and select the Open option The Open pis Open gels window will be displayed Fig 2 1 Hold down the Shift or Ctrl keys and in the Open gels BEI Chercher a Gels Si eH m Figure 2 1 The Open window allows the selection of the gels that will be opened and loaded into Melanie Melanie 3 User Manual 17 2 MEE GELS File L gt Import File L gt Save File L gt Revert from File File L gt Save As Files column click on each of the gel file names you want to open Click Open The gels will be opened Melanie format The Melanie program works with Melanie file formats as
101. h equal items are rearranged according to the second criteria To do so first sort the column of secondary choice by clicking on its header Then click in the header of your principal criterion to sort In Fig 5 5 the principal criterion is the spot ID of the reference gel and the second criterion is the volume values Melanie 3 User Manual 75 76 5 EEEE REPORTS AND NAVIGATION Organizing a table display You may re organize the columns of a table directly from the report window Drag the header of the column to another chosen column Fig 5 6 The first one will be inserted before the second one Gels Report iof E gi amp sl ele Information on selected Gels 6 Gw ee 59 uge Mas 1 93004ve w 1011 00 2 b 3300330 2362 01 c 330027c w Class 1856 0 1715 0 5 3300Bve w 1696 0 1728 0 1858 0 1726 0 383 0 oO oO OO e e pr sc Cl ae tns aj e e Figure 5 6 Re organizing columns in the report window You may also enlarge or reduce the columns size Drag the separation between two columns to the right to enlarge the column or to the left to reduce its size While dragging you will notice that the cursor changes its format To enlarge or reduce all columns to the same size at once hold the Shift key and drag the separation of two columns To resize columns so that their whole content is displayed
102. have been matched to it Set the reference gel select spots then choose Show ID Fig 7 1 Inversely to hide the group IDs select Hide All ID Figure 7 1 Spot IDs of the reference gel group ids are displayed on all matched gels 86 Melanie 3 User Manual Select La Groups SELECTING GROUPS Menu Select Selecting groups The group is the basic element for analyzing and searching changes in protein expression across gels Groups allow you to produce reports or histograms and to analyze sets of gels In such cases you first have to select the groups of interest To select and display all groups choose All To select one specific group take By ID and specify the group ID when requested If the reference gel is not set Melanie will request you to select one To select the groups one or more spots belong to select spots using the Spots tool then For Spots You may also select groups directly from a displayed Report On Groups see Reports and navigation on page 69 Finally if some groups have already been selected you have the possibility to do Inverse Selection This will unselect the selected groups and select all unselected ones Restriction of groups selection Melanie allows you to restrict the groups selection according to the presence or absence of selected spots in the groups Select spots choose Select Groups Restrict Selection and one of the options in the Restrict Groups Selection d
103. he Spot tool lets you select individual spots Click on any spot to select it The selected spot will be highlighted in green To select more than one spot select the first one then hold the Shift key and select additional spots by clicking on them To select all spots in a given region position the cursor at the top left position of the desired region hold down the mouse button then move the cursor to the bottom right position All spots in the selected region will be selected and highlighted in green Spots may also be selected from the Select Spots sub menu Choose By ID and enter a spot ID to select the corresponding spot If colors have been previously assigned to spots you may choose them by taking By Color and selecting the desired color Choose Only Gaussians to only select spots that have been mod eled by a Gaussian function Select a region on a gel using the Region tool then In Region to select all spots in the selected region or All to select all spots in all selected gels If you have already saved a Spois Report you may reselect all the spots by choos ing From Report File A dialog box lets you choose the name of the file which con tains the report 46 Melanie 3 User Manual Window L gt Information CURSOR INFORMATION Finally if some spots have already been selected you have the possibility to do Inverse Selection This will unselect the selected spots and select all unselected ones You may also select spo
104. he data could not be approximated by a straight line For example e 10 x 0 with 1 correlation indicates that all paired spot values are the same in the two gels e 12 x 0 with 0 95 correlation indicates that almost all paired values are 20 higher in the reference gel Melanie 3 User Manual 89 90 EE GRours 1 0 x 0 2 with correlation 0 95 indicate that almost all paired values are shifted by 0 2 in the reference gel In general when the data show a high correlation and the fitting line is not the iden tity 1 0 x 0 you should check for possible reasons your values are systemati cally slanted perhaps staining difference of protein load or image acquisition problem Information related to the scatter plots may be found in the Reports menu button in the toolbar Fig 7 2 The gels legend can be obtained by choosing Gels Report on the menu The Fitting Report displays for each plot its corresponding slope and off set values of the regression line its coefficient correlation value and the number of spot pairs The Scatter Report displays for each pair of spots their corresponding values in the gels and the fitting error in relation to the regression line The latter may be used for example to verify abnormalities between pairs Statistics for Groups Considering groups allows you to analyze variations of proteins among gels and statistics help you to analyze these variations Melanie provides some descripti
105. histograms on classes may also be selected as any other object and be saved printed or exported to other software You may re select the corresponding groups on the gels by using the Select on Gel Image button and navigate through groups with Select Next and Select Previous buttons In the Histograms on Classes window you may find short ways to useful operations You may also find report and histogram short cuts The Heports menu button Fig 8 3 allows you to display reports correlated to the classes The Gels Report item is a reduced version of the original Report on Gels to be used for example as a legend to the classes The Groups Heport item separately displays a Report on Groups for selected groups for each class The Information displayed is the original information usually found in the Report on Groups see Heports on groups on page 96 You may also create from the Histograms on Classes window a Report on Classes for the selected corresponding histograms as well as a more complete histogram of 104 Melanie 3 User Manual Analyze L gt Classes HISTOGRAMS ON CLASSES GROUPS classes groups Histogram button in the toolbar In the same way as Histograms and Report on Groups Histograms and Report on Classes are dual elements they have a reciprocal relationship To simplify the visual search of non overlapping intervals you may display the classes values sorted in ascending order in this case select the Sorted values
106. ia log box Use the sliders to choose the threshold on the restriction The groups to the corresponding selected spots will be restricted according to e selected spots is gt to keep groups that have selected spots in at least the chosen number of gels e selected spots is lt to keep groups that have selected spots in at most the chosen number of gels unselected spots is gt to keep groups that have unselected spots in at least the chosen number of gels unselected spots is lt to keep groups that have unselected spots in at most the chosen number of gels For the ease of understanding two possible usages will be studied while these func tions are described Case number 1 Suppose that you have 6 gels and you would like to study the groups of AT LEAST 3 spots and with a Vol value higher than 0 02 In other words you would like to analyze spots according to two criteria Melanie 3 User Manual 87 88 EE GRours 1 have a Vol value higher than a certain threshold representing meaningful spots AND 2 be present in at least half of the selected gels In this case you may select this special set of spots by performing the following steps e Select all the 6 gels make Select Spots All e then Select 4 gt Spots Restrict by value choose Vol Keep selected only the spots that have a value 20 02 and click OK Spots corresponding to the first criteria will remain selected To rest
107. ic histograms are described in Histograms on groups on page 93 and Histograms on Classes on page 104 Reports menu Class separation Classes Groups Histograms Dispersion Miel mee interval 7 Histograms on Classes vol The chosen statistics are Mean 100 and 5 0 Central value abcdefghijklmnopar abedefghijk lmn 168 Spot values Sorted values Figure 8 6 Histograms on Classes Groups The histograms on classes may also be selected as any other object and be saved printed or exported to any other software You may re select the corresponding groups on the gels by using the Select on Gel Image button and navigate through groups with Select Next and Select Previous buttons You may also decide to display the chosen classes displayed values sorted in ascending order In this case select the Sorted values check box at the bottom of the histograms window Fig 8 6 The spot values are sorted inside the classes and Melanie 3 User Manual Analyze L gt Classes Report REPORT ON CLASSES the classes are also sorted according to the central value i e the Displayed value chosen for the Histogram or the Report on Classes Fig 8 7 06 0 6 0 4 0 4 0 2 0 2 0 0 abcdefghijk efdjkcbaihg 374 374 Figure 8 7 Histograms on Classes Groups with unsorted and sorted in ascending order values The classes are sorted according to their central values and the spots are sorted
108. ies of gels is to pro duce a synthetic gel by merging spots from these gels In this case the synthetic gel is used as a path connecting the studied gels in analysis operations As well side effects of gel runs such as staining running differences sample varia tion may be overcome by merging all gels from the same sample into an averaged gel Besides Melanie allows you to choose the spots which are included in the synthetic gels To create a synthetic gel perform the following steps 1 Select the spots to be included in the synthetic gel 2 Choose Tools Create Synthetic Gel 3 The Create Synthetic Gel window then lets you specify the new file name Melanie 3 User Manual 119 NENNEN CREATING IMAGES The synthetic gel contains all selected spots If spots belong to a group only one spot per group will be present in the synthetic gel which is the closest to the aver age quantification value of the group In this case Vol Area and Od values assigned to the spots in the synthetic gel correspond to the chosen spot The Od and Vol values are recalculated according to the total Od and Vol respectively of the new synthetic image If some gels are matched against others you may decide to keep only triple spots in the synthetic gel In this case only the groups whose spots form a triangle are considered in the synthetic image Fig 10 1 Figure 10 1 Triangle of pairs The synthetic gel is saved in the
109. ift H 27 Front to Back Ctrl F 25 Next Page Up 25 Previous Page Down 25 Align Gels Align 31 Unalign 31 Other Alignment 31 Grid Lines Show 31 Hide 31 Gray Levels Adjust Contrast 33 Pseudo Colors 35 Zoom In Shift Up 28 Out Shift Down 28 1 16 32 28 Move Gels Same Location 27 Up Ctrl Up 27 Down Ctrl Down 27 Left Ctrl Left 27 Right Ctrl Right 27 Top Left Right 27 134 Melanie 3 User Manual Table 10 8 Edit menu Short cuts See page Gels Calibrate Intensity 44 Reset Calibration 44 Spots Auto Detect 39 Edit Shift E 42 Copy from Gel 42 Delete Shift X 42 Annotations Add Label 55 Modify Alt E 59 Copy Alt C 59 Copy Paired Labels 59 Paste Annotations 59 Paste Labels Alt V 59 Duplicate Labels 59 Delete by Category 59 Delete Alt X 59 Link with Spot Alt L 59 Unlink from Spot Alt U 59 Pairs Match Annotations 81 Auto Match Gels 80 Add Ctrl Shift L 81 Delete Ctrl Shift U 81 Groups Set Reference Gel 86 Group Spots Shift G 86 Ungroup Spots Shift U 86 Classes Add to Class 101 Remove from Class 101 Preferences 36 Melanie 3 User Manual 135 Table 10 9 Show menu EEEE SHORT CUTS AND MENU STRUCTURE
110. images are exported as they appear on the Screen i e with their associated components such as spots and annotations But the saved file will contain a flat rasterized image without any structure 18 Melanie 3 User Manual File L gt Export File L gt Print File L gt Page Setup File Raw Image PRINTING GELS Choose Gel to File In the Export Image as dialog box choose the type of the file in the Type list and type the file name in the Name field In the same way you may save Melanie display s window in one of the supported foreign file formats Choose Gels Window to File By defining a region you can export only part of the gel To the clipboard You may paste images directly to another software Define a region in the gel then make Gel Region to Clipboard Activate the program to which you want to copy your image to and paste You may also copy and paste the whole Melanie screen In this case choose Gels Window to Clipboard Printing gels Melanie provides printing options You may print selected gels or regions of gels one image per page or print the whole Melanie window The image will be printed as it is displayed on the screen i e holding objects and properties such as Spots Annotations Contrast Adjustment Alignment Zoom Grid etc Printing selected gels Select some gels then choose Gels The selected gels will be printed in full size You may also print only regions of gels by defining regions o
111. ings will also be created Programs and files distributed with the Melanie package e Melanie program e Example images e Three images in directory Gels Ecoli e Same images after complete analysis in directory Analyzed Ecoli Other files e Manual files in directory Doc On line html files in directory Help 2 25 Note about Master protein maps The SWISS 2DPAGE database of two dimensional polyacrylamide gel electro phoresis 2 D PAGE reference maps used to be freely available on the server http www expasy ch and distributed with the Melanie system Since September 1998 the Swiss Institute of Bioinformatics decided to adopt the solution chosen for the SWISS PROT database namely to ask non academic users to financially contrib ute to the maintenance of these databases No license fee is charged to academic users for non commercial use and the master gels may be downloaded from the ftp site ftp www expasy ch databases swiss 2dpage MASTERS Non academic users are kindly requested to contact GeneBio http www genebio com for the purpose of concluding the necessary license agreements Melanie 3 User Manual INSTALLATION Licence hardware protection key To run Melanie you need to install the provided hardware protection key HASP key on the parallel port Before proceeding please turn off your computer and the attached printer if any The HASP key must be attached to the parallel port on the back of your compu
112. ional gel electrophoresis images Electrophoresis 1993 14 1232 1238 32 Golaz O Hughes G J Frutiger S Paquet N Bairoch A Pasquali C Sanchez J C Tissot J D Appel R D Walzer C Balant L Hochstrasser D F Plasma amp Red Blood Cell Protein Maps The 1993 Update Electrophoresis 1993 14 1223 1231 33 Hughes G J Frutiger S Paquet N Pasquali C Sanchez J C Tissot J D Bairoch A Appel R D Hochstrasser D F Human Liver Protein Map The 1993 Update Electrophoresis 1993 14 1216 1222 Melanie 3 User Manual 149 D C 150 Proteome and ExPASy 34 The ExPASy WWW server http www expasy ch 35 Wilkins M R Williams K L Appel R D Hochstrasser D F Editors Proteome research new frontiers in functional genomics Springer Verlag Berlin Heidelberg 1997 36 Binz P A Miller M Walther D Bienvenut W V Gras R Hoogland C Bouchet G Gasteiger E Fabbretti R Gay S Palagi P Wilkins M R Rouge V Tonella L Paesano S Rossellat G Karmime A Bairoch A Sanchez J C Appel R D Hochstrasser D F A Molecular Scanner to Highly Automate Proteomic Research and to Display Proteome Images Analytical Chemistry Vol 71 1999 37 Hoogland C Sanchez J C Walther D Baujard V Baujard O Tonella L Hochstrasser D F Appel R D Two dimensional electrophoresis resources available from ExPASy Electrophoresis 1999 in press 38 Gras R Miller M Gay
113. is item The results will be displayed in the report and by sorting the values in this column you may select the most important groups values for the factorial discrimination What happens when you open your old Melanie files When you open a file in Melanie that was created in Melanie 2 3 or earlier it is con verted to a new file format In this new format the existing information on the gels is converted to annotations by using the following rules Landmarks are converted to the category Landmarks Landmarks with Pi and Mw are also copied to the category Pi Mw Labels with Accession Numbers are converted to the category Ac Labels with short ID are converted to a category called ProteinName Gel comments are converted to a category called GelComment Its label displays the text Gel Comment and is positioned in the top left corner on the gel By double clicking on the label a dialog box shows the comment text If you work with Melanie 3 and save the files in the older format for Melanie 2 3 or earlier portions of your work in particular the new categories of annotations will not be retained Save As You may also decide to save files created in Melanie as earlier Melanie versions by using the command File Save As and by choosing Melaniell gel in the Type field In this old format the existing annotation information is converted by using the following rules Category Landmarks is converted to Landma
114. ith the help of the following tables Melanie 3 User Manual 131 ENIE SHORT CUTS AND MENU STRUCTURE Menu structure Table 10 6 File menu Short cuts See page Open Ctrl O 17 Close Ctrl W 20 Raw Image Load 19 Unload 19 Select Gels with Loaded Image 19 Settings 19 Save All Changes Ctrl S 18 Contrast Mapping 18 Calibration 18 Spots Shift S 18 Annotations Alt S 18 Pairs Ctrl Shift S 18 Save AS Revert from File All Changes 18 Contrast Mapping 18 Calibration 18 Spots 18 Annotations 18 Pairs 18 Import Images 18 Labels 68 Annotations 68 Calibration 44 Export Gel to Clipboard 18 Gel to File 18 Gels Window to Clipboard 18 Gels Window to File 18 Page Setup 19 Print Gels 19 132 Melanie 3 User Manual Table 10 6 File menu Short cuts See page Gels Window 19 Exit 20 Melanie 3 User Manual 133 EINE SHORT CUTS AND MENU STRUCTURE Table 10 7 View menu Short cuts See page Layout Settings 24 Stack Selected Gels Ctrl G 25 Unstack Selected Gels Ctrl U 25 Set Stack Reference 25 Show Overlapped 27 Hide Overlapped 27 Show Pairs Ctrl Shift Y 27 Hide Pairs Ctrl Sh
115. ivided by the total Vol over the whole image The Od and Vol values depend on pixel intensities calibration see Calibrating spots on page 44 Calibrating spots Melanie enables you to calibrate pixel intensities Rather then displaying gray level values taken from the scanner device you may display image values indicating the real world values for example optical density values This may also be useful to compensate for variations due to image scanning Melanie provides three different ways to calibrate gels By adding calibration annotations to gels By using calibration annotations from the reference gel Byimporting a calibration For example you may calibrate the image of a cali bration strip tablet using Melanie Generate and save a report on gels or any other Melanie report containing the columns Gray Slope and Gray Offset which may be imported to calibrate all your gels For this purpose add calibration values to known spots using any existing category of labels For example create one category called Calibration and add calibration values to their labels see Creating annotations on page 55 Melanie 3 User Manual Edit L gt Gels L gt Calibrate File L gt Import L gt Calibration Edit gt Gels L gt Reset Show L gt Spots SHOWING SPOTS Select gels which have calibration values and choose Calibrate Intensity Choose the label category which contains the calibration values
116. lay gels significant spots or both and the graphical results of the correspondence analysis will be displayed Melanie 3 User Manual 97 98 EE GROUPS Figure 7 12 shows the correspondence analysis plot for 6 gels and 18 significa tive group IDs Gels that are found close to each other apart from the scaling factor may correspond to the same class OR ie nid it ie zm i rl o maz St hs mi Zerf Le Se E 8 3 Figure 7 12 Plot of correspondence analysis The Reports menu button Fig 7 11 allows the display of reports correlated to the groups The Gels Report item is a reduced version of the original Report on Gels to be used for example as a legend for the histograms Spots Report The Spots Report item in the Reports menu button Fig 7 11 is useful to compare one spot against the other spots of the same group The available measures to quantify the spot values relatively to their groups are the same as above chosen value in the Displayed values list Therefore you can select the spots which are out liers or representative of a group You can select the most representative spots of a group by for example choosing the spots whose values are close to the mean difference below 20 In this case take Mean 100 and 0 8 lt ratio lt 1 2 As well a median ratio 1 will select the spots that are in the middle of the group You can select outliers whose values are outside the ra
117. lectrophoresis pictures by heuristic clustering analysis A step toward machine learning Electrophoresis 1988 9 136 142 10 Pun T Hochstrasser D F Appel R D Funk M Villars Augsburger V Pelle grini C Computerized classification of two dimensional gel electrophoretograms by correspondence analysis and ascendant hierarchical clustering Applied and Theo retical Electrophoresis 1988 1 3 9 11 Armitage P Berry G Statistical Methods and Medical Research Blackwell Sci entific Publications Oxford London 1987 12 Miller M J Olson A D Thorgeirsson S S Computer analysis of two dimen sional gels Automatic matching Electrophoresis 1984 5 297 303 Two dimensional electrophoresis 2 DE 13 2 D PAGE protocols http www expasy ch ch2d protocols 14 Link A J Editor 2 D Protocols for Proteome Analysis Methods in Molecular Biology Vol 112 Humana Press Inc Totowa NJ 1998 15 Herbert B R Sanchez J C Bini L Two Dimensional Electrophoresis The State of the Art and Future Directions In Proteome research new frontiers in func tional genomics M R Wilkins K L Williams R D Appel D F Hochstrasser Eds Springer Verlag 1997 2 DE databases 16 The SWISS 2DPAGE 2 DE database http www expasy ch ch2d 17 WORLD 2DPAGE index to federated 2 DE databases http www expasy ch ch2d 2d index html 18 Hoogland C Sanchez J C Bairoch A Hochstrasser D F Appel R D The SWISS 2DP
118. lity to interactively adjust the results see Editing spots on page 39 e Semi automatic spot editing facilities see Adding modifying spots on page 42 Accurate spot quantification using either boundary contours or Gaussian modeling see Spots quantification on page 44 Matching or how to find similar protein patterns across many gels Powerful automatic matching and editing facilities see Automatic matching on page 80 Wide variety of tools are available to monitor the accuracy and to fine tune effectively the detection and matching see Quality control on page 14 Navigation and display Extended navigation inside and across gels including various zooming modes easy moving of gels customizable grids and overlay displays see Gels on page 17 e Contextual windows help you to navigate and instantly gives you access to related information see Magnifying window on page 29 and Cursor information on page 47 e Simple procedures to select and display data subsets to work on Full control over what and how the information is displayed Melanie 3 User Manual 11 1 NENNEN INTRODUCTION Spots marking with different colors and the choice of spot shapes display see Coloring spots on page 46 and Showing spots on page 45 Large choice of the information type to display and how the associated labels are displayed see Showing annotations and labels on page 64 Comprehensive and intuitive visual comparison Tile mod
119. lle a a sumy pf kram p uzru procu beer Arsd Ze WM MIr MAPFINS CH GEL MEDLINE RTM ISCH Be ay duel lal ZABCHEZI C APTEE BD COLATOGO PASCHTATIC BEAVIER P EANOCH A HOSZTHETKASZEB LE Zreck FUE SP AE Ekurepkai ki 16115 1 TSO AUNT MDCOOPOMLZER GEESS Hr facra ai pire MAP LOCATIONS SPOT SCH ek FE Men S TM Pad OT DAHF pes Merite e SPOT SHEET pi e 11 Mee u EPOT oppor pled 4T him 2 35 A Figure 9 1 The SWISS 2DPAGE entry for human Loading labels from a master On most 2 DE databases the accession number is the unique entry identifier while the protein name may change It is therefore strongly recommended to use Acs Melanie provides a way of automatically updating the protein names in your gels Select a gel containing labels with valid Acs Set it as the master gel Select all labels you want to update Choose Load Protein Name from Swiss 2D For each selected label Melanie will request its short name from SWISS 2DPAGE database using the Ac as identifier and then update the ProteinName label accordingly In addition to labels containing data from the SWISS 2DPAGE database spots in SWISS 2DPAGE master gels also present the particularity of possessing serial numbers an identification number that uniquely links a spot on the gel and protein data in the SWISS 2DPAGE database to one spot on a specific SWISS 2DPAGE maste
120. ls excluding single pairs take Multiple Pairs You may also select pairs directly from a displayed Heport on Pairs see Reports and navigation on page 69 An algorithm to predict the accuracy of pairs has been implemented in Melanie It is based on the length of the vectors between paired spots You may select bad pairs by choosing Bad Pairs the Vector length and Score parameters Select L gt Pairs This measure of bad pairs is computed by Melanie for each pair according to the fol lowing steps 1 aneighboring region is considered around each pair 82 Melanie 3 User Manual Reports L gt REPORTS ON PAIRS 2 corresponding regions are superimposed in order to minimize the vector lengths in the region using Least Squares 3 for each pair of spots in the region the score is increased by 1 when its vector length in pixels is below the threshold and decreased otherwise 4 a pairis considered to be bad if its final score is smaller than the second thresh old the Score parameter On the other hand you may also select good pairs In this case perform the follow ing steps 1 Choose two gels and then Select Pairs All Pairs 2 Select Show Spots Only Selected 3 Choose Select 4 Pairs Bad Pairs 4 Choose Select Spots Inverse Selection Menu Reports Reports on pairs Melanie can display a report on pairs among matched gels Select matched gels and a number of pairs using one of th
121. m match 63 ON THE 83 ONESPOIS Gi eme b eus 49 organizing sess 76 PINUNO EE 71 SAVING esse 71 selecting objects 72 sorting data 74 restricting groups 87 reverting gels 18 rotating gels 124 running Melanie 3 S saving foreign image formats 18 histograms 71 Melanie images 18 ee eee ees 71 ET re DEE 18 scaling gels 124 scatters 88 fitting report 90 scatter report 90 Select Annotations I ODRA 61 By Category 62 BY Golof zazna ii 61 By Content 61 BY MS a aio da yt aa 63 Combine Difference 63 Exclusion 63 Intersection 63 Union Macnee 63 Common Labels 63 From Report File 62 In Region ase 61 Inverse Selection 62 Gels Aligned ata 21 Alliance n 21 By Name 21 For Classes
122. n 5 Bending parameter However you may choose other mapping functions e g in order to highlight small light spots Melanie lets you change the gray level mapping in two different manners You can define the minimum and maximum gray levels i e you may look at only the light or the dark regions of the images or you can change the default linear mapping function Melanie 3 User Manual 33 2 MEE GELS View 34 L gt Gray Levels L gt Adjust Contrast The Bending parameter expands or compresses the contrast range at the dark or light ends of the range When bending is higher than zero the image is lighter and it is darker when bending is lower than zero Fig 2 16 Figure 2 16 Region of a gel showing a contrast adjustment the left image shows the original image the central and right images show the same gel area after transformation by setting the bending parameter to 2 and 2 respectively To adjust the gels contrast select some gels then choose Adjust Contrast In the Adjust Contrast window change the minimum and maximum gray levels respec tively left and right bar corners item 3 Fig 2 15 or the bending slider item 5 Fig 2 15 Once the parameters have been changed the resulting gray level of the modified display can be visualized in the Display sub window item 4 Fig 2 15 from the Adjust Contrast window By choosing the Only in Region check box in the Adjust Contrast window the image gray level dis
123. n about the selected spots their identification number ID quantification values gel name their center position on the gel image X and Y values circularity value p and Mw and all attached labels You can also have information taken from the paired spots in the reference gel see Groups on page 85 The circularity value is calculated according to the spot perimeter and area The higher the value the more circular the spot As usual reports may be saved printed exported used to navigate through data etc see Heports and navigation on page 69 Customizing reports You may choose the spot attributes to build your personalized reports on spots and their order of appearance In the report window click on the Settings button in the toolbar In the Settings Spots report dialog box Fig 3 7 to add attributes to the report select their names in the Gels list left column and transfer to the Visible attributes list right column using On the other hand you can remove spot Melanie 3 User Manual 49 3 EE 5007 attributes from the report by selecting the items on the Visible attributes list and clicking The order is preserved on the display apri Tal ex amp do Iren on siipi fools stem BF i Lag be hrein pon irk Settings E button si 24 Figure 3 7 Spots report and the settings window You may al
124. n selected gels Printing the Melanie window Select Gels Window The image of the whole Melanie window is sent to the printer Page setup The Page Setup command calls the standard Windows dialog where you may set printing parameters such as printer name paper size and orientation etc Loading unloading gels You may control Melanie s use of memory by loading or unloading gel images raw pixel values The default Unloaded mode uses less memory but as a consequence the real pixel values are not displayed in the Cursor Information window Window Melanie 3 User Manual 19 2 MEE GELS File gt Close File L gt Exit Information You may choose to keep their raw images in memory Of course as a consequence pixel values information will be displayed but more memory will be needed too To load raw images select gels and choose Load You may also decide to free up the memory used by the loaded images by choosing Select Gels with Loaded Image to determine those that are loaded and then choosing Unload to finally liberate the memory At this point you can decide whether or not to keep raw data from any gels opened subsequently In this case select the check box in the pop up dialog of Settings When using the default mode of unloaded images Melanie will in any case auto matically and implicitly load their raw images when needed for example prior to per forming the Auto Detect Spots and Adjust Contrast operations
125. n with a spot click on the anno tation basis and drag to a spot You may also select the annotation and choose Link with Spot On the other hand you may select the annotation and drag outside the spot or choose Unlink with Spot to separate one annotation from a spot One spot may have many annotations One annotation may have many labels but only one label from each category Melanie 3 User Manual 55 NENNEN ANNOTATIONS Edit L gt Annotations Creating label categories To create your own category of labels you must add a label to the gel Type the name of the new category in the upper field of the displayed window You may then type the content of the label in the pop up editing field and click OK The label con tent will be displayed on the gel This new category will be available on the list of categories while there is at least one label of this category in the open gels Creating sets The pre defined category called Set was created to help you mark spots indicat ing that they belong to a set It contains no information except the fact that it belongs to a special set The label is defined by the word Set followed by an identification name To create a set select one or many spots choose Add Labels click on the cate gory Set and append a keyword which will identify this set to the string Get Click OK For example to mark spots reminding you that they need to be sequenced type Set To be seq
126. nd Mw grid may also be displayed over gels which don t have this information but have been matched against the reference gel containing pl and Mw In this case the grid values will be relative to the reference gel Fig 2 13 Helen CIGLE E je Dude Name Ini dra Wee See Cm r sa fb S Ze eg Inch HME ru ia Hah 2 ipri Besch rege f Figure 2 13 Grid lines over gels Select a gel and choose Show In the displayed window choose a grid unit from centimeters inches or pl Mw Also choose the number of sub divisions horizontal and vertical in the sliders The information displayed in square brackets in the top right corner and in the bot tom left corner of the gel indicate the coordinates of the visible portion in chosen units To hide the grid lines of selected gels select the gels and choose Hide Aligning gels Gel alignment is a very useful technique for comparing 2 DE images that present too many variations in protein migration In general while all Melanie programs may work with unaligned gel images alignment might prove helpful when comparing stacked gels or for matching distorted 2 DE gel images You may align two or more gels at once You must select a reference gel i e an image the other gels will be aligned to At least four annotations containing labels from the category Landmarks for the default alignment algorithm must have been defined and must be well distributed over the
127. nge given by its mean and two times the standard deviation In fact with a normal distribution at most 5 of the values should be outside this interval Other examples to find out outliers can be summarized as Melanie 3 User Manual Analyze c Heuristic Clustering HEURISTIC CLUSTERING Median ratio value gt 2 selects spots which are at least 2 times higher or lower than half of the spots from the group MidRange 100 Half Range 100 normalized value 1 selects spots which are the highest or the lowest in their groups MidRange 50 Half Range 50 normalized value gt 2 selects spots which are at least 2 times higher or lower than the 75 of spots from their groups By restricting the selected groups you can for example remove the groups which contain too many outliers You can also use this functionality to search for peculiar gels For example a gel containing more outliers than the others could indicate a peculiar gel and you should check the reasons before continuing the analysis Heuristic clustering Melanie proposes a powerful analysis method to automatically create sets of gels and highlight significant groups of spots Heuristic clustering a machine learning algorithm from the artificial intelligence is used to describe the characteristic spots of 2 D PAGE gels while using heuristics to speed up the search see reference 9 The Heuristic clustering algorithm is an alternative to blin
128. not only due to chance This chapter describes how the analysis and visualization of differently expressed proteins through populations of gels can be highlighted with statistical tests reports and histograms Menu Edit Specifying classes To identify variations between two populations of gels you need to specify which gels belong to which population by creating classes To create a class select the gels which belong to it and choose Add to Class In the dialog box enter a name for the class for example class A or choose among the existing names Repeat the same procedures to create the other classes Melanie 3 User Manual 101 8 WEN CLASSES Edit L gt Classes 102 If you want to cancel the class of one or many gels select the gels and then choose Remove from Class Menu Analyze Overlapping measures As for groups of spots each protein class may be summarized by two statistical val ues namely central tendency and dispersion see Statistics for Groups on page 90 By computing an overlapping measure between the classes range summary you can find the proteins which are expressed differently in one population against the others Histograms and reports on classes display for each class of gels and for each selected group two values that describe classes and indicate their overlap accord ing to the Displayed value list on the toolbar see Fig 8 3 or Fig 8 8 Center Raw central tendency of the cur
129. ns Delete Annotations Alt Y Show Annotations Show All Alt Z Show Annotations Only Selected Pairs handling Table 10 17 Short Cut Menu Command Ctrl ShifttA Select gt Pairs All Ctrl Shift H View Stack Hide Pairs Gtrl Shift L Edit Pairs Add Gtrl Shift M Select gt Pairs For Spots Ctrl Shift S File Save Pairs Ctrl Shift U Edit Pairs Delete Ctrl Shift Y View Stack Show Pairs 144 Melanie 3 User Manual If you are using Melanie with a graphics display set to 256 colors you may feel that the colors are badly displayed If printed reports are truncated you may change the paper orientation to land scape in the printer set up Directories cannot be destroyed when a Explorer window has their names dis played Accentuated characters are not correctly supported by the english API Melanie 3 User Manual 145 C EEE KNOWN Bucs 146 Melanie 3 User Manual REFERENCES The Melanie project 1 The Melanie II 2 DE analysis software WWW page http www expasy ch melanie 2 Appel R D and Hochstrasser D F Computer Analysis of 2 D Images In Meth ods in Molecular Biology Vol 112 2 D Protocols for Proteome Analysis A J Link Eds Humana Press Inc Totowa NJ 1998 pp 363 381 3 Appel R D Palagi P M Walther D Vargas J R Sanchez J C Ravier F Pasquali C Hochstrasser D
130. o decide to keep the image alignment Another dialog box then lets you specify the new file names You may also add an extension or replace the extensions of the original image files Click OK New gels will be created con taining only the parts of the gels that lie within the specified regions plus the speci fied objects Filtering gels Melanie has been optimized for analyzing 2 DE gel images without prior image pro cessing or enhancement If you analyze low quality images Melanie provides image processing filters that may help you to enhance the quality of your images Melanie 3 User Manual 121 NE CREATING IMAGES Nevertheless keep in mind that after filtering images spot quantification values will change and will be hard to interpret Select the gels you want to filter then pick a filter in the Filter Image options This will create copies of the gels after they have been filtered leaving the original images unaffected The following filters are available 1 Smoothing Smooths the raw gel image You have the choice between the fol lowing four smoothing algorithms Gaussian smoothing Diffusion smoothing e Polynomial fitting Adaptive smoothing You will also be asked to choose how many times you wish the smooth filter to be applied 2 Histogram Equalization Performs histogram equalization on the selected gels Various options are available and the following five equalization algorithms are provided
131. o use the Next button in the toolbar item 5 Fig 5 2 to select on screen the corresponding following object listed in the reports or histograms In the same way use the Previous button item 6 Fig 5 2 to select the preceding object An example of navigation through the data existing on a Report on Groups is shown in Fig 5 4 Some groups of spots which in this case also have common landmarks displayed on screen were selected to verify their match and a Report on Groups was generated The selection was limited to group 352 the corresponding line was selected and the Select on Gel Image button was pressed Fig 5 4 item a Notice that all the spots belonging to group 352 are selected on the screen and the gels are displaced to show the spots Fig 5 4 item b By clicking the Select Next button the following group on the report group 513 was selected on the screen Fig 5 4 items c and d And so on all the other groups were verified by means of the report Melanie 3 User Manual 73 5 EEEE REPORTS AND NAVIGATION This is a simple example to illustrate the endless possibilities of the usage of reports as well as the navigation through data Figure 5 4 Navigating on selected groups through the report a Selection of group ID 352 in the report b Group ID 352 is displayed on screen c Selection of the following line group ID 513 d Group ID 513 is displayed on screen Sorting data in tables Data may be sorted in th
132. on page 53 e 2D Calibration uses known pi mw to calculate standards for unknown spots and allows automatic propagation through all matched gels see Pre defined label categories on page 53 Besides the usual categories such as calibration pi Mw and intensities and landmark Melanie has integrated advanced new data types such as database access number MS spectrum link to external data of any format located on your disk network or Internet A simple click on the link displays the associate data text html spreadsheet multimedia see Creating specific links on page 56 12 Melanie 3 User Manual FEATURES OVERVIEW Analysis and protein characterization studies The richness and robustness of the analysis tools give you access to the most sophisticated and reliable analysis techniques A wide range of tools allows to efficiently and easily browse the protein expression to extract markers by highlighting subtle changes You can either browse the data in comparative tables or in several graphical reports such as Scatter Histograms Plots and pointing out the involved proteins in gels The intuitive interface encapsulates both the classical and innovative techniques to reach superior accuracy The available tools are based on e Robust statistics and measures see Statistics for Groups on page 90 e Statistical tests see Statistical tests on page 108 e Automatic classification by Artificial Intelligence techniques
133. ories of labels they are automatically created using different background colors Ac is displayed on the gel in pink Land mark in blue Pi_Mw in pale green Comment in pale pink Set in yellow and all user defined categories are displayed in gray Menu Edit Creating annotations To create an annotation pick the annotation tool and double click on a e gel s pixel to create a pixel annotation e gel s spot to create a spot annotation at the position in a gel where you want the annotation to be located The Annotation window pops up Enter the name of a new category or choose one of the existing categories by clicking on its name Type the wanted label word or text in the dialog box The annotation is created and its label is displayed on the gel Labels may be added to an existing annotation Select the Annotation tool and dou ble click at the basis of an annotation In the dialog box type the name of a new cat egory or choose categories by clicking on its name Type the content of the label Alternatively you may add labels by selecting annotations or spots and choosing the Add Labels option Annotations may be linked to spots in this case they have small squares at their basis Fig 4 3 Otherwise annotations may simply be positioned over pixels on the LIVJ ECOLI Figure 4 3 Annotation detail on one spot square basis and on a pixel cross basis gels small cross at their basis To link an annotatio
134. orrelation coefficients between gels Melanie 3 User Manual Analyze L gt Scatters Plot SCATTER PLOT Select two or more gels and groups of spots Choose Scatter Plot Select one of the spot value types Od Vol Area Od Vol to calculate the correlation The Scatter Plot window plots the pairs values and the fitting line of each selected gel against the reference gel Fig 7 2 Reports Es Hegression line Regression line equation Correlation coefficient Number of pairs Figure 7 2 Scatter graphic on groups As usual you may save and print the graphics using the corresponding buttons of the window toolbar The relationship between the spots values from two gels can be estimated by searching the linear dependence between the spot values from one gel against the values taken in the other gel The linear dependence is defined as the best fitting straight line through the pair values The fitting line is defined by a slope and its off set i e the equation y slope x offset In Melanie s case variable x is the spot values from one gel and y is the corresponding value in the reference gel The goodness of fit of this approximation is given by the correlation coefficient Corr This coefficient can vary between 1 and 1 where an absolute value near 1 indi cates a good fit i e the spot values of one gel can be predicted to some extent by the values of the other gel while a low value indicates that t
135. oth gels will be stacked immediately The Gels option in menu Show also provides the following choices to this purpose Show L gt Gels e Only Selected displays all selected gels and hides the other ones Hide Selected hides all selected gels Show All displays all opened gels including the currently hidden ones e Sorted by Name order the cells sorted by the gel names e Sorted by Classes order the cells sorted by the gels classes names Menu View Changing the display gels order Melanie displays gels on the screen through two options The Adaptive option retains the same number of cells on the screen as the View 7 number of visible gels This is Melanie s default To select this option take Layout L gt Layout Settings Settings and choose Adaptive in the dialog box To display visible gels with a fixed number of cells i e independently of the number of visible gels take Layout Settings select Number of cells and choose the number of cells with the slider Fig 2 5 Dim es L i hoe aeri eegen Deen ese dene pier Figure 2 5 Display area with fixed number of cells equal to 6 24 Melanie 3 User Manual View gt Stack STACKING GELS To insert one gel into one specific cell on the screen click on its banner and drag it to the desired cell You may change the order in which cells are arranged on the screen Click in the border of the cell you want to displace and drag to another cell The dragged
136. pick out the refer ence gel provided it has not yet been selected You then have to select one of the spot value types for the report Od Vol Area Od Vol and choose the statistics values to be displayed in the report In the Report on Groups in addition to the central and dispersion values described earlier other measures for groups analysis are also available Fig 7 10 Additional values Iv Coef Variation Range ratio al 100 Separability Figure 7 10 Additional values for the Report on Groups Coefficient of Variation is deviation divided by the central tendency It mea sures the relative variability by correcting the magnitude of the data values thus giving a measure that has no units When you choose the statistics Median and Mean absolute deviation this measure is also known as the Coefficient of Dis persion e Range Ratio is the maximum value divided by the smallest value in the sample specified by the percentage slider Melanie 3 User Manual REPORTS ON GROUPS e Separability is the highest difference among two consecutive sorted values in the whole sample It measures the greatest gap that you can have if you want to split the group values in two classes Correspondence Analysis C A which shows the amount by which each spot contributes to the constitution of the first two principal axis on the factorial space see reference 10 To simplify the comparison between groups
137. played You may also add a spot by double clicking in the desired location and choosing Dif fusion in the Add Spot window The suggested spot displayed in the Edit spots win dow corresponds to the surrounding pixels which have a grey value darker than the clicked pixel Melanie 3 User Manual Edit gt Spots L gt Edit Edit gt Spots L gt Copy from Gel Edit L gt Spots Delete ADDING MODIFYING SPOTS You may add or modify spots inscribed in a pre selected region 1 Define a small region using the Region tool 2 Choose Edit A dialog box is displayed with an enlarged representation of the selected region Fig 3 4 3 Add or modify spots if necessary 4 Click OK The new spots will be added to the gel You may add spots from one specific gel to other gels provided that they were pre viously matched Perform the following steps 1 Select spots 2 Select one or more gels you want to copy the spots to 3 Choose Copy From Gel Pairs will be automatically created between the selected spots and the newly cre ated spots In this case all gels must be in the same directory To delete spots select them then choose Delete Edit Window In this window the selected spots are shown in a grey level related transparency mode instead of opaque which facilitates spot editing The grey levels are repre sented in gradations from blue to pink dark pixels are blue and light pixels are pink 5 Clerks KR ba
138. r Note that other smoothing algorithms are available in the Tools Gels menu see Fil tering gels on page 121 To avoid smoothing set the parameter to zero Laplacian threshold The detection algorithm looks for spot peaks by cal culating the Laplacian for each pixel in the image To detect as many spots as possible set a small value for this parameter If you set high values only very curved spots will be detected and flat spots will be ignored Partials threshold Melanie also checks the spot curvature in both the X and Y axes in order to separate spots with flat sections or to eliminate streaks Augmenting this value increases the separation power of the detection algorithm Saturation Some very dark spots may be saturated especially when digi tized with older scanners and may therefore be flat on the top To insure that these spots are detected Melanie artificially increases the peakedness curva ture of all pixels whose OD are above a given threshold The threshold is expressed as a percentage of the maximum grey level in the image A value of 90 is usually considered correct e Peakedness increase This is an internal value expressing how much Mel anie increases the curvature of saturated pixels 100 is the default value and should only be changed with care Minimum perimeter With this parameter you may decide to ignore spots that are too small Melanie will ignore all spots whose perimeters in pixels a
139. r gel The serial number has the form 2D xxxxxx where x is a digit or an Melanie 3 User Manual SWISS 2DPAGE MASTER GELS upper case letter For example 2D 000C5W and 2D 000C6F identify two spots on the Human Plasma protein map that have been identified as Apo E Fig 9 1 The serial numbers are embedded in the SWISS 2DPAGE master gels and may not be changed To display the serial number information of SWISS 2DPAGE master gels using Melanie select a master gel then choose Load Swiss 2D Serial Num ber The serial numbers are placed in labels from the SerialNumber category which by default are not visible To make this category visible choose Show Annota lions Hidden Categories and select the category name in the proposed list of categories Tools Database gt Load Swiss Melanie 3 User Manual 117 9 MS DATABASES 118 Melanie 3 User Manual Tools gt Create Synthetic CREATING IMAGES The functions found in the Tools Gels menu processes existing 2 DE gel images on the screen but affects image files on disk From this menu you may create new gel images from the selected ones You can duplicate crop and filter gels create new synthetic and flip or scale as well as erase gels Except for the Erase option all options in this menu create new gels that are automatically opened and displayed in the Melanie window Creating synthetic gels One way to study variations in protein expression among a ser
140. rasser D F Multiple parameter cross species mprotein identification using Multildent a world wide web accessible tool Electro phoresis 1998 19 13199 3206 44 Sanchez J C Wilkins M Appel R D Hochstrasser D F Identifying Proteins for Proteome Studies In Protein Function a practical approach second edition E T Creighton Ed IRL Press 1997 pp 1 27 45 Wilkins M R Lindskog l Gasteiger E Bairoch A Sanchez J C Hochstrasser D F Appel R D Detailed peptide characterization using PeptideMass a World Wide Web accessible tool Electrophoresis 1997 18 403 408 46 Hochstrasser D F Appel R D Golaz O Pasquali C Sanchez J C Bairoch A Sharing of Worldwide Spread Knowledge Using Hypermedia Facilities amp Fast Com munications Protocols Mosaic amp World Wide Web The Example of ExPASy Meth Inform Med 1995 34 75 78 Melanie 3 User Manual 47 Appel R D Bairoch A Hochstrasser D F A new generation of information retrieval tools for biologists the example of the expasy WWW server Trends in Bio chemical Sciences TiBS June 1994 222 19 6 pp 258 260 Melanie 3 User Manual 151 D C 152 Melanie 3 User Manual SUPPORT Bio Rad provides technical support on the features of the Melanie software The staff of Bio Rad is receptive to your suggestions Many of the new features and enhancements in this version of Melanie are a direct result of conversa tions with customers biologists
141. re smaller than the given threshold Melanie 3 User Manual Additional criteria affect the way spots are represented You may choose between the following three options Detect spot This is the default Spots are detected using the Laplacian and spot shapes are represented that match the original spot shapes Detect then fit with Gaussians Spots are detected with the Laplacian then Gaussian modeling is performed on all detected spots Fit existing spots with Gaussians Spots have to already be detected and modeling is performed on them Note that the parameters are of no use here First try to detect spots using the default parameter values then tune the parame ters for your data You may save your chosen parameters by clicking Save and typ ing a file name You may also load an existing parameters file by clicking Load and choosing the file name Click OK Spots will be detected according to the parameter values you have set After detection the spot shapes will be displayed over the gels Fig 3 2 p a o ef 9729 D QUU ad aoo g oH e e a BE z a Figure 3 2 Left Detected spot shapes Right Spots modeled as Gaussians Default parameters are adapted to 12 bit images When using 16 bit images a dia log box lets you choose before deciding the detection parameters whether you want to change the grey levels to correspond to 12 bit images You must note that parameters may not be th
142. rent class Dispersion Raw dispersion values of the current class These two values define the interval which summarizes the proteins sample for the class You can also characterize a class only by the central value by setting the dispersion per centage slider to 096 The following values are different measures to indicate the overlapping degree between the current class against one of the other classes Gap Maximum difference between the range of the current class and the range of one of the other classes Negative values indicate overlapping intervals and positive values indicate no overlapping intervals Fig 8 1 Melanie 3 User Manual OVERLAPPING MEASURES e Ratio Maximum ratio between the range of the current class and the range of one of the other classes Values smaller than 1 indicate overlapping and higher indicate no overlapping Fig 8 1 No overlapping case Interval class A 1 Interval class ap Gap gt 0 and Ratio gt 1 Overlapping case Interval class A Interval class B lt gt Gap lt 0 and Ratio lt 1 Figure 8 1 Schema of the Gap and Ratio values e Normalized Minimum overlapping between the range of the current class and the range of the other classes A value smaller than 1 indicates overlapping For example 0 25 indicates that 25 of the range is not recovered by one of the other classes In the same way 1 5 indicates a
143. rict the selection according to the second criteria e make Select Groups Restrict selection Keep the groups whose num ber of selected spots is gt move the slider to choose 3 gels and click OK The remaining groups of spots verify both specified criteria Case number 2 Suppose now that you would like to study the groups in which ALL spots have a Vol higher than 0 02 This choice would restrict the selection of groups to the two criteria defined in case number one plus a third criteria 3 all present spots have a Vol value higher than a certain threshold Without unselecting the preceding case e make Select Groups Restrict selection Keep the groups whose num ber of unselected spots is lt select value 1 in the slider and click OK The remaining selected groups contain spots that verify the three specified criteria The following logical step could be to analyze the remaining spots with statistical cri teria like Student T test or Wilcoxon test also available in Melanie to highlight meaningful spots As you may notice there are endless possibilities for combining selections of spots and groups and your own experiments and needs will lead you to the adapted use of this function Menu Analyze Scatter plot In order to analyze gel similarities or experimental variations such as stain intensitie variations or sample loading you may plot scatter graphics on groups with regres sion lines and c
144. rks Category Pi Mw is converted to Landmarks Melanie 3 User Manual 129 A ES DIFFERENCES BETWEEN MELANIE II AND MELANIE OR WHERE TO Category Ac is converted to Labels with Accession Numbers e Category ProteinName is converted to Labels with short ID Category GelComment is converted to the textual comment associated to a gel All the other existing annotations and labels will not be saved 130 Melanie 3 User Manual The following menus are provided in the melanie software File to handle whole gels and data on disk open and close gel images save data print images import and export data View to modify the window display fixed or variable number of cells and gels display stacked aligned zoomed grid lines Edit to edit add modify delete specific objects e Show to show or hide specific objects Select to select specific objects Analyze to compute differences and similarities among gels statistical and artificial intelligence data analysis Reports to display information on selected objects and to navigate in gels by means of the data Tools to create new gel images from selected ones duplicate crop filter gels create synthetic gels flip and scale gels as well as erase gels Window to display information and zoom windows and to list open report windows Help to obtain on line help The descriptions of menus and sub menus may be easily found w
145. s possibilities of their usage You may also visually compare spots or even navigate through pairs and groups Histo grams on Groups and Report on Groups are dual elements they have a reciprocal relationship Histograms are a more visual way to look at groups To get histograms on groups select groups on the gels or by using the Report on Groups then take Groups His tograms The descriptive statistics directly available in the subsequent dialog box are location and dispersion values explained in Statistics for Groups on page 90 Melanie 3 User Manual 93 EE GROUPS In the displayed histograms the brown lines correspond to the spot values the blue line corresponds to the chosen Central value and the red lines to the range defined by Central Dispersion Central Dispersion Sort by Similarity Reports Groups Histograms exremes M ll ree e sej Histograms on Groups Moll The chosen statistics are Mean 100 and M S D Central abcdefg 206 Gel index Group ID Sorted values Figure 7 6 Histograms on Groups Displayed values Figure 7 6 shows some histograms for selected groups The slider on the bottom right corner of the window allows you to visualize the rest of the groups which are equitably disposed in pages The histograms may also be selected as any other Melanie object and be saved printed or exported to other software To select one histogram click on the histo gram windo
146. set of labels Each label belongs to pre defined categories or user defined categories The label contents may be any textual data examples in Fig 4 1 Pre defined label categories Some pre defined label categories are provided with the Melanie software to help you organize your data and to mark spots which have common characteristics These categories are 1 Ac This category is provided to hold the proteins accession number AC taken from a user selected database and it is the link to Melanie s remote database query engine A double click on this label category displays the corresponding pro tein entry in the chosen database with the chosen browser software See Querying a remote database through the Internet on page 9 114 2 Landmark This category is used to mark pixels in the gels as reference points for the operations of gel alignment and calculation of corresponding locations between gels Two annotations are considered to refer to the same point in different gels when they have the same label Melanie appends the word AMBIGUOUS to a landmark label when you add an existing landmark to a gel Melanie 3 User Manual 53 NENNEN ANNOTATIONS 3 Pi Mw This category is supplied to contain the pl and Mw information which is used to compute approximated p and Mw value for any point in a gel You may enter the p and or Mw separated by a space of the gel at the given position A value of 1 means that no value
147. so choose to display spots information of the corresponding pairs in the reference gel provided that there is a selected reference gel and the gels are matched together Select the reference gel information in the Settings Spots report dialog box in the Reference gel list as described in the previous paragraph Click Apply to confirm your choice and the selected attributes will be displayed on the report You may also save the current chosen report template by clicking Save and typing a file name or load an existing file by clicking Load and choosing the corresponding file name The template name will be added to the menu command Reports Spots Report Fig 3 8 Tools Window Help Gels Report he Rad Categories Report Mysettings2 Mysettings3 Hare Hepat Matches HENO All Matches Report Figure 3 8 Templates of Report on Spots 50 Melanie 3 User Manual REPORT ON SPOTS You may have as many report templates as you wish and the saved formats may be re used by choosing their format file names on the menu item Reports Spots Report The template names available when Melanie starts are those that exist in the Menus subdirectory of the Melanie installation directory Melanie 3 User Manual 51 3 EE 5007 52 Melanie 3 User Manual Individual points pixels and spots in a gel image may be marked by annotations An annotation is defined by its position row and column number and its
148. spots such as spots ID quantification values attached labels etc This report is completely customizable i e you may choose the information to be displayed and the order in which they will be displayed Report on Labels Displays information about selected labels such as con tent category spots ID and positions on the gel X and Y Report on Annotations Displays for selected annotations information about all categories and their labels e Report on Categories Displays information about all labels belonging to chosen categories The displayed information is the same as for the Report on labels e Report on Pairs Displays the list of selected paired spot IDs among selected gels Report on Matches Displays information on matching between the selected gels and all open gels that is the number of paired spots between each pair of gels Melanie 3 User Manual 69 5 EEEE REPORTS AND NAVIGATION e Report All Matches Displays information on matching between all gels in a given directory In the Analyze menu Report and Histograms on Groups Displays information on each selected group see Pairs and groups on page 8 such as group ID chosen value type of each spot in the group and chosen statistical values calculated on all spots in the group Report and Plot on Scatters Displays information on scatter plots such as slope offset and the correlation coefficient values the pair values and the fitting error
149. tack in the form of blue vectors linking the locations of paired spots Fig 2 8 Choose Show Pairs To hide vectors choose Hide Pairs Moving gels The hand tool moves gels in order to show other parts Pick the Hand tool in the toolbar put the cursor inside a gel hold down the left mouse button and move the mouse The gel image will follow your movement If you hold down the Shift key and the mouse button simultaneously then all visible gels will be displaced by the same amount once the button has been released By double clicking inside a gel all visible gels will be positioned at the same loca tion with the same magnification The corresponding location from the clicked gel to the others gels is estimated by interpolation between the two nearest common landmarks i e landmarks with the Melanie 3 User Manual 27 2 ES GELS View L gt Move Gels same name When the gels have no common landmarks they are aligned at the same image location In addition to the Hand tool Melanie provides other useful operations to move gel images in order to show other image parts The Move Gels option enables you to scroll through visible parts of selected gels To scroll up select Up or alternatively hold the Ctrl key and press the Up Arrow key Use the respective commands to scroll down left or right The Move Gels option also lets you choose to display the top left top right bottom left or bottom right part of the selected gels
150. tant Annotations may only be selected and manipulated when they are visible on the gels Labels may only be selected and manipulated when the annotations are visible Annotations may be displayed with Show All menu option and hidden with Hide All You may show only selected annotations with Only Selected annotations or hide with Hide Selected Select a region on a gel using the Region tool then In Region to show all annota tions in the selected region If you have selected spots before selecting annotations you may take For Spots and all the annotations associated to the selected spots will be selected You may hide a certain number of categories among those that are visible on the gels Choose Visible Categories and in the dialog box click on the desired cate gory names to hide them Fig 4 3b shows for some of previously chosen spots Fig 4 3a categories Ac Pi Mw and Comments while categories Landmarks ProteinName and others are hidden 64 Melanie 3 User Manual SHOWING ANNOTATIONS AND LABELS You may also choose to display labels belonging to a certain category Make Hid den Categories to display the categories which are not visible on the gels In the dialog box click on the desired category names to make them visible p H duma lt ECOLI Pi 5 20 Mw 43000 FABD ECOLI 5 20 43000 q Be 0509 ST UCC ECOLI ECOLI 00509 a 90 AT 5 00 1 dmk34 13857 A 04816 e ML EC
151. ter If a printer cable is attached to this port disconnect it After you have attached the HASP key you can attach the printer cable to the key itself and restart your computer The installer will install the system driver that allows the Melanie software to detect the HASP key Netscape Navigator and Internet Explorer In order to use the on line manual as well as to access remote databases over Internet you must install the Netscape Navigator World Wide Web browser from Netscape Communications Corp or Internet Explorer from Microsoft Corporation Running Melanie To run the Melanie program double click on the Melanie icon You may also run the Melanie program by double clicking on the icon of a gel image file Fig 1 1 In this way the given gel will automatically be opened upon running the program ter a EA b Figure 1 1 a Melanie icon b Gel icon Upon loading the program the Melanie logo appears Click on the logo to get rid of it Melanie 3 User Manual 3 1 ES INTRODUCTION Interface The Melanie window is divided into four main parts shown in Fig 1 2 and respec tively called menu bar 1 toolbar 2 gel display zone 3 and status bar 4 255 Melanie Ele View Edit Show Select Analyze Reports Tools Window Help Gels 0 Spots fr Annotations o Figure 1 2 Melanie display 1 Menu bar 2 toolbar 3 display zone 4 status bar 5 reference gel zone 6 hidden gels zone 7
152. the Database Settings window to display in your cho sen browser a list of federated 2D PAGE databases and servers which may be que ried from Melanie or consult the page http www expasy ch ch2d 2d index html The World 2D button is a reminder of the possible servers Copy the desired data base format name from the displayed window and paste it to the field Database URL in Melanie The query formats are specific to the databases Some formats must include the character others must include the character Melanie concatenates the Ac label to the text typed in the field Database URL These settings may also be made permanent from the Edit Preferences Database menu option 114 Melanie 3 User Manual Show L gt Annotations SWISS 2DPAGE MASTER GELS Querying the database To query the remote database double click one spot that has a label with a valid accession number Ac This will call the chosen browser program on your computer that is going to automatically retrieve the corresponding entry from the remote data base Alternatively you may select the label and choose Show Annotations Linked Data For example if you have set the database SWISS 2DPAGE on the www expasy ch server with the query engine Nice2DPAGE by double clicking the spot label which has the accession number P02649 you will obtain the entry for Human Apo E in your chosen browser see Fig 9 1 SWISS 2DPAGE master gels SWISS
153. the statistical value will be calculated for the remaining values Dispersion measures the variability of the sample data how clustered or scattered data are about its center There are numerous measures of variability standard deviation range interquartile range and many others 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 restrict the sample to the central values by trimming out the extreme values By selecting Mean squared deviation M S D the square root of the average squared difference of each sample value to the center loca tion is calculated Melanie 3 User Manual 91 92 EE GROUPS By selecting Mean absolute deviation M A D the mean of the absolute difference between each value and the central value is cal culated It is not affected by outliers as much as the Mean squared deviation since the differences are not squared By selecting Half range size the difference between the largest and the smallest values divided by 2 is calculated As for the central tendency the percentage slider allows you to remove extreme val ues from the dispersion statistics A 100 value means that all the values are used to calculate the dispersion and on the other hand 0 will always represent no dis persion zero Some examples Mean 100 and Mean squared deviation 100 are the most commonly
154. their optical density area and volume are computed Alternatively spots may be modeled by Gaussian functions In this case their quantification values will be the Gaussians peak values and volume as well the area of the Gaussian s half height section Menu Edit Editing spots Select gels to detect spots on then take Auto Detect The Detect Spots window appears Various detection parameters are available and may be adjusted The default parameters have been optimized for silver stained 2 D PAGE images run Edit gt Spots Melanie 3 User Manual 39 40 3 EE 5007 according to the SWISS 2DPAGE protocol see reference 13 You may tune the parameters to adapt the detection program to your images The following parame ters may be set Fig 3 1 Detect Spots Select detection parameters File Not saved Parameters Number of smooths _ gt 2 Laplacian threshold All gt 3 Partials threshold gt 1 Saturation aj jJ 30 Peakedness increase gt 100 Minimum perimeter A gt 10 Detection Detect spots C Detect then fit with Gaussians Fit existing spots with Gaussians Load Save Default Cancel Figure 3 1 The spot detection parameters Number of smooths Before detecting spots Melanie will smooth the image using a smooth by diffusion algorithm in order to eliminate parasite noise Select the number of times the smoothing operation should occu
155. tion ty adi an drm K Dh igi a r za Age ani kwa and He kl ze k kdl zadna Figure 3 4 The spot editing window To add modify spots in the Edit spots window hold down the left mouse button and draw the outline of the area that you want to add When the button is released the outline is closed by a line joining the first and the last point of the outline and it is filled Hold down the right mouse button and draw the outline of the area to delete or split a spot You may also use the left mouse button to add pixels to the spot the right mouse button to remove pixels and the middle mouse button to fill in holes Melanie 3 User Manual 43 44 3 IN SPOTS Spots quantification Melanie automatically computes the spot values once they have been detected The following quantification values are calculated for each detected spot Od the highest calibrated pixel intensity in the spot e Area the spot s area mm Vol the spot s volume i e the integration of Od over the spot s area If spots have been modeled as Gaussians the following quantification values are computed e Od the optical density of the Gaussian s peak Area the area of a section of the Gaussian taken at the peak s half height e Vol the Gaussian s volume The following normalized values are also available e Od the relative Od i e the Od divided by the total Od over the whole image Vol the relative Vol i e the Vol d
156. tion will be selected according to Fig 4 2 Set A Set B Set A Set B C SO Intersection AND Union OR SetA Set B Difference XOR Exclusion A and Not B Figure 4 2 Combining sets of spots on Melanie Intersection selects spots that are in the intersection of two criteria Melanie 3 User Manual 63 EEEE ANNOTATIONS Show L gt Annotations Union selects spots that belong to either one criterion or the other Difference select spots that are unique in one criterion plus those that are unique in the second criterion Exclusion select spots that are found only in the first criterion and are not found in the second criterion For example you may select spots that are in the intersection of two analyses like Student T test and Differential Analysis Make the first analysis and mark the out coming spots with the label Set St Make the second analysis and mark the out coming spots with the labels Set DA Select spots from the first analysis by choosing Select Annotations By Category and in the dialog box select cate gory Set St Then make Select Annotations Combine Intersection and choose category Get DAT in the dialog box Only spots that belong to both sets will remain selected Menu Show Showing annotations and labels Annotations and categories may be displayed hidden packed unpacked and selected Annotations may also be colored with different colors Impor
157. tions of potentially differently expressed proteins Student t test The Student t test is the most used test to determine whether two populations are significantly different and this based on the assumption that the data follow the normal probability distribution the standard deviation of the two classes are similar The statistics are based on the difference between the two classes means normal ized by the standard deviation 108 Melanie 3 User Manual STATISTICAL TESTS The displayed value for each group of spots in the Report on Statistical Test corre sponds to the probability p of confidence on the Student t test hypothesis i e to affirm with p probability that both populations are significantly different When the normal distribution assumption is not followed non parametric tests like Wilcoxon or Kolmogorov may provide a better analysis Wilcoxon or Mann Whitney test The Wilcoxon or Mann Whitney Test investigate the hypothesis that two populations differ only with respect to their median values The characteristics of this test are that no assumptions are made about the data distribution It considers only that they have the same distribution The statistics are based on the number of shifts needed to order the values from the two populations so that they don t overlap For the example given in Fig 8 9 the population A population B p a as b agb 0 a b bs D sample values
158. to one class may be compared to a class of gels of healthy tissues from other patients Melanie 3 User Manual 9 10 1 ES INTRODUCTION The definition of classes in Melanie allows statistical and meaningful considerations about populations of gels Reference gel Class A Class B Figure 1 8 Two classes Spots from Class A S5 S3 may be compared to spots from Class 54 55 Sg The analysis and visualization of differently expressed proteins through populations of gels can be highlighted with statistical tests and several reports and histograms Selecting and showing objects Melanie can manage and display a huge amount of objects thus congesting clut tering your display In this case you may decide to display only some chosen objects at a time i e you may filter the data which you would like to work on As a consequence an object may at a given time have any of the three statuses shown hidden or selected For consistency most operations in Melanie may only be applied to selected objects on selected gels Objects may be selected using tools from the toolbar or using cer tain menu options Once selected an object is marked in green The green color is always used to highlight selected objects As a general rule objects may only be selected when they are visible on the screen For example you may only select a spot if it is visible see Showing spots on page 45 Nonetheless as an exception
159. tributions are restricted to the selected region Any changes made with the Adjust Contrast command will only affect how the image is displayed on your screen They will not affect the underlying data the spot detection and quantification Adjust contrast preview Each change of the gray levels of an image when in the Adjust Contrast window may immediately be reflected in a given region if such a region has been defined prior to adjusting contrast Select one region in a gel In the View menu choose Melanie 3 User Manual GRAY LEVELS Adjust Contrast Each time you change one of the mapping parameters the change will immediately be reflected in the selected region Fig 2 17 IM fee a Hes get ju um D Figure 2 17 The Adjust Contrast option The modifications are directly reflected in the selected region Note that if the small rectangle temporarily disappears from the display when the Adjust Contrast window pops up slightly change one of the parameters T on The gray levels will be adjusted in all selected gels If you save gels after having performed color adjustments the latter will be saved with the gel images see Saving gels on page 18 Displaying gels with pseudo colors View Choose Pseudo Colors Select one of the color lookup tables for example FiveR L gt Gray Levels amps Click OK The lookup table will be changed accordingly Fig 2 18 To go L gt Pseudo Colors Melanie 3 User
160. ts directly from a displayed Report on Spots see Reports and navigation on page 69 Note that by selecting annotations or labels their linked spots are also selected Restrict spots selection You may select spots which have a value inside a certain interval Make a first selec tion of spots and then Restrict by Value In the dialog box select a spot value type among Vol Area Od Vol and Od In the Restrict by Value dialog box click gt and type the value which will represent the lower limit of the interval Click also lt and type a value to define the higher limit of the interval The spots whose values are included in the defined interval remain selected Menu Window Cursor information You may at any time display information of spots and pixels directly on the screen In this case choose Window Information Available information for pixels are their X and Y coordinates on the gel as well as pl and Mw Pixel grey values are also available when raw images of gels are loaded see Loading unloading gels on page 19 Otherwise the Value field displays Melanie 3 User Manual 47 3 Edit 48 L gt Preferences L gt Cursor unloaded Information for detected spots also include their spot ID and quantifica tion values Vol Area Od Vol and QOd Figure 3 6 Cursor information and settings window You may choose what information to display as well as the order in which this infor mation
161. u may also export graphics to software like Word or Photoshop for example Copy the selected graphics to the clipboard by choosing the Copy to clipboard but ton in the toolbar or in the contextual menu Paste into the desired software Selecting objects You may select objects like spots gels labels annotations groups and pairs directly from their reports You may also select groups of spots directly from histo grams Double click on a report line or on a histogram to select and display the cor responding object Alternatively you may use Shift and Ctrl keys to select report lines or histograms Click the Select on Gel Image button in the toolbar item 4 Fig 5 2 or use the con textual menu The corresponding objects will be selected on the screen and the gels will be displaced to show at least one of them An example of refining the selection of spots is shown in Fig 5 3 In this example some spots were first selected then a Report on Spots was generated In the report the values in the column Area were sorted in descending order The five first lines i e the five largest spots were retained in a new selection of spots which is displayed on the gel Figure 5 3 Re selection of spots through the report Note that the Area values were sorted in descending order and the re selected spots are the first 5 spots on the sorted report Melanie 3 User Manual NAVIGATING THROUGH DATA Navigating through data You may als
162. uenced Fig 4 4 A label named To be sequenced will be attached to the selected spots 4 17 To be sequenced be sequenced mcs be sequenced Figure 4 4 Creating a Set To be sequenced set of spots in Melanie Pies gela a roin gr Note that the keyword Set is not displayed on the labels At the time when a new category is created it is immediately shown on the screen except for the new cate gory Set In this case it is visible or hidden depending on the current state of the generic category Set Creating specific links Many interesting possibilities to link your gels to 2 DE databases mass spectrome try files text files or any other files are available in Melanie Using specific keywords in labels of any category creates links to specific objects In this case the keyword and the follower text are not visible on gels and are replaced by three dots a visual help to indicate a linked label 56 Melanie 3 User Manual CREATING SPECIFIC LINKS by double clicking on link labels labels are not edited but the link is opened You may have links in any label category but you can only have one link per label The available link types are Http links you may link your gels to specific web pages To create an http link add an annotation to a gel In the text field type your text which must contain the word http followed by a web page A doubl
163. ve statistics of location and dispersion which may be calculated and displayed in the Report and Histograms on Groups Descriptive statistics are summary values which describe spot values from a group They allow you to locate groups composed of spots whose quantification values are peculiar These variations may be due to an inadequate detection matching opera tion or may result from protein expression changes among gels Therefore these statistics are useful for controlling the extracted data as well as for analyzing them The statistics tool includes all the options that the expert may expect Nevertheless the reluctant user towards statistics may simply use the default settings The available statistics are part of the Robust Statistics and include the most com mon measures currently used Melanie extracts from each group of spots the cen tral tendency which allows you to localize the central value representing the data and the dispersion which indicates how closely the data fall about this center Melanie 3 User Manual STATISTICS FOR GROUPS The descriptive statistics can be chosen with the following interface Fig 7 3 Central tendency Mean al D 100 z C Midrange Reference gel values Dispersion Mean squared deviation al gt 100 C Mean absolute deviation f Half range size Figure 7 3 Statistics common to Report on Groups Histograms on Groups Report on Classes and Histograms on Classes
164. w To select many histograms use Shift or Ctrl keys You may re select the corresponding groups on the gels by using the Select on Gel Image button and navigate through groups with Select Next and Select Previous buttons By using the Reports menu you may display the numerical values of selected histo grams when choosing Report on Groups and the latter will create a new groups report with the selected groups You may as well display the histograms legend by selecting the Gels Report item U 7 tion 94 Melanie 3 User Manual Use the Report on Groups to change the order or to refine your histograms selec HISTOGRAMS ON GROUPS Comparing protein expression changes Melanie provides facilities in the histograms window to visually compare protein expression changes across different groups normalize values sort values select unselect adaptive gradations and search by similarity You may simplify the comparison across groups by normalizing the spot values rela tively to their groups statistics values Displayed values item Fig 7 6 The available normalizations are Value Raw spot value Relative Spot value Central tendency Ratio Spot value Central tendency e Normalized Spot value Central tendency Dispersion To highlight spot variations you can display the spot values sorted in ascending order in this case select the Sorted values check box at the bottom of the histo grams window
165. w you may change the labels contents and add new categories The Delete menu option deletes all selected labels The Delete Categories option deletes from selected annotations labels which belong to chosen categories Select the labels take this menu option and choose the categories to delete You can also manually change an annotation position Select an annotation and drag the annotation basis to the desired location To interactively change an annotation s tag position select an annotation and drag its tag to the desired position Fig 4 8 e R TIG_ECOLI 2 D FTIG_ECOLI 4 VW Figure 4 8 Default annotation tag position left and modified tag position right Editing annotations We have already described how to create modify and delete annotations and labels Some other convenient options of Melanie are the copy paste and dupli cate facilities Copying pasting annotations You may select annotations in a given gel image then copy them to another gel This is a simple means of creating a series of similar annotations in a set of gels Perform the following steps 1 Select annotations 2 Choose Copy This will copy the selected annotations into memory 3 Select only the gels you want to copy the annotations to 4 Choose Paste Annotations The copied annotations will be pasted into each of the selected gels to their corresponding locations The corresponding location
166. w enlarged parts of gels momentarily In this case select the Magnify tool press Ctrl and click on the region you want to enlarge Fig 2 10 Figure 2 10 Magnify glass Magnifying window Another way to enlarge parts of gels is to use the Zoom Window which enables you to see the region under the cursor examples in Fig 2 11 and 2 12 To display the Zoom Window choose the menu Window Zoom You may change the magnifi Melanie 3 User Manual 29 2 ES GELS cation of the zoom window by pressing the and the buttons located at the top of the window and you may also resize this window Pe we L we jek E ee Bed SI len Figure 2 11 Zoom window used to enlarge the focused to region The zoom window displays the visible objects like annotations and spots as they appear on the screen It also displays one green rectangle indicating the visible part on the display area of the pointed to gel the gel which has the cursor Figure 2 12 Zoom window used to locate the focused to region on a gel displaying spots and labels The small rectangle indicates the visible area of the pointed to gel 30 Melanie 3 User Manual View L gt Grid Lines DISPLAYING GRID LINES Displaying grid lines You may display grid lines over selected gels to indicate their dimensions in centi meters inches or in pl and Mw units provided that this information is available in the annotation category Pi Mw The pl a
167. when you request the selection of a hidden object it may be momentarily displayed on the screen but it will immediately be hid den once it is unselected again The Shift and Ctrl keys enable you to select more than one object Melanie 3 User Manual FEATURES OVERVIEW The Shift key enables you to select consecutive objects select the first object then hold the Shift key and select the second object All objects between the first and the second one will be selected The Ctrl key enables you to select discontinuous objects It also enables you to select and unselect objects i e to add or remove objects to or from an existing selection hold the Ctrl key and click on the chosen object it will be added to the selection Click once more on the selected object and it will be removed from the selection Note When all objects from a certain type are hidden like spots annotations pairs and groups their manual selection is also possible In this case they will remain visible by the time they are selected Features Overview Melanie is a complete state of the art software application designed to analyze images of two dimensional electrophoresis 2 DE Melanie provides interactive as well as automatic gel analysis Its high flexibility and richness of the functionalities allow Melanie to behave and respond according to your specific requirements Protein detection and quantification e Automatic detection with the possibi
168. your gels suc cessfully This manual is divided into four parts Part gives an overview of the Melanie soft ware as well as the basic procedures for analyzing 2 DE images Part II details the necessary steps for comparing images as well as analyzing resulting data Part III presents features such as image creation and working with remote databases Part IV provides additional information such as menu short cuts and technical support Important points in the manual as well as useful hints are marked by a small hand in the margin in order to draw your attention Melanie 3 User Manual 1 1 EE INTRODUCTION Hardware and software requirements Melanie is a PC compatible 32 bit application that runs under the Windows 95 Win dows 98 and Windows NT 4 0 environments We recommend 64 or even 128 MB of memory to process many gels simulta neously The amount of memory required is mainly determined by the file size of scanned gel images Installation Insert the Melanie CD ROM into the CD ROM drive on your computer and the installer will start automatically The Melanie installation program will guide you through a series of screens The installer will create a default directory tree under Program Files on your hard disk called Bio Rad Melanie3 you can select a different directory if you wish The main Melanie program will be placed in the Melanie 3 directory Additional directo ries for storing sample images and Melanie s sett

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