Melanie 7.0 offers a unique and flexible interface for the comprehensive visualization, exploration and analysis of 2-D gel data from both DIGE (Fluorescence Difference Gel Electrophoresis) and non-DIGE technologies. It will improve your analyses of 2-D gels and identify protein markers of interest.
Now, many of the functions found in earlier versions have been enhanced and simplified, and significant new features have been added for greater convenience and speed. The user interface is even easier to use and new interaction modes simplify even the most complex analysis.
Melanie 7.0 offers:
Well established methods
Melanie is a solution that can be applied to a wide range of 2-D gel and blot experiments. The software has comprehensive tools that are well suited to the traditional gel analysis workflow as well as to the most challenging data sets.
Melanie is also designed for DIGE applications - through the use of the recognized DeCyderTM 2-D Differential Analysis Software (DeCyder 2-D) co-detection algorithm, support of the internal standard approach, and a set of specific analysis tools.
Fig 1. Melanie 7.0 can be used for a wide range of 2-D gel experiments, including DIGE.
A single workflow
A unique analysis pathway is used for all your gel studies, both for conventional 2-D electrophoresis and DIGE gels. The main workflow is exemplified by the three folder types in the workspace:
- Image Pool - This is where your imported images will appear. Gels and images in this folder are displayed in the Image Pool sheet dedicated to viewing, exploring, editing, and calibrating images before adding them to a project for further analysis.
- Match - This is where you define your sets of gels, called match sets, to be matched together. Images of this folder are displayed in a MatchSet sheet in order to be detected and matched.
- Classes - This is where you assign images to populations, or classes. When displaying these classes in a Class sheet, statistical analyses can be performed.
In the workspace, you can control and verify the status of your gel analysis, and have direct access to the desired step in the workflow.
Easy import and visualization
Melanie automatically recognizes all 2-D gel image formats during import, including TIFF images and *.GEL, *.MEL, *.IMG, *.GSC, and *.1SC format files.
All images of a DIGE experiment can be imported in a single operation. If they comply with the common sense DIGE file naming conventions, the software automatically suggests the correct combinations of co-run images that can be validated with a single click.
The software therefore provides excellent image viewer functionality, without limiting the number of images that can be displayed simultaneously.
Before performing analysis, the images can be edited (rotated, flipped, cropped, and inverted) or calibrated to remove image-scanning variations. The contrast settings and color palettes can also be adjusted at any time.
The desired gels can subsequently be inserted into a project, by simple drag and drop, for further analysis.
Minimal spot editing
The cornerstones of a fast and successful 2-D gel analysis are efficient spot detection, accurate spot quantitation, and robust gel matching. In Melanie, these functionalities integrate effective solutions to avoid or minimize editing of the results.
Melanie offers two spot detection algorithms:
- For conventional 2-D images, the Melanie algorithm provides only a few easily adjustable spot detection parameters, for distinguishing real spots from noise.
- For DIGE images, the powerful co-detection algorithm from DeCyder 2-D simultaneously processes one, two, or three images derived from a single gel.
The advantage of the individual spot detection for conventional 2-D images is that the image data from every single gel is fully exploited. However, spot detection differences can occur. In particular, some spots are split in different ways among gels to be compared.
Although Melanie 7.0 offers simplified spot editing tools, it also provides elegant solutions to deal with detection variations between gels without calling for spot editing:
- By exploiting the capability to define multiple matches, you can create "composite spots" that are treated as unique entities in the quantitation (Fig 2).
- You can propagate spots from one image to the other matched images. This allows you to quantify identical areas on all gels.
Fig 2. By creating a multiple match for the selected spots, you can create composite spots that are treated as unique entities in the quantitation.
Hierarchical population matching
All images in an experiment are not equally easy to compare, even when the gels are run in a highly controlled way. Typically, gels belonging to the same biological group are easier to match than images from different biological populations. This information is not exploited when matching all images against a unique arbitrary reference image and often results in missed spots in the analysis.
By combining Melanie's powerful parameter-free matching algorithm with hierarchical population matching, you can create more efficient match designs. Figure 3 shows and example of a match hierarchy, where images are first matched within a match set, and two of these match sets are then compared at a higher level.
The advantages of hierarchical population matching are substantial:
- With one or two easily created landmarks (tie points), the entire experiment is matched in a matter of seconds, and matches are automatically propagated at each level of the unrestrained match hierarchy.
- By first matching gels within biological populations, one minimizes the number of difficult match combinations. This significantly reduces time spent on match editing.
- The likelihood of a spot being matched is much higher when matching with a gel from the same biological population. Spots that are represented in a single population are therefore included in the analysis. This considerably reduces the number of spots missed in the analysis.
- Any protein spot can be matched against equivalent spots in other images, without the need to copy spots to the reference.
Fig 3. Scheme of a match hierarchy A, with sub match sets AT1 and AT2, and its display in the workspace and MatchSet sheet.
Versatile analytical methods
The ultimate goal of your 2-D image analysis is to answer questions such as:
- Are there proteins or protein patterns that might be characteristic of a biological state (e.g. tumor versus normal tissue)?
- Are there proteins that might be used for the development of noninvasive tests (i.e. diagnostic markers)?
- How many classes exist in a given data set?
The various analytical methods in Melanie can be used to answer these questions and select proteins for picking, digestion, and subsequent analysis by mass spectrometry. Selection of protein spots can be based on multiple criteria such as statistical significance of change, magnitude of change, spot volume, or any combination of criteria.
The available methods are:
- Scatter plots, to analyze gel similarities or experimental variations.
- Descriptive statistics of central tendency and dispersion, to summarize the magnitude and variability of the spot values within a population. These values are calculated and displayed in various tables and histograms. They are also exploited in the calculation of the Overlapping measures described below.
- Factor analysis, to help identify underlying sources of variation and indicate whether, and how well, spot groups and populations might be separated. This type of analysis is very sensitive to outliers and can help to identify possible mismatches.
- Statistical tests, to perform differential expression analysis. The statistical significance of change can be used to reduce the data set to only those proteins that show changes in expression level. The tests include one-way ANalysis of VAriance (ANOVA) for comparison of multiple gel populations, and the non-parametric Mann-Whitney/Wilcoxon, and Kolmogorov-Smirnov tests for the comparison of two populations.
- Overlapping measures, to identify significant protein expression variations without making any restrictive assumptions, as do the various statistical tests. Overlapping measures summarize each gel population by an interval and compute the overlap between these intervals.
- Histograms, to visualize expression profiles.
Focus on your problem
Melanie has a full set of dedicated tools to focus the analysis on your specific problem.
Based on the quantitative and qualitative filters in the reports, protein spots that are not of interest can be disabled or tagged in order to concentrate the analysis on a specific subset of spots. Such spot sets are saved for later retrieval or combination.
In Melanie, further focus is achieved by continuously synchronizing the current selection in all the different views, to allow a clear overview of all critical information (Fig 4).
Fig 4. All views in the interface are linked to allow a clear overview of the critical information. Pink spots have been temporarily disabled so that they do not appear in the reports.
This also means that when you select a spot belonging to a group, any matched spots of the group are automatically selected as well.
User-friendly interface with flexibility
Whether working with 10, 50, or 500 images, Melanie allows users to display, manipulate, and process gel data with unmatched flexibility and ease.
The application layout and gel images can be freely reorganized to optimize space and visibility in accordance with personal preferences. The intelligent multi-worksheet display guarantees a structured and consistent presentation, where related images always remain grouped and you can instantly view the desired gel subsets.
Melanie 7.0 offers fully dynamic tables, histograms, plots, and 3-D views in which both content and selection are continuously updated to stay up-to-date with the corresponding sheet that contains the gel images.
Each user can customize his or her personal work environment, for instance by creating custom toolbars, menus or report templates.
Adaptable visualization tools
Melanie offers numerous features to visualize different aspects of your gel images (Fig 5).
The various modes to move and zoom your images allow you to become familiar with the spot patterns or to navigate to the desired location in your image, possibly aided by the Overview feature.
Signal intensity can be inspected using the Adjust Contrast feature, the brand new 3-D View, or the Profile curves.
The software further provides specific tools to visually compare different images:
- Automatically displayed match vectors allow easy verification of match results, while superimposing spots can be viewed in a different color.
- Automatic warping aligns gel images, eliminating differences in spot position. Aligned image are then overlaid to produce dual channel images with clearly visible differences in protein expression.
- Synchronized and simultaneous 3-D views of multiple gels can be displayed.
Fig 5. Different images can be compared by displaying synchronized 3D views and dual color images (red spots correspond to those from the sheet reference A_T1_Gel1). The Overview at the bottom right of each gel image shows the location of the currently visible area.
Grid lines can be used to visualize deformations in aligned gels, or to evaluate distances between spots, in terms of pixel coordinates, pI/MW units, or real world units (cm or inch). The pI/MW calibration grids use pI and MW values of known standards to automatically calculate values for all other spots and propagate these through all matched images.
Seamless integration
To support the collaborative efforts of researchers both within and outside your company, Melanie 7.0 ensures seamless sharing of project data within a network and provides import/export features that allow you to send analyzed results (including images, spots, matches, annotation, spot sets, etc,) to external partners.
Many additional features enable the seamless integration of our software into your laboratory workflow:
- Support of multiple recommended image formats, TIFF images, GEL, MEL, IMG, GSC, and 1SC files.
- Direct image acquisition from Twain-compatible scanners.
- Export spot data in Text, ExcelTM, and XML format for further downstream analysis.
- Fully automated integration with spot-picking robots.
- Clipboard support to copy gel images, graphics, and data tables to other programs.
- Annotation capabilities that allow gel objects to be linked to external search engines or databases.
Quality and data integrity
It is important to protect all your gel data, not only by ensuring data integrity and consistency, but also by allowing you to reversed undesired user manipulations. Melanie therefore offers:
- Unique identifiers for each object to assure data consistency and database integration.
- Sophisticated multiple undo/redo function.
- Project backup and restore function.
- History function to keep track of all operations carried out during a work session, for control and quality assurance.
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