Features & Benefits
Melanie 7.0 simplifies and improves analyses of 2D gels and blots, and identification of protein markers of interest. It can be applied to data from both DIGE (Fluorescence Difference Gel Electrophoresis) and non-DIGE technologies.
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 has been improved and new interaction modes simplify even the most complex analysis.
Melanie 7.0 offers:
Melanie is the fruit of more than one decade of collaborations with eminent partners and users worldwide. The application has attained a high level of maturity over the years and has evolved into a solution that can be applied to a wide range of 2D 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 (Fig 1).
Melanie is also designed for DIGE applications, which allow the separation and co-migration of more than one sample per gel, using size- and charge-matched CyDyeTM DIGE Fluor dyes to label the different samples, and the inclusion of an internal standard for every spot on every gel. It uses the patented co-detection algorithm of the DeCyderTM 2D Differential Analysis Software (DeCyder 2D) to fully exploit the advantages offered by multiplexing.
A unique analysis pathway is used for all your gel studies, both for conventional 2D electrophoresis and 2D DIGE gels. The three steps in the workflow are:
- Import & Control - Import images and view, explore, edit and calibrate them before further analysis.
- Organize & Process - Allow gel comparisons by detecting and matching images.
- Analyze & Review - Define treatment groups, perform statistical analysis and review the results.
In the workspace, you can control and verify the status of a gel analysis, and have direct access to the desired step in the workflow.
Fig 1. Melanie 7.0 can be used for a wide range of 2D gel experiments, including DIGE.
Melanie automatically recognizes all 2D gel image formats during import, including TIFF images and *.GEL, *.MEL, *.IMG, *.GSC, and *.1SC format files. For DIGE gels, it furthermore detects probable combinations of co-run images that can be validated with a single click, while importing from *.DS files allows fully automatic gel naming and grouping.
The software therefore provides excellent image viewer functionality, without limiting the number of images that can be displayed simultaneously.
Prior to 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.
The cornerstones of a fast and successful 2D 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.
MINIMAL SPOT EDITING
Melanie provides spot detection algorithms optimized for DIGE and non-DIGE applications:
- For conventional 2D 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 2D simultaneously processes one, two, or three images derived from a single gel.
The advantage of the individual spot detection for conventional 2D 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.
In addition to simplified spot editing tools, version 7.0 provides robust 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.
- Spots can be propagated from one image to the other matched images. This allows for quantification of identical areas on all gels.
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 condition or treatment group are easier to match than images from different groups. 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, more efficient match designs can be created. Figure 2 shows an 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:
- By first matching gels within populations, one minimizes the number of difficult match combinations. This significantly reduces time spent on match editing.
- 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.
- This strategy for comparing populations reduces the number of spots missed in the analysis, by minimizing the influence of the global match reference.
- Any protein spot can be matched against equivalent spots in other images, without the need to copy spots to the reference.
Fig 2. Scheme of a match hierarchy A, with sub match sets AT1 and AT2, and its display in the workspace and MatchSet sheet.
Typical questions for 2D image analysis are:
- 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. The Fold change can be used to compare expression levels between different populations.
- Factor analysis, to help identify underlying sources of variation and indicate whether, and how well, spot groups and populations might be separated.
- 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), Mann-Whitney/Wilcoxon, and Kolmogorov-Smirnov.
- Overlapping measures (summarize each gel population by an interval and compute the overlap between these intervals), to identify significant protein expression variations without making any restrictive assumptions, as do the various statistical tests.
- Histograms, to visualize expression profiles.
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 3).
Fig 3. 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.
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 can be instantly viewed.
Melanie 7.0 offers fully dynamic tables, histograms, plots, and 3D 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.
Melanie offers numerous features to visualize different aspects of gel images (Fig 4).
The various modes to move and zoom the images allow the user to become familiar with the spot patterns or to navigate to the desired location in the image, possibly aided by the Overview feature.
Signal intensity can be inspected using the Adjust Contrast feature, the new 3D 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 images are then overlaid to produce dual channel images with clearly visible differences in protein expression.
- Synchronized and simultaneous 3D views of multiple gels can be displayed.
Fig 4. 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.
Grids can be used to visualize deformations in aligned gels, or to evaluate distances between spots, in terms of pixel coordinates, pI/MW units, or linear measures (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.
To support the collaborative efforts of researchers, Melanie 7.0 ensures seamless sharing of project data within a network and provides import/export features that allow users to send analyzed results (including images, spots, matches, annotations, spot sets, etc.) to external partners.
Many additional features enable the seamless integration of our software into the 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, Excel®, 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.
It is important to protect all gel data, not only by ensuring data integrity and consistency, but also by allowing the user to reverse undesired 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.