Quality Control and Experiment Creation | Image Alignment | Prefiltering
Analysis / Review | Advanced Statistical Analysis | Spot Picking | Reporting | Explore Data

 

Quality Control and Experiment Creation Specification

Experiment set up

• Analysis of single stain and DIGE experiments
  
• Supports multiple recommended image formats
  
  • .tiff
    
  • .img
    
  • .png
    
  • .gel
    
  • .mel
    
• Add images from multiple folders
  
• Ability to add additional gel images to an analysed experiment
  
• User controlled selection of reference image for the next stage of analysis

Quality control

• Loads and examines a wide range of image formats
  
• Unlimited number of images in an experiment
  
• Automatically examines images using the following QC checks:
  
  • Image format
    
  • Image compression
    
  • Level of saturation
    
  • Greyscale
    
  • Dynamic range
    
  • Intensity bit depth level
    
  • % intensity level
    
  • Previous image editing
    
• Provides feedback and recommendations following QC check
  
• Highlights images which have not passed the quality control process and reports on all reasons for failure
  
• Tick / cross buttons to individually include / exclude images from further analysis
  
• Image manipulation tools
  
  • Rotate
    
  • Flip
    
  • Invert
    
  • Crop

Mask of disinterest

• Ability to draw a mask of disinterest which is excluded from the analysis
  
  • Exclude area outside rectangle
    
  • Exclude area within rectangle
    
  • Exclude area within an ellipse
    
• Ability to edit a mask of disinterest
  

 

Image Alignment Specifications

• Enables a near pixel level correction of image distortion through the addition of alignment vectors
  
• Enables correction of dye related offsets in DIGE image multiplexes
  
• Automatic placement of alignment vectors
  
• Assisted placement of manual vectors
  
• Coordinated views and tools for rapid verification / correction of alignment
  
• Exported images retain the grey level calibration in .gel and .img file formats

Image display

• Preset image enhancement options access a wide range of gel image grey levels
  
• Automated contrast stretching using Enhanced Grid squares option

Alignment visualisation

• 4 simple integrated and interactive image views on User Interface (UI)
  
  • Zoomed alignment view
    
  • Alpha blend view with auto zoom for accurate vector placement
    
  • Whole Image overlaid view
    
  • Zoomed Chequerboard view
    
• Quick, simple image focus using one-click selection from displayed list

Alignment controls

• Adjustable alignment focus grid size
  
• Automatic and manual control of positioning the alignment focus
  
• Systematic mouse/keyboard driven navigation of alignment focus
  
• Drag and drop placement of alignment vectors
  
• Show aligned view with option to:
  
  • Apply alignment change
    
  • Always show aligned
    
• Show unaligned to view vector length and orientation
  
• Flexible options for vector removal
  
  • Single vector removal by right mouse click
    
  • Remove automatic vectors in current square
    
  • Remove automatic vectors from the whole image
    
  • Remove all vectors in the current square
    
  • Remove all vectors

Viewing and information options

• Option to change alignment overlay colours
  
• Display alignment vectors
  
• Display aligned grid
  
• Tick / cross buttons to individually include / exclude images from further analysis

 

Prefiltering Specifications

• Filters out spots from the SameSpots analysis
  
• Range of selection criteria for filtering
  
  • Filter by average normalised volume
    
  • Filter by spot area
    
• Filter out spots in a selected area of the image
  
• Right click on selected spot to set average normalised volume limit or spot area limit for filtering
  
• Undo / redo filtering

 

Analysis / Review Specifications

Performing SameSpots analysis

• Automatic analysis of aligned image set
  
  • Spot detection using SameSpots method
    
  • Background subtraction
    
  • Normalisation
    
  • Matching of all spots across experiment

Group set up

• Group images according to experiment structure
  
• Ability to set up multiple experimental groupings, e.g. male v female, control v treated
  
• Image search facility to assist with group set up in large experiments
  
• Rename image groups
  
• Colour coding of image groups
  
• Ability to add and remove images from image groups
  
• Ability to delete groups

Review spots of interest

• Automatic identification of interesting spots according to fold change and ANOVA p-values
  
• Spots ranked by ANOVA p-value and fold change
  
• Switch between experimental groupings and all views are automatically updated
  
• Spot IDs remain consistent when experiment groupings are changed
  
• Tick / cross buttons to individually include / discount interesting spots
  
• Click and drag to select multiple spots to be ticked / crossed
• Notes field for each spot
  
• Visible count of number of spots ticked / crossed
  
• Automatically advance through spot list

Spot tags

• Colour coded tags to assist with data exploration
  
• Right click on highlighted group of spots to tag
  
• Add name label to spot tag
  
• Filter spot list by tags
  
• Tag editing
  
• Spots can be tagged multiple times
  
• Spot tags maintained throughout the workflow

Viewing options

• Whole image view
  
  • Click and drag of focus area automatically updates the review spots list, 2D montage and 3D montage view
    
  • Click on spot for zoom view
    
• 2D montage view
  
  • Show current spot or all spots
    
  • Adjustable contrast
    
  • Adjustable montage view size
    
• 3D montage view
  
  • Select images for showing in 3D
    
  • Show current spots or all spots
    
  • Click and drag to reposition 3D view
    
  • Rotate option
    
  • Adjustable peak scale
    
  • Contour display option (where supported by graphics card)

Spot editing tools

• Spot edit performed on a single image propagated across all the images in an experiment
  
• Editing tools:
  
  • Split spot
    
  • Merge spots
    
  • Delete spot
    
  • Add spot
    
• Undo / redo spot editing
  
• Automatic recalculations and update of measurements table following spot editing
  
• Single click recalculation of spot ranking by ANOVA p-value and fold change following editing

Expression profile

• Plot of mean log normalised volume for each experiment group
  
• Error bars showing 3 standard errors within groups

 

Advanced Statistical Analysis Specifications

Further statistical analysis of spots of interest

• Calculation and display of q-values for indication of False Discovery Rate (FDR)
  
• Calculation and display of spot power
  
• Selected spot(s) shown in standardised expression profile view
  
• Selected spot(s) highlighted on Principal Components Analysis (PCA) plot
  
• Spot tags can be added and are maintained throughout the workflow
  
• Filter spot list by tags and all views are automatically updated

Principal Components Analysis

• Calculate principal components for all groups
  
• Calculate principal components for a selection of spots
  
• Graphical visualisation of calculated components with groups highlighted for visual confirmation of clustering
  
• Selection of principal components to display on axis
  
• Zoom functionality

 

Spot Picking

• Supports a selection of spot picking robots
  
  • GelPix
    
  • ProPic
    
  • Ettan
    
  • Generic robot (output of spot picking coordinates)
    
• Select picking image from within the experiment or browse for separate picking image
  
• Image manipulation tools for picking image
  
  • Rotate
    
  • Flip
    
  • Invert
    
  • Crop
    
• User controlled selection of image which best corresponds to picking gel
  
• SameSpots pixel level alignment for alignment of experimental image to picking gel
  
• Manual adjustment of spot pick location
  
• Spot tags can be added and are maintained in throughout the workflow
  
• Spots selected for picking shown on whole image view
  
• Tagged spots shown on whole image view

 

Reporting

• Spot selected for spot picking shown in experiment report
  
• Whole image view showing locations of spots selected for picking
  
• Spot information for each selected spot:
  
  • Spot position
    
  • Spot notes
    
  • Spot ID
    
  • 2D montage view showing spot outline
    
  • Spot expression profile showing mean log normalised volume for each experiment group and error bars showing 3 standard errors within groups
    
• Print and save report
  
• Open report in new window

 

Explore Data Specifications (using Progenesis PG240)

Spot Measurement

• INCA image filtering and noise reporting with Data Quality Control measurements
  
• Statistical comparisons using normal and paired t-test
  
• Error information viewed as:
  
  • Coefficient of Variation
    
  • Standard Error of Mean
    
  • Percentage SEM
    
• Data displayed in measurements (single gel)
  
• Data displayed in comparisons (multiple gel)
  
• Displays multiple spot fields including difference field
  
• Spot annotation
  
• Show/hide spot numbers
  
• Control annotation display options
  
• Advanced parameters for display options

Calibration

• 1D calibration method
  
• MW and pI ladder creation
  
• Flexible ladder placement
  
• Multiple curve fitting options
  
• Arbitrary user defined units
  
• 2D calibration method
  
• Protein list for marker entry
  
• Markers assigned only to reference gels
  
• All matched gels automatically calibrated
• Spot normalisation - choice of methods
  
• Normalise to single or group of spots
  
• Normalise to spot group average or collective volume

Data Visualisation

• 2 main image windows
  
• Montage window
  
• Animation window
  
• Histogram window
  
• Expression table
  
• 3D gel window
  
• 3D spot view enhancement
  
• 3D view including INCA calculations applied to surfaces
  
• 2 / 3D scatter plots
  
• Difference map
  
• Saturation map
  
• Profile window
  
• Zoom window
  
• Unique resizable scroll bars for zoom control
  
• Filter and rank spot measurements within tables
  
• Dynamically linked tables and image views
  
• Customisable table column display
  
• No. of matches for each reference spot displayed in comparisons window
  
• Data +/- INCA values displayed within tables
  
• Data QC results displayed within tables
  
• Sort data by error values in tables

Intensity Calibration

• Map image pixel values to known optical densities
  
• View calibration curve
  
• 5 curve-fitting algorithms

File Navigation

• View all files and directories using tree structure for easy navigation
  
• Create experiments
  
• Unlimited number of gels per experiment (RAM dependent)
  
• Open / close experiments
  
• Reorganise Files

Internet Facilities

• Internet querying of Federated databases
  
• Automatic HTML web page builder

Import/Export

• All windows can be copied to the Windows clipboard
  
• Tables can be exported as .csv files or direct to Microsoft Excel®
  
• Images can be exported as .bmp or .jpeg files
  
• Reports can exported in RTF or HTML format
  
• An entire experiment can be exported in a publicly available XML format

Additional Features

• Adjust image brightness/contrast /colour scheme
  
• Customise display options
  
• Printing options
  
• All tables can be reordered
  
• Access analysis options and common commands through main tool bar
  
• Tool tips on all features
  
• Status bar to display additional information
  
• Shortcut keys