SameSpots has a simple workflow that allows quick and objective differential expression analysis. Alignment removes positional variation between gels and allows a single spot pattern to be detected, allowing multiple experimental designs to be analysed.
SpotCheck is a workflow within SameSpots that allows objective validation of gel running within the lab.
Simple workflow of SameSpots for differential expression
Images – Alignment – Spot Detection – Automatic Analysis – Statistics – Reporting
Images are automatically quality checked when uploaded to the software, giving you instant feedback before you start analysis. In the case of analysing a DIGE or multi-stained gel experiment, images are grouped according to the gel.
An alignment reference image is then selected and any areas of the images to be excluded from the analysis, e.g. damage, air bubbles, or edges of the gel, are masked from the analysis.
Alignment removes any positional variation between the spot patterns of the gels introduced during the electrophoresis and imaging processes. Alignment is performed at the pixel level to provide direct and accurate comparison of images allowing a single spot pattern to be detected.
Alignment is often completed using vectors which are automatically created, manual vectors can be added to aid the alignment.
Spots are automatically co-detected on all images in the analysis, creating a single spot pattern of the analysis. Normalised spot volumes are automatically calculated following detection.
The filtering stage allows removal of spots based upon position, area, normalised volume or a combination of these. Normalised spot volumes are automatically recalculated following filtering.
Experimental Set Up and Automatic Analysis
Images can be analysed by three experimental designs:
- Between subject – samples from a single subject appear only in one condition.
- Within subject – Samples from a given subject were taken under different conditions, e.g. a time series study
- Two-way ANOVA – Samples from a given subject appear in two conditions.
Images are grouped into conditions to be analysed and multiple experimental designs can be set up.
ANOVA p-values and fold change between each of the conditions are automatically calculated. Each spot can be visualised in a number of ways with expression profiles and spot details available.
Spots can be sorted by p-value or fold change, spots of interest can be tagged and the list can be filtered to include only the spots of interest. Additional details can be added to the spots including MS data, pI and MW information or notes.
SameSpots has two major advantages for statistical analysis of spots due to alignment of the images followed by detection of a single spot pattern:
- No missing values.
- Consistent spot outlines.
Statistical analyses which can be completed within SameSpots include:
- Principle component analysis (PCA),
- Correlation analysis – dendrogram,
- ANOVA p-value calculation,
- Power analysis and
- q-value calculation.
Spot Picking and Reporting
Spot picking files can be created for use with most of the common picking robots and information for manual picking can be exported.
A HTML report of results can be created from selected fields.
Clip Gallery is a tool which allows any images, tables or graphs from the analysis to be exported in high quality for use in reports and presentations.