The Nucleic Acid Analysis Toolbox enable you to:
- locate regions that have a high probability of coding for a highly expressed protein
- determine which reading frame is the coding frame, if such a region is found.
- Perform simple profile analysis with up to two transfac profiles.
MacVector offers a range of algorithms for calculating coding preferences and locating protein-coding regions in a sequence:
- Open reading frames
- G+C % composition
- Fickett's TestCode
- Uneven positional base frequencies
- Positional base preference
- Gribskov codon preference
- Staden codon preference
- MacVector codon preference.
Most algorithms generate three plots, one for each possible starting codon position. These can be displayed separately, or combined in a single panel to accentuate the differences between plots.
To run this analysis, a nucleic acid sequence window must be the active window; and a codon bias file for the organism under investigation must be available. Several codon bias files are provided with MacVector, or you can make your own if you have access to the Entrez databases.
The NA Analysis Toolbox also allows you to scan a sequence with up to two profile files. MacVector reads profile files using the transfac profile format. A set of profile files of the Jaspar database of transcription factors is provided in the Profiles folder. As is a profile file of Donor and Acceptor sites for RNA splicing. The plot shows the probability that the target sequence has a significant match to the profile. The boxes are a simple search for matches to the profile consensus sequence. With a highly conserved profile, the results will be essentially identical, but with more degenerate profiles you may get many high probability matches that don't match the consensus. The consensus is calculated so that the base at each position is the least degenerate IUPAC ambiguity that equals or exceeds the threshold. See MSA Views for how to generate a profile.
1. Choose Analyze | NA Analysis Toolbox.
The Codon Preference Plot dialog box is displayed.
2. Scroll through the Algorithm list and select the checkbox for an algorithm you want to use.
You can browse through descriptions and parameters for the algorithms without selecting their checkboxes. Simply click on a name in the algorithm list to display this information.
3. In the Parameters panel, adjust the settings as required. Note that window size is set independently for each algorithm.
4. Repeat steps 2 and 3 to add more algorithms, as required.
5. Select Codon Bias File and choose the appropriate file, using the file selection dialog box that is displayed.
6. Choose the genetic code by selecting from the genetic code drop-down menu.
7. From the drop-down menu in the Plotting options panel, choose either:
- Combined plots, to combine the plots into a single panel
- Plot separately, to plot each frame in a separate panel.
8. To adjust the display of codons in the plots, select as required the checkboxes for:
- Show start codons
- Show stop codons
- Show rare codons.
To specify where the codons should appear in the display, choose Above, Center or Below from the Location drop-down menu. They will only be displayed where separate plots have been selected and where frame-specific data has been generated.
9. To restrict the search to a certain region of the sequence, type in the sequence numbers that bracket the region in the Region panel. Alternatively, choose a region from the features table drop-down menu at the right of the text boxes.
10. Select Options if you want to alter the plot colors, codon symbols, or the definition of rare codons.
11. Select OK to generate the plot.
Alternatively, select the Defaults button to restore the default settings, or Cancel to close the dialog box without performing any analyses.
Plot colors, codon symbols, and the definition of rare codons are specified using a separate subdialog box, NA Analysis Toolbox Plot Options.
1. In the NA Analysis Toolbox Plots dialog box, select the Options button.
The NA Analysis Toolbox Plot Options dialog box is displayed.
2. In the Plot colors panel, set colors as required. You can do this either by selecting an arrow button and choosing from the drop-down color palette, or by clicking on the color swatch and using the system Color Picker.
- Single sets the color for all separate plots
- Frame +1 /-1 sets the color for the first frame in a combined plot, Frame +2 /-2 the second, and Frame +3 / -3 the third.
3. In the Codon appearance panel, set the symbols and colors for Start, Stop and Rare codons, using the appropriate drop-down menus and arrow buttons.
4. In the Define rare codons as less than or equal to panel, select a radio button to choose the way rare codons will be defined:
- % of all codons
- % of synonymous codons - i.e. all codons that generate the same amino acid as that codon.
Then type the required threshold value in the selected text box.
5. Select OK to apply the settings.
Alternatively, select the Defaults button to restore the default settings, or Cancel to close the dialog box without saving the settings.