To generate the Somatic Mutation Comparison Tool report
1. On the Comparisons menu, select Somatic Mutation Comparison Tool.
The Somatic Mutation Comparison Tool window opens.
2. To load the files that are to be compared, do one of the following:
• On the Somatic Mutation Comparison Tool main menu, click File > Load Projects.
• On the Somatic Mutation Comparison Tool toolbar, click the Load Projects icon 
.
. The Load Projects dialog box opens.
3. For each project (Tumor, Matched Normal, and Pool), click the Load File icon 
to browse to and select the appropriate sequence alignment project file (Aligned Sequence Project (*.Pjt)) for loading.
to browse to and select the appropriate sequence alignment project file (Aligned Sequence Project (*.Pjt)) for loading.4. Specify your report settings.
Setting | Description |
|---|---|
Maximum Contamination | Independently compares the normal sample to the tumor sample to account for the possibility of the contamination of the normal sample with tumor DNA. If the frequency of the variant in the matched normal sample is less than the indicated threshold, then the variant is not filtered from the tumor sample. |
Number of Pooled Samples | The number of samples that are included in the pool. Used in conjunction with the Maximum contamination threshold to consider possible contamination in the pool, such as low level tumor DNA. Sets an acceptable low level frequency that determines if a variant should be filtered out from the tumor sample. If the variant falls below this frequency, then it is not filtered out from the tumor sample. Note: Four to five samples is the recommended value for the pool. |
Somatic Allele Count | The minimum coverage that is required for the variant in the tumor sample to be included in the Somatic Mutation Tool report. |
Relative Directional Balance (T/N) | Selected by default. The ratio of the Read Balance for the variant in the tumor sample to the Read Balance for the reference allele in the normal sample.If the value for a variant falls below this ratio threshold, then it is filtered out from the report. Note: This option is useful for filtering out variants that are less directionally balanced in the tumor sample than in the normal sample. |
Somatic Allele Frequency (T/N) | The ratio of the frequency of the variant in the tumor sample to the frequency of the variant in the normal sample. If the ratio is less than the indicated threshold, then the variant is filtered out from the report. |
Pooled Allele Count Ratio (T/P) | The ratio of the number of reads with the variant in the tumor sample to the number of reads with the variant for the pool. |
5. Optionally, do any or all the following as needed in the Filter and Display Settings pane:
• To specify the information that is to be displayed for each variant, click Mutation Report Filter/Display Settings.
Because the Variant Comparison Tool report settings are identical to those used in the Sequence Alignment Mutation report, the Mutation Report Settings dialog box opens. See Sequence Alignment Project Mutation Report Settings.
• To specify the tracks that are to be used in the comparison, click Tracks Filter/Display settings to open the Query Tracks dialog box and select and/or import the appropriate tracks. You can also set the display and filter settings for the selected tracks at this point.
 | For detailed information about loading track data for previously run projects, see the Geneticist Assistant User’s Manual or Online Help. |
• To generate a CNV (SNP-Based Normalization with Smoothing) report for the data, select CNV report, and then click CNV Filter/Display Settings to open the and specify the appropriate settings for the report. See The CNV (Copy Number Variation) Tool.
| | If you select this option, then the report is displayed on a CNV Table tab in the report. You can toggle the report view between the SNP Table tab and the CNV Table tab. |
6. Click OK.
The Somatic Mutation Comparison Tool report is generated. It is displayed on the SNP Table tab.
The Somatic Mutation Comparison Tool report is interactive.
• To view alignments for selected projects, click View > Check Projects to View Alignments, or on the report toolbar, click the Check Projects to View Alignments icon 
.
.The Sequence Display Settings dialog box opens. The dialog box displays all the projects for which you can view the alignments.
At a minimum, you must select the projects for which you want to view the alignments. You can also set any or all of the following options for the selected alignment projects:
Option | Description |
|---|---|
Base Display Size | Indicates the font size for the base display. You can leave this option set to the default value of 8, or edit as needed. |
Mark Center Lines | Selected by default. Indicates whether to show the center line (a green vertical line) in each alignment display. |
Show Translation | Select this option to add the Translation pane to the Variant Comparison Tool report. The pane displays the amino acid sequence in IUPAC nomenclature for each alignment display. |
Show Mutation Calls | Select this option to add the Mutation Calls pane to the Variant Comparison Tool report for each alignment display. Tick marks are displayed at the locations where mutation calls where made. |
After you set your options and click OK to close the Sequence Display Settings dialog box, a window that is linked to the report table for the selected projects opens. You can do the following in this window:
• To change the focus of the report to the selected variant, double-click on a variant in the alignment view.
• To change the focus of the report to the selected variant, right-click on a variant in the alignment view, and on the context menu that opens, select Go to position in Mutation report.
• To change the focus in the corresponding alignment view to the selected variant, double-click on a variant in the Mutation report.
• To automatically save the Sequence Display Settings that you selected, click View > AutoSave Display Status. The next time you run a comparison in the Variant Comparison tool, these setting are automatically applied for the display.
• To copy a sequence in the Pile-up pane for an alignment project, press and hold the [Shift] key and the [Ctrl] key, and then click and hold the left mouse button and draw a box around the region of the sequence that you are copying. The selected region is filled with black. Right-click and select Copy Sequence or Copy As Picture to copy the sequence or image to your clipboard. Use standard keyboard commands or menu commands to paste the copied sequence or image into an application.
• To search the displayed alignment, click Search > Sequence Search, or on the report toolbar, click the Sequence Search icon 
. The Search dialog box opens, where you can indicate how you want to search the displayed alignment—by Sequence, by Position (chromosome, chromosome position (for example, 1, 20000)) or by Gene Name. You can also click Option to search by a reverse complement sequence.
. The Search dialog box opens, where you can indicate how you want to search the displayed alignment—by Sequence, by Position (chromosome, chromosome position (for example, 1, 20000)) or by Gene Name. You can also click Option to search by a reverse complement sequence. | | The Search Sequence function is enabled only when the Check Projects to View Alignments option is selected. |
• To change the current Mutation report display, click Settings > Settings to open the Mutation Report Settings dialog box. Select the Filter and Display options for the report.
| | For information about the available settings on each of the tabs on the Mutation Report Settings dialog box, see Sequence Alignment Project Mutation Report Settings. |
• To select the tracks that are to be queried for the projects, click Settings > Tracks Settings > Query to open the Query Tracks dialog box.
| | For detailed information about loading track data for previously run projects, see the Geneticist Assistant User’s Manual or Online Help. |
• To change the display and filter settings for the tracks that are included with the projects, click Settings > Tracks Settings > Filter/Display to open the Variation Tracks Settings dialog box. Select the Filter and Display options for the report relative to the imported tracks.
| | For detailed information about the available settings on each of the tabs on the Variation Tracks Settings dialog box, see Variation Tracks Settings dialog box. |
• To save the report and/or related information in a variety of formats, click the indicated option on the File menu:
• Save Report - To save the report to a tab-delimited text (*.txt) file.
A default name and location are provided for the file, but you can change both of these values.
| | You can also click the Save Report icon  on the report toolbar. |
• Save VarMD Report - To save the report as a VarMD report, which is a format that you can use in the third party VarMD tool.
• Save as Project Link - To save all the information for the currently displayed comparison (the samples, the comparison settings, and the report settings) in an .ini file. You must specify the file name. By default, the file link is saved in the project folder for the project that was loaded last for the comparison, but you can always select a different location.
• Load Project Link – To load a previously saved comparison, after you click this option, scroll to and select the appropriate project link. The comparison is loaded into the Variant Comparison tool. The comparison display is determined by the information (the samples, the comparison settings, and the report settings) that was saved for the project link.
• Save SNP Consensus – To save the consensus sequences for all the variants that are displayed in the Variant Comparison tool report. The sequences are saved to a .fasta file in the project output folder for the first loaded project. The default name for the file is based on the name of the first loaded project appended with _SNP_Sequences, but you can change one or both of these values.