To generate a Structural Variation report
 | The following procedure describes how to set up a new Structural Variation report. Optionally, on the Structural Variation Report Settings dialog box, you can click Load Settings to browse to and select a Settings file (.ini file) to generate the report based on the saved settings in the file. |
1. On the NextGENe Viewer Reports menu, click Structural Variation.
The Structural Variation Report Settings dialog box opens. The General tab is the open tab.
2. Specify the type of data that is being analyzed:
Option | Description |
|---|---|
• Structural Variation/Fusion Report - Selected by default. Initially, all events that pass the Breakpoint (primary) filter settings are selected for inclusion in the Structural Variation report. The option that you select here is a secondary filter that determines which of the reads that passed the Breakpoint filter settings should still be considered for inclusion in the report before applying any of the final (tertiary) filter settings that are specified on the Filters tab. | |
• Linked Reads >= [ ] – Selected by default. The default value is identical to the Linked Reads value that was set in the Structural Variation Settings dialog box. (See Detecting structural variations in NextGENe.) The number of Linked Reads for the event must be greater than or equal to the specified threshold for the event to be included in the report. | |
• Scaled Links Reads >= [x] if Total Aligned Reads >= [y] – Scaling can be applied to Linked Reads if false positives are a concern. Both thresholds must be met for an event to be considered for inclusion in the report; otherwise, the reads are filtered from the report. | |
• Long Reads Report (> 75 bp) | |
• Paired Reads Report | |
3. Optionally, if you are carrying out targeted sequencing, then to view the possible structural variations in specific regions, select Input Region of Interest (*.bed), and then browse to and select the appropriate BED file.
4. Optionally, open the Display tab and select the columns that are to be included in the report (by default, all columns are included), or clear the options for the columns that are not to be included.
Column | Description | ||
|---|---|---|---|
Basic | |||
Variant Type | Selected by default. The type of structural variant such as deletion, duplication/insertion, translocation (gene fusion), and so on. | ||
Distance | The number of bps between the breakpoint of Position 1 (left image in the Alignment viewer) and the breakpoint of Position 2 (right image in the Alignment viewer). | ||
Orientation | Position 1 orientation is determined by evaluating the direction of the read end to the breakpoint. • The orientation is + when the breakpoint position is greater than the read end. • The orientation is - when the breakpoint position is less than the read end. Position 2 orientation is determined by evaluation the direction of the breakpoint to the read end. • The orientation is + when the breakpoint position is less than the read end. • The orientation is - when the breakpoint position is greater than the read end. Note: In the Alignment viewer, arrows that point from left to right indicate a positive orientation while arrows that point from right to left indicate a negative orientation. | ||
Chromosome | Selected by default. The name of the chromosome where the structural variation is found. | ||
Breakend ID | Unique system-generated ID that is generated for every breakpoint. | ||
Statistics | |||
Coverage | The maximum count of forward/reverse-oriented reads at positions 1/2 in each region of the breakpoint. This count includes reads that span the breakpoint and reads that do not. | ||
Link Reads | Selected by default. The total count of forward/reverse-oriented fragments (pseudo paired reads) that map to positions 1/2 divided by the total coverage at positions 1/2. | ||
Ratio | The total number of read fragments that span the breakpoint (pseudo paired reads) that map to positions 1/2 divided by the total coverage at positions 1/2. | ||
Median Length | The median length of fragmented reads (pseudo paired reads) that map at positions 1/2. | ||
Median Score | Selected by default. The median of the matching percentage for all pseudo paired reads that match at positions 1/2. See Median Score for a Structural Variation event. | ||
Position | |||
Breakpoint | Selected by default. The actual position of the breakpoint. | ||
Start/End | The positions in the reference sequence where the Structural Variation event begins and ends. | ||
Nomenclature – Select the options to display the breakpoint start and end positions relative to the selected annotation. | |||
Genome | The chromosome position. | ||
RNA | The RNA position (r.#) | ||
CDS | The CDS position (c.#). | ||
Protein | The protein position (p.#). | ||
Annotation - If the breakpoint overlaps any of the indicated regions, then the corresponding annotation is displayed in the Structural Variation report. | |||
Check all transcripts | Selected by default. NextGENe dynamically selects the transcript with the intron-exon junctions that best align to the breakpoints. If you clear this option, then the transcript that has been defined as the preferred transcript is used to annotate the r.#, the c.#, and the p.#. Note: The preferred transcript is pre-defined in the reference. You can always override this value. See Set Preferred Transcript | ||
5. Open the Filters tab, and then specify the criteria for filtering specific Structural Variation events from the report. By default, all columns are selected.
Option | Description |
|---|---|
Variant Type – Select the variant type that is to be filtered from the report. | |
Merge Overlapping Events – If both sides of a Structural Variation event match both sides of another Structural Variation event, then the events are merged. | |
Within same exon pair | Selected by default. Merge the duplicate events if the duplication occurs within the same exon pair. |
Within [ ] bps | Merge the duplicate events if the duplication occurs within the indicated number of bps. The default value is ten. |
Events | |
Unique Breakpoints | Selected by default. If both sides of a Structural Variation event align to a unique position, then the event is considered to be unique. |
Ambiguous Breakpoints | If one or both sides of the Structural Variant event align to multiple locations, then the event is considered to be ambiguous, and the event is aligned to the first location. |
Limit Regions - Both sides of a Structural Variation event must meet all the specified criteria, or the event is filtered from the report. | |
Genes | The breakpoint must be contained fully within a gene, or plus/minus the indicated number of bps on either side of the gene. |
Exons | Selected by default. The breakpoint must be contained fully within an exon, or plus/minus the indicated number of bps on either side of the exon. |
CDS | The breakpoint must be contained fully within a CDS, or plus/minus the indicated number of bps on either side of the CDS. |
6. Optionally, open the Summary Report tab, and then do any or all of the following:
• Specify an alternate name for the Structural Variation report when it is displayed in the Summary report. The default name is Structural Variation.
• By default, the Structural Variation report summary is selected for display in the Summary report. Clear this option if the report summary should not be displayed in the Summary report.
• By default, the Structural Variation report is selected for display in the Summary report. Clear this option if the report is not to be displayed in the Summary report.
| | If you change any of these options, then you must save these settings in a Settings file (.ini file). These settings are applied to the Structural Variation report only if you select this Settings file during the setup of the Summary report. See Summary Report. If you do not change any of these options, then it is optional for you to save the settings to a Settings file. |
7. Optionally, open the Output tab, and then specify the formats (TXT and VCF) in which the Structural Variation report is to be automatically saved and what type of consensus sequence is to be saved. By default, all formats and the consensus sequence are selected.
Setting | Description |
|---|---|
Save TXT report | Saves the Structural Variation report as a text (*.txt) file. Note: If you clear this option, then this is the only format in which you can manually save the report. You must use the Save icon on the report toolbar to do so. |
Save VCF Report (filtered) | Saves the Structural Variation report in a format that adheres to Variant Call Format (VCF) specifications. The report contains only those Structural Variation events that passed the Structural Variation Filter settings. See Step 5. |
Save VCF Report (Unfiltered) | Saves the Structural Variation report in a format that adheres to Variant Call Format (VCF) specifications. The report contains all Structural Variation events, whether the events passed the filter settings or not. See Step 5. |
Save consensus sequence | Saves the consensus sequences for all the Structural Variation events that passed the filter settings to a .fasta file. To specify the settings for the saved file, click Edit Settings. See Save consensus sequence. |
8. Optionally, click Save Settings to save the settings for this report in a Settings file (.ini file). You can use a saved Settings file to specify the post processing options for a project in:
• The Project Wizard. See To specify the post-processing options for a Sequence Alignment project.
• The NextGENe AutoRun tool. See The NextGENe AutoRun Tool.
• The Summary report. See Summary Report.
9. Click OK to generate the report.
The report is interactive:
Option | Description |
|---|---|
View a position or region in the Alignment viewer | Double-click a value in any column. |
Save the report to a .txt file | On the report toolbar, click the Save Report icon  A default report name (<project_name>_Structural_Variation_Report) and location (the project output folder) are provided, but you can change one or both of these values. |
Modify report settings | On the report menu, click Settings > Settings to open the Structural Variation Report Settings dialog box and modify the report settings as needed. The report display is dynamically updated after you click OK to save the settings. |
Copy selected text or text in a range of cells in the report to your clipboard | 1. Press and hold the left mouse button, and then drag your cursor over the continuous report cells that you are copying. The selected cells are highlighted in light blue. 2. Right-click anywhere in the selected cells. and on the context menu that opens, select Copy to copy the selected cells to your clipboard. 3. Use standard keyboard commands or menu commands to paste the copied cells into a third-party application. |