Coverage Curve Report
The Coverage Curve displays the coverage distribution of sample reads along the reference sequence without directional information and reports low coverage regions. The report is useful for identifying regions that were not adequately sequenced because of low coverage. If the project used condensation, then the report displays the condensed coverage information. If you are carrying out targeted sequencing, then to view the coverage distribution for specific regions, use the option to load a BED file. If you used PCR amplicons to obtain sequencing data, you can create and upload amplicon text files for analysis.
 | The following procedure describes how to set up a new Coverage Curve report. Optionally, on the Coverage Curve 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 Coverage Curve.
The Coverage Curve report opens. Two options are possible:
• If post-processing options were not used to specify a Settings file for the report, then by default, the first time that the report opens for a sequence alignment project, it displays all the low coverage regions across the entire reference with a low coverage threshold that is equal to the total coverage threshold that was specified in the Mutation Filter settings for the project regions. See Mutation Filter settings.
• If post-processing options were used to specify a Settings file for the report, then by default, the first time that the report opens for a sequence alignment project, the settings that are specified in the loaded settings file are applied. If multiple Coverage Curve reports were selected in the post-processing settings, then the first loaded Settings file is applied.
After you change any of these default values for a project, NextGENe “remembers” these values and generates the report accordingly.
| | Reference sequence regions that are highlighted in red indicate regions where the coverage falls below the user-set mutation filter coverage threshold. The highlighted regions are useful for identifying large deletions or regions where PCR failed. Detailed information for each highlighted region is displayed in the report table below the graph. |
2. On the report menu, click Settings > Settings.
The Coverage Curve Settings dialog box opens. The General tab is opened by default.
3. To define the regions of the reference that are to be used for reporting low coverage regions, do one or both of the following as applicable:
Action | Step |
|---|---|
Load an amplicon text file for analysis | Select Input amplicon TEXT File (*.txt), and then click Add to browse to and select the appropriate Amplicon text file. You can load multiple Amplicon text files. An Amplicon text file must be a tab-delimited text file with the following format: • From left to right, the column headings are: Amplicon ID, Start, and End. Each column heading must be separated by a tab. • Enter the values for each amplicon in a separate row, with a tab between each value. Use reference positions for Start and End fields. • Save the file as a tab-delimited text file. |
Load Regions of Interest for analysis | Select Input region of interest, and then do one of the following: • To load a BED file, select Use ROI defined in BED files, and then click Set to browse to and select the appropriate BED file. • To use Regions of Interest that are defined in GenBank reference files, select Use ROI defined in reference files. • To use Regions of Interest that are relative to the contigs of the reference, click Use contigs. Note: This option is appropriate if you are using a reference that was recreated from a BED file for custom amplicons. |
4. Define the Coverage settings for the project.
Option | Description |
|---|---|
Define the low coverage threshold for including regions in the report | Enter the cut-off value in the Highlight Coverage field. |
Use Original Coverage | Available only for Condensation projects. Select this option to use original coverage values for generating the Coverage Curve report instead of condensed reads coverage. |
5. Optionally, open the Display tab and select the columns that are to be included in the report, or clear the options for the columns that are not to be included.
Column | Description |
|---|---|
Length | Selected by default. The total length of the low coverage region. |
Description | Selected by default. If this option is selected and you have loaded: • A BED file, then information in Column 4 for the file is displayed if it is available. • An amplicon text file, then any description that you have entered in the amplicon text file is displayed. |
Reference Position Start | The starting location for the low coverage region in the reference. |
Chr | Selected by default. The name of the chromosome on which the low coverage region is located. |
Chr Position Start | Selected by default. The base number that indicates where the low coverage region starts in the chromosome. |
Gene Start | The name of the gene where the low coverage region starts. |
Exon Start | The exon number where the low coverage region starts. This number includes both coding and non-coding exons. |
CDS Start | The CDS number where the low coverage region starts. |
HGVS Start | The HGVS nomenclature for the start of the low coverage region. |
RNA Accession Start | The RNA accession from NCBI for the gene at the start of the low coverage region. |
Protein Accession Start | The protein accession from NCBI for the gene at the start of the low coverage region. |
Reference Position End | The ending location for the low coverage region in the reference. |
Chr Position End | The base number that indicates where the low coverage region ends in the chromosome. |
Gene End | The name of the gene where the low coverage region ends. |
Exon End | The exon number where the low coverage region ends. This number includes both coding and non-coding exons. |
CDS End | The CDS number where the low coverage region ends. |
HGVS End | The HGVS nomenclature for the end of the low coverage region. |
RNA Accession End | The RNA accession from NCBI for the gene at the end of the low coverage region. |
Protein Accession End | The protein accession from NCBI for the gene at the end of the low coverage region. |
6. Optionally, open the Summary Report tab and specify how the Coverage Curve report is to be named and which of its information is to be displayed in the Summary report.
Setting | Description |
|---|---|
Report Name | The name that is displayed for the Coverage Curve report in the Summary report. The default value is Coverage Report but you can change this value. |
Display target region statistics | Selected by default. Display the target region statistics in the Summary report. |
Display coverage curve | Selected by default. Display the coverage curve in the Summary report. |
Display coverage report | Selected by default. Display the coverage information in the Summary report. |
| | If you change any information on the Summary Report tab, then you must save these settings in a Settings file (.ini file). These settings are applied to the Coverage Curve report only if you select this Settings file during the setup of the Summary report. See Summary Report. |
7. 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.
8. Click OK to generate the report.
The report is interactive:
Option | Description |
|---|---|
Zoom in the graph view | Hold down the left mouse button and draw a box from the upper left hand corner of any region in the graph towards the lower right hand corner. A box is formed around the area that is being reduced for viewing. Note: After you zoom in on a region, you can use the use right mouse button to scroll the region. |
Zoom out the graph view | Hold down the left mouse button and draw a box from the lower right hand corner of any region in the graph towards the upper left hand corner. The magnification for zooming out is always 100%. |
Save Low Coverage Region information to a text file | Do one of the following: • On the report menu, click File > Save Coverage Report. • On the report toolbar, click the Save Report icon  . |
Display summary coverage information for the BED file regions | |
Modify the report settings and dynamically update the report display | 1. On the report menu, click Settings > Settings. The Coverage Curve Settings dialog box opens. 2. Modify the report settings, and then click OK to close the dialog box. |