To generate the Expression Comparison report
1. On the NextGENe Viewer Comparisons menu, click Expression Comparison Report.
The Expression Comparison Report Settings dialog box opens. The General tab is the only tab.
2. Indicate how to define the segments that are to be analyzed and reported on by the tool.
Option | Description |
|---|---|
Use segments as defined in reference files. | • Contig - Report coverage levels for each contig. Note: This option is appropriate if you are using a reference that was recreated from a BED file for custom amplicons. • Gene - Report coverage levels for each gene region. • Continuous Exon - Report coverage levels for the entire mRNA region for a gene, one region per gene. • ROI - Report coverage levels based on Regions of Interest that are defined in a GenBank reference file. Note: For information about defining ROIs in a GenBank reference file, see Advanced GBK Editor tool.. • Exon - Report coverage levels for each mRNA region. (Coding and non-coding exons.) • Continuous CDS - Report coverage levels for the entire coding region for a gene, one region per gene. • Amplicon - Available only if an amplicon BED file was loaded during the Load Data step for the project. (See To set ROI regions from a BED or GBK file.) For overlapping amplicons, each read is counted only for its intended amplicon, where the intended amplicon is determined by the percentage of the amplicon that the read covers. The amplicon with the higher coverage is selected as the intended amplicon. • CDS - Report coverage levels for each coding region. |
Set incremental segment length | Only complete segments (requiring the minimum number of bases specified) are included in the report. The very ends of contigs or chromosomes are excluded from the report when the segment is less than the number of bases that are specified. Tip: Some genome references are built from contigs (each chromosome consisting of several contigs) while others are built from assembled chromosomes (each chromosome is essentially a single contig). If the selected reference was built from contigs, then you should select Contig so that the next segment starts at the beginning of the next contig. |
Input region of interest (*.bed) | You can upload a Region of Interest file in a BED format. |
3. Optionally, select one or both Limit options and if needed, modify the default limits (200 bp) for reporting the coverage for only the first or last '”x” number of bases of the selected segment type.
 | If any Limit option and CDS are selected, then the coverage levels for the first or last “x” number of bases in each CDS region is reported. |
 | Optionally, at this point, you can click Save Settings to save the settings for this report in a Settings file (.ini file), which can be used to generate the Expression Comparison report for another project based on the settings in the file. |
4. To limit the saved report to a smaller subset of regions, select Filter Saved Report, and then click Set to browse to and select the appropriate BED file.
| | Even if you select this option, the graphical report display is still based on a larger selection of regions. |
5. Click OK to open the Load Project Files dialog box.
6. Click Set to browse to and upload the reference project file (the control sample, for instance).
7. Click Add to browse to and select an alignment project file that is to be included in the comparison. Repeat this step until you have added all the necessary project files.
 | You can load a maximum of ten projects. |
8. Click OK to close the Load Project Files dialog box and generate the report.
The report is interactive:
Option | Description |
|---|---|
Change the report view | The default report view is “Max Coverage. Click View, and then on the View menu, select a different viewing option. The report can display either “Min Coverage,” “Max Coverage,” “Average Coverage,” “Minimum Forward Read Coverage,” “Minimum Reverse Read Coverage,” “Read Counts,” “Forward Read Counts,” “RPKM,” “RPK,” or “FPKM” for each region. Note: FPKM is available only if paired end data was analyzed for the projects. |
Change view for a condensation project | If the loaded project used condensation, then the view is based on the condensed reads. To change the view so that is based on the original reads, click Display original information |
Do normalization on the coverage values in the graphical report display and the saved report. Note: Applicable only for autosomal chromosomes. | Click Do Normalization. For example: Comparing Sample A and Sample B: • On chromosome 1, Sample B has fewer reads count than Sample A. • The coverage counts of Sample B for the chromosome (Max coverage, min coverage as set in menu->View) is amplified by multiplying by the following ratio: A's reads count for the chrom/B's reads count for the chrom. • If the ratio is larger than 10,000, then multiply all coverage counts by 10,000. Note: This calculation does not affect RPKM and FPKM. |
Move the report view to the indicated segment | In the View Segment field, enter a position, and then click Go. |
Save the report to a text file | Do either of the following: • On the report menu, click File > Save. • On the report toolbar, click the Save Report icon  A default name (<project_name>_ExpressionComparisonReport) and location (the project output folder) are provided for the file, but you can change one or both of these values. Note: The saved report is a table that lists the gene name and description for each region as well as the actual expression values for each region for every loaded project. The differential expression values of Ratio, log2Ratio, z-value, and p-value are also calculated and displayed for each gene in the report. |
Change the report settings and dynamically update the report display | 1. On the report menu, click Settings > Settings. The Expression Report Settings dialog box opens. 2. Edit the report settings as needed, and then click OK to close the dialog box. |
Carry out detailed differential expression pathway analysis for the sample projects | Note: This option is available only if your report settings specify to use Gene for the coverage regions. If your report settings are not set to Gene, then before you can use this option, you must change your report settings accordingly. See Change the report settings and dynamically update the report display. |