The Identify Candidate Variants and Genes from Tumor Normal Pair tool identifies somatic variants and differentially expressed genes in a tumor normal pair. One tumor normal pair can be compared at the time. If you would like to compare more than one pair you must repeat the analysis with the next tumor normal pair.
To run the ready-to-use workflow:
Toolbox | Ready-to-Use Workflows | Whole Transcriptome Sequencing () | Human, Mouse or Rat | Identify Candidate Variants and Genes from Tumor Normal Pair ()
- Double-click on the Identify Candidate Variants and Genes from Tumor Normal Pair tool to start the analysis. If you are connected to a server, you will first be asked where you would like to run the analysis.
- Specify the RNA-seq reads from the tumor sample (the panel in the left side of the wizard shows the kind of input that should be provided as in figure 18.15). Click Next.
- In the next step you will be asked to select the RNA-seq reads from the normal sample (see figure 18.16). Click Next.
- Configure the parameters for the RNA-Seq Analysis (figure 18.17), first for the tumor sample, and then for the normal sample in the following step.
If you wish to use spike-in controls, add the relevant file in the "Spike-in controls" field.
You can also specify that the reads should be mapped only in their forward or reverse orientation (it is by default set to both). Choosing to restrict mapping to one direction is typically appropriate when a strand specific protocol for read generation has been used, as it allows assignment of the reads to the right gene in cases where overlapping genes are located on different strands. Also, applying the 'strand specific' 'reverse' option in an RNA-seq run could allow the user to assess the degree of antisense transcription. Note that mate pairs are not supported when choosing the forward only or reverse only option.
- Specify in the next 2 windows a target region for the analysis of the sample with the Indels and Structural Variants tool, first for the tumor sample, followed by the normal sample (figure 18.18).
The targeted region file is a file that specifies which regions have been sequenced. This file is something that you must provide yourself, as this file depends on the technology used for sequencing. You can obtain the targeted regions file from the vendor of your targeted sequencing reagents. Remember that you have a hg38-specific BED file when using hg38 as reference, and hg19-specific BED file when using hg19 as reference.
- In the next wizard step (figure 18.19) you can adjust the settings for the Create fold change track tool. This tool calculates for each transcript or gene the ratio between the expression values in the normal and the tumor sample. It becomes then possible to filter on fold changes and expression values, which makes it easy to identify differentially expressed transcripts or genes. The parameters that can be adjusted in this wizard step are described in Create Fold Change Track.
- Set the parameters for the Low Frequency Variant Detection step (see figure 18.20). For a description of the different parameters that can be adjusted in the variant detection step, see
Low Frequency Variant Detection.
- The next wizard step (figure 18.21) concerns removal of germline variants. You are asked to supply the number of reads in the control data set that should support the variant allele in order to include it as a match. All the variants where at least this number of control reads show the particular allele will be filtered away in the result track.
- Finally, for the Remove Variants Found in HapMap, you can also specify which specific Hapmap population(s) characterize(s) best the samples.
- Click Next to go to the last wizard step. Preview All Parameters allows you to view all parameters but not to edit them. Choose to save the results and click Finish.
Thirteen types of output are generated:
- Gene Expression Normal and Gene Expression Tumor () A track showing gene expression annotations. Hold the mouse over or right-clicking on the track. A tooltip will appear with information about e.g. gene name and gene expression values.
- Transcript Expression Normal and Transcript Expression Tumor () A track showing transcript expression annotations. Hold the mouse over or right-clicking on the track. A tooltip will appear with information about e.g. gene name and transcript expression values.
- RNA-Seq Mapping Report Normal and RNA-Seq Mapping Report Tumor () This report contains information about the reads, reference, transcripts, and statistics. This is explained in more detail in the Biomedical Genomics Workbench reference manual in section RNA-Seq report (http://resources.qiagenbioinformatics.com/manuals/biomedicalgenomicsworkbench/current/index.php?manual=RNA_Seq_report.html).
- Read Mapping Normal and Read Mapping Tumor () The mapped RNA-seq reads. The RNA-seq reads are shown in different colors depending on their orientation, whether they are single reads or paired reads, and whether they map unambiguously. For the color codes please see the description
in (see Mapping view settings).
- Differentially Expressed Genes file () A track showing the differentially expressed genes. The table view provides information about fold change, difference in expression, the maximum expression (observed in either the case or the control), the expression in the case, and the expression in the control.
- Variant Calling Report Tumor () Report showing error rates for quality categories, quality of examined sites, and estimated frequencies of actual to called bases for different quality score ranges.
- Annotated Somatic Variants with Expression Values () A variant track showing the somatic variants. When mousing over a variant, a tooltip will appear with information about the variant.
- Amino Acid Track
- Genome Browser View RNA-Seq Tumor_Normal Comparison () A collection of tracks presented together. Shows the annotated variants track together with the human reference sequence, genes, transcripts, coding regions, and variants detected in ClinVar and dbSNP (see figure 18.22).
Figure 18.22: The Genome Browser View is a collection of a number of tracks. The Genome Browser View makes it easy to compare the different tracks. Each track kan be opened individually by double-clicking on the track name in the left side of the View Area.