Identify variants and add expression values

The Identify Variants and Add Expression Values ready-to-use workflows can be used to identify novel and known mutations in RNA-seq data, automatically map, quantify, and annotate the transcriptomes, and compare the mutational patterns in the samples with the expression values of the corresponding transcripts and genes.

To run the ready-to-use workflow:

        Toolbox | Ready-to-Use Workflows | Whole Transcriptome Sequencing (Image rna_seq_group_closed_16_n_p) | (Human (Image human_folder_closed_16_n_p), Mouse (Image mouse_folder_closed_16_n_p) or Rat (Image rat_folder_closed_16_n_p)) | Identify Variants and Add Expression Values (Image identify_variants_and_add_expression_values_wts_16_n_p)

  1. Double-click on the Identify Variants and Add Expression Values 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.

  2. Specify the RNA-seq reads to analyze. The reads can be selected by double-clicking on the reads file name or clicking once on the file and then clicking on the arrow pointing to the right side in the middle of the wizard (figure 16.25).

    Image rnaseq_identify_variants_expression_step2
    Figure 16.25: Select the sequencing reads to analyze.

    Click on the button labeled Next.

  3. Specify a target region for the Indels and Structural Variants tool (figure 16.26).

    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.

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    Figure 16.26: Specify the target region for the Indels and Structural Variants tool.

  4. Set the parameters for the Low Frequency Variant Detection step (see figure 16.27). For a description of the different parameters that can be adjusted in the variant detection step, see http://clcsupport.com/biomedicalgenomicsworkbench/current/index.php?manual=Low_Frequency_Variant_Detection.html. If you click on "Locked Settings", you will be able to see all parameters used for variant detection in the ready-to-use workflow.

    Image wts9
    Figure 16.27: Specify the parametes for transcriptomic variant detection.

  5. If you are working with the workflow from the Human folder, specify here the relevant 1000 Genomes population from the drop-down list (see figure 16.28). Choose the population that matches best the population your samples are derived from.

    Under "Locked settings" you can see that "Automatically join adjacent MNVs and SNVs" has been selected. The reason for this is that many databases do not report a succession of SNVs as one MNV as is the case for the Biomedical Genomics Workbench, and as a consequence it is not possible to directly compare variants called with Biomedical Genomics Workbench with these databases. In order to support filtering against these databases anyway, the option to Automatically join adjacent MNVs and SNVs is enabled. This means that an MNV in the experimental data will get an exact match, if a set of SNVs and MNVs in the database can be combined to provide the same allele.

    Note! This assumes that SNVs and MNVs in the track of known variants represent the same allele, although there is no evidence for this in the track of known variants.

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    Figure 16.28: Select the relevant population from the drop-down list.

  6. Click on the button labeled Next to go to the last wizard step (shown in figure 16.29).

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    Figure 16.29: Check the selected parametes by pressing "Preview All Parameters".

    Pressing the button Preview All Parameters allows you to preview all parameters. At this step you can only view the parameters, it is not possible to make any changes (see figure 16.30). Choose to save the results and click on the button labeled Finish.

    Image rnaseq_identify_variants_expression_step6preview
    Figure 16.30: Preview all parameters. At this step it is not possible to introduce any changes, it is only possible to view the settings.

Seven different output types are generated:

  1. Gene expression (Image rnaseqtrack_16_h_p) 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 expression values.
  2. Transcript expression (Image rnaseqtrack_16_h_p) 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 expression values.
  3. RNA-Seq Mapping Report (Image proteinreport_16_n_p) 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://clcsupport.com/biomedicalgenomicsworkbench/current/index.php?manual=RNA_Seq_report.html).
  4. Read Mapping (Image read_track_16_n_p) 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 http://www.clcsupport.com/biomedicalgenomicsworkbench/current/index.php?manual=View_settings_in_Side_Panel.html).

  5. Annotated Variants with Expression Values (Image variant_track_16_n_p) Annotation track showing the variants. Hold the mouse over one of the variants or right-clicking on the variant. A tooltip will appear with detailed information about the variant.
  6. RNA-Seq Genome Browser View (Image trackset_16_n_p) 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 16.15).
  7. Log (Image table) A log of the workflow execution.