3. The Filter View Interface¶

We first want to look at high quality variants, so the first filter we will apply is that of Read Depth. Now this field is not shown by default, so we will show it by selecting the eye icon in the Variant tab, expand the Sample Fields category, and select Read Depths (DP), and then OK to close this dialog.

New project view

Figure 3-1: New project view.

Column visibility dialog

Figure 3-2: Column visibility dialog.

Next, returning to the Variant tab, this field can be added to the filter chain by locating it under the Sample field category, right click on it, and select Add to Filter Chain.

Add read depth filter

Figure 3-3: Adding Read Depth to filter chain.

Next, enter a value of 100 in the Read Depths card and select the Equal to and Greater than filter card options. Next, add the Genotype Qualities (GQ) field to the filter card by right-clicking on the column title in the Variant tab and selecting Add to Filter Chain. For this filter, we will input a value of 20 and select the Greater than option on the filter card. Now the VarSeq project and filter chain should look like the image below.

Add genotype quality filter

Figure 3-4: Adding Genotype Quality to filter chain.

Next, we would like to filter further using specific annotation sources. To add annotations, click on Add in the title bar and select Variant Annotation….

Adding variant annotation

Figure 3-5: Adding a variant annotation.

This will bring up the Select Data Source dialog where you want to select the Public Annotations header in the file tree.

Select data source dialog

Figure 3-6: Select Data Source dialog.

We are going to select 2 different annotation sources, NHLBI ESP6500SI-V2-SSA137 Exomes Variant Frequencies 0.0.30, GHI, and ClinVar 2019-02-01, NCBI. To find these in the list of annotations, one can simply scroll through the list, or type title snippets in the Filter box to limit the annotations displayed. To select this specific older version of ClinVar, you must un-check the Current checkbox in the top-right of the window as previous versions of sources are filtered by default.

Note

We are using these specific allele frequency and ClinVar tracks here to demonstrate manually annotating and filtering variants, not as examples of a production workflow. At the end of this tutorial we will discuss better filters and how to save your project as a template. We also suggest using the ACMG Guidelines Gene Panel template as a starting point for production workflows.

Adding NHLBI annotation

Figure 3-7: Adding the NHLBI variant annotation.

Adding clinvar annotation

Figure 3-8: Adding the ClinVar variant annotation.

The annotation sources are selected by checking the box next to the annotation title and then clicking on Select. At this point, the user may be asked to download the selected sources to the local system for faster recall in the future, so select, Yes.

Annotation source download dialog

Figure 3-9: Annotation source download dialog.

The annotation sources are added to the variant table and can be found by scrolling to the right. We are going to add a filter to the variant Classification field in the ClinVar track and All MAF from the NHLBI track. This is done that same as above by right clicking on the field column title and selecting, Add to Filter Chain.

Adding clinvar classification filter chain

Figure 3-10: Adding ClinVar Classification to the filter chain.

Adding MAF to filter chain

Figure 3-11: Adding MAF to the filter chain.

Since we are trying to filter down to problematic variants, we are going to select Likely Pathogenic, Pathogenic, Uncertain Significance, and Missing in the ClinVar Classification filter card, and enter a Minor Allele Frequency value of 0.3 and select every option EXCEPT the Greater than in the filter card.

Final filter chain

Figure 3-12: Final filter chain.

Keep in mind that we can rearrange the filters to change the order in which the filters are applied. The filter cards can be rearranged by left clicking on the card title and dragging and dropping the card in a different order. While moving cards, users can notice that the variant totals will change accordingly as the filters are evaluated top-to-bottom. We will use the filter cards in the order of Read Depths, Genotype Qualities, Classification, All MAF to match the image below.

At this point the extensive filtering has led us to 986 variants. We will order these in decreasing Read Depth (DP) by right clicking on the Read Depth column and selecting Sort Descending.

Read depth ordering

Figure 3-13: Project with ordered read depth.

From this point, we would like to run the ACMG Classifier algorithm.