As mentioned at the beginning of this example (see section 2.1), there is a known 15 bp deletion Variant in exon 19 whose frequency we want to evaluate in our Sample. The corresponding sequence is in the “artificial” ‘EGFR_Exons_18-22’ Reference Sequence we already defined in our Project, so we can proceed with the definition of the Variant.

First, we click on the ‘Variants’ sub-tab on the right-hand panel of the ‘Project’ tab (the Variants Definition Table) and then choose the ‘Add’ button at the left margin. This creates a new entry in the Variant Definition Table. We decide to use the generic name, Var_1, for our Variant and to not enter any ‘Annotation’. We are thus ready to associate the Variant to its Reference Sequence by click-and-dragging it to the ‘EGFR_Exons_18-22’ node on the References Tree. This fills in the ‘Reference’ field for this Variant and sets the Status to “Accepted” in the Definition Table, and also adds the Variant as a sub-node of the References Tree (Figure 2‑15).

To complete the definition of our Variant, we must enter a ‘Pattern’ of variation for the Variant with respect to the Reference Sequence. To do this, we double-click on the ‘Pattern’ cell for the Variant in the Variants Definition Table. This opens the ‘Edit Pattern’ window, pre-loaded with the Reference Sequence to which the Variant is associated (Figure 2‑16).

We decide to type the Pattern directly into the ‘Pattern’ text area of the window, using the AVA software’s Variant Definition Syntax. [Since the location of the deletion was initially defined relative to exon 19 (positions 93-107), we must first calculate its position in the artificial Reference Sequence we are using: 328-342.] So, we type: ‘d(328-342)’ and press ‘Enter’. This highlights the Variant pattern in the Reference sequence according to the ‘Legend’ at the lower right corner (in this case highlighting a string of gaps in gray to represent the deletion; Figure 2‑17).