Defining the Amplicons

2.2.4

Now that we have a Reference Sequence, we can enter our Amplicons. To do so, we click on the ‘Amplicons’ sub-tab of the table-view. The ‘Add’ button on the left margin is now capable of creating new Amplicons, as this is its new context. For our EGFR Project, we need to add 11 Amplicons, so we click the ‘Add’ button 11 times, which adds 11 rows in the Amplicons Definition Table, with generic Amplicon Names. As before, the information is entered by double-clicking into the various fields for each Amplicon; the fields are: ‘Name’, ‘Reference’, ‘Annotation’ (optional), ‘Primer 1’, ‘Primer 2’, ‘Start’, and ‘End’).

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Since we have only one Reference Sequence, we can associate all our Amplicons with it at once by multi-selecting all the rows from the Amplicons Definition Table and dragging the selection to the proper (in our case, only) node in the References Tree, on the left. This adds a branch of Amplicon nodes hanging off the Reference node; and the ‘Reference’ fields on the Amplicons Definition Table are updated so that each Amplicon now has this field populated with this Reference Sequence.

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Primer 1 and Primer 2 are those used to prepare the Amplicon library (or libraries); for our example, these are listed in Table 2‑1 (above), along with the Amplicon Names. We enter the sequence of all the Primers by double-clicking in each of the ‘Primer’ fields; this opens a sequence editor window into which the sequence can be typed or pasted (always 5’-->3’; the software will compute the reverse complement of Primer 2 to align it to the Reference Sequence). For the Amplicon Names, we double-click in the ‘Names’ fields and type or paste the Amplicon Names in the Table cells.

•	We choose not to enter any Annotations for our Amplicons.

After adjusting the width of the column (by dragging of the separation lines between the headers) so that all the fields are completely readable, the application view looks as shown in Figure 2‑9.

Figure 2‑9: The AVA window after associating all 11 Amplicons to the single ‘EGFR_Exons_18-22’ Reference Sequence, and entering the Amplicons’ names and Primer sequences

Next, we define the ‘Targets’ by specifying their Start and End (i.e. by positioning the Primers along the Reference Sequence), for each Amplicon. To do this, we double-click in either the ‘Start’ or the “End’ field of each Amplicon. This opens the Edit Start/End window for this Amplicon and carries out an automatic search of its Primer 1 and the reverse-complement of its Primer 2 along its Reference Sequence (Figure 2‑10). As long as no errors were made when entering the Primer sequences, each Primer in our EGFR example will find an exact unique match and be displayed with a yellow background, and the Start and End of the Target will appear in the corresponding fields in the Amplicons Definition Table.

Figure 2‑10: The Edit Start/End window for Amplicon EGFR_19_1, showing the two Primers with yellow background and the Target with blue background

After setting the Start and End points for the Targets within all Amplicons, the Amplicons Definition Table of out Project looks as shown in Figure 2‑11.

Figure 2‑11: The completed Amplicons Definitions Table for the EGFR example experiment