Creating the Sequencing Plates

Usually two sequencing plates are created from each PCR plate (forward and reverse direction), in which the samples again retain the same well assignments.

Load and select the corresponding PCR plate by using the search function. Click again on New Reaction in the navigation bar.

This time set Type of reaction = Cycle Sequencing in the pop-up window and again make sure that Create plate from existing document is selected so that the sample IDs are taken over from the PCR plate.

A view of the sequencing plate opens. The plate name has the format CaBOL_SeqF_xxxx (forward direction) or CaBOL_SeqR_xxxx (reverse direction). The plate number is the same as that of the extraction and PCR plate.

The Edit All Wells icon should be used to enter the laboratory information for the plate:

Enter the Locus, Date (date of dispatch/ date of sequencing), Primer and Direction, and the Sequencing company under Technician.

Please find the primer in the shared database in the folder Primer.

Using the Bulk Add Traces function, the raw sequences (traces) are loaded in and connected to the sample:

• In the pop-up window, specify the location of the raw sequences.
• Specify the part of the file name of the raw sequences by which it can be assigned to the sample. This is usually the sample number/ CaBOL-ID. (When assigning via the well instead of Field: AccessionNumber, make sure that the well number which the sequencing company put in the trace name is the same well number like on your plate)
• For the option Only match files with names containing, enter the primer name so that only the sequences of the correct direction are attached to the plate.

Close the window with OK, the raw sequences are loaded and you return to the plate view .

Similarly, create the second sequencing plate for the reverse direction.