How to run the Create Amplicons tool

To run the Create Amplicons tool:

        Toolbox | Genome Finishing Module (Image finishing_tools_folder) | Create Amplicons (Image forward_inner_primer_region_16_h_p)

This opens the dialog shown in figure 5.1.

Image amplicon_creator_step1
Figure 5.1: Select a contig or sequence.

Select a sequence or contig and click Next.

Amplicon creation is directed by annotation types. This means that it is possible to create amplicons to e.g. all regions with a certain annotation (such as "scaffolds" or "genes") in the input sequence. However it is also possible to narrow down the region to be used for amplicon creation to for example single gene level. This is done using the "Restrict by qualifiers" function. The dialog shown in figure 5.2 allows specification of which regions should be used for amplicon creation.

Image amplicon_creator_step2
Figure 5.2: Specify parameters for the Create Amplicons tool.

The parameters to be specified in this step are:

Amplicon options
  • Amplicon length. Allows specification of the desired length of the amplicon annotations to be created.
  • Overlap size. A positive value specifies of the number of nucleotides by which the amplicon annotations should overlap (if tiling amplicons are desired). A negative overlap designates the number of nucleotides by which amplicon annotations should be separated.
Amplicon placement
  • Annotation type. Contains a drop-down list that makes it possible to annotate the type of problematic regions the amplicons are created to.
  • Offset relative to annotation ends. A positive value will extend each amplicon by that number in both directions and a negative value will shrink.
  • Restrict by qualifier. Enables restriction of annotations by qualifier (figure 5.2 and figure 5.3).
            Qualifier key. Amplicons are only applied to annotations when the selected qualifier key (e.g. gene, product etc.) has the specified qualifier value.
            Qualifier value. Amplicons are only applied to annotations when the selected qualifier key has the specified qualifier value (e.g. TIMP2, metalloproteinase inhibitor 2 precursor etc.)

Image amplicon_annotations_qualifiers
Figure 5.3: Annotation type, qualifier key, and qualifier value.

Amplicon annotations are created back to back on the sequence within the start and end positions that were specified in the algorithm (figure 5.4).

Image amplicon_output
Figure 5.4: Amplicon annotations are added to the sequence, back-to-back.