Primer Design toolsΒΆ

The Primer Design service uses the mopo16S tool to infer the best primer pair able to amplify the ITS1 belonging to the taxonomic clade settled by the user.

../_images/image12.jpg

Supplementary Figure 13: A snapshot of the tool submission mask.

Initially, the user needs to apply the Prepare primer input inference file tool, allowing to extract all the ITS1 sequences available for a specific clade in ITSoneDB. The user must select the taxonomic clade of interest. In order to streamline the taxon name searching an interactive drop-down menu is available. The sequence clustering (Enable clustering button) is enabled by default and may be disabled only for datasets with less than 1000 sequences. Once executed, the tool will generate a fasta sequences collection that will constitute the mopo16S tool input.

Following, the primer inference is performed by using the mopo16S tools.

The tool execution requires 2 mandatory inputs:

  1. Reference dataset file: a fasta collection of target sequences uploaded by the users or generated by using the Prepare primer input inference file tool;

  2. Reference primer file: mopo16s requires an initial primer set to eventually infer new primers. Otherwise a primer pair can be provided by the system [PMID:25077016].

It is also possible to set advanced parameters, for whose description we suggest to visit the developer site.

Mopo16S starts by inferring the efficiency (a measure of the amplification capacity of each primer), the coverage (virtually amplified sequences in input collection) and the matching-bias (mismatches between primers and input sequences) of the submitted primer pairs. Then infers a new primer pair (or a set of pairs) that maximise the efficiency and coverage and minimize the matching-bias.

Three outputs are produced:

  1. Efficiency, coverage and matching bias of the input primer pair;

  2. A list of new primers;

  3. Efficiency, coverage and matching bias of the new designed primers;

Tools