TestPrime Tutorial

1. Submitting a Primer Pair for Evaluation

At the top of the TestPrime page you find the submission form:

  1. Enter your primers under "Sequence Data" just as you would order them. Spaces will be automatically removed and IUPAC encoded wobbles (ambiguities) resolved into multiple oligos.
  2. Select the database matching your gene (LSU or SSU). If available, choose the RefNR dataset.
    While the Ref is significantly larger than the RefNR, the distribution of sequences across taxa depends only on which organisms are most popular for sequencing. The RefNR is derived from the Ref using a clustering process and therefore has a more uniform distribution of sequences across taxa.
  3. Configure the desired "stringency" of the in silico PCR. If you decide to allow one (or more) mismatches, you have the option to configure a "perfect match" zone at the 3' end of the primer. "0 mismatches" is a conservative setting which leaves room for as yet unobserved sequence variations and other adverse conditions. "1 mismatch" with "5 bases" 0-mismatch zone a more realistic simulation of PCR behavior.
  4. Click "Run TestPrime". Your job will appear in the Tasks table where you can follow its progress.

 

2. Wait for your analysis to complete

When your job is finished, the page will extend to show your results (use the "show" link to switch between results if you submitted multiple evaluation requests).

 

3. Overall Results

At the top of the results, you will see a row of buttons:

The first button will bring you to the taxonomy browser where you can inspect how well your primer pair covers different taxa. The second button will add the "amplified" sequences to the cart, allowing you to use this set of sequences with the other tools on the website (e.g. applying filters with the search or using it as a target group for TestProbe). The last button will create a file containing the amplified sequences for download.

Below this, four pie charts show you how the primer pair performed on the whole database:

The pie charts in the top row show you the overall coverage. The left chart shows only those sequences that were long enough to determine whether they would be amplified or not. The right chart also shows those sequences for which the position at which the primer would match has not been sequenced ("short sequences").

The bottom row shows breakdowns of the mismatches and the short sequences into forward and reverse primer. In this case, you can see in the left chart that 48.4% of the mismatches were caused only by the reverse primer, indicating that it might be worthwhile to look for alternative reverse primers to increase coverage. In the right chart, you can see that the reverse primer also matches at a position which was not sequenced in many cases (97.8%). This does not indicate a bad primer, it merely means that little data is available to measure its performance (and thereby much less data to evaluate the performance of the combined primer pair).

Below this, you find two tables containing the raw results. The first one shows the per-taxon summaries, the second one the individual, amplified sequences. The contents of both tables can be downloaded ("Export Table to CSV") for further analysis or just to include the data with your notes for future reference.

You can hover the mouse over any of the column headers to see a description of the data contained in that column.

 

4. Taxonomy Drill-Down

To inspect the results of your TestPrime job taxonomically, switch to the taxonomy browser. The drop-down menu "Show" allows you to select which data should be visualized (if you click on the "Inspect Results in Taxonomy Browser" the current TestPrime job will already be selected).

 

The browser will now show additional percentages in green and two pie charts as in the following screen shot:

The green numbers show the amount of amplified sequences relative to the number of those that were long enough for analysis. This is the coverage. The grey numbers are relative to the total amount of sequences in the respective clade.

The pie charts at the bottom of the page show the coverage in the same manner as the top-row in the overall summary do, with the exception that these consider only the currently selected clade. The right chart also shows one further group of sequences: "excluded data". These are the sequences that were not in the dataset selected for evaluation, i.e. sequences that are not in the Ref or RefNR datset.