Probe Match (Cart contains no sequences)

  1. Type in or paste your probe or primer into the probe sequence window. You'll see that your probe will automatically be pasted into the search field of the "ProbeBase Search" formular on the right. More information Opens internal link in current windowhere.
  2. Adjust your parameters. If you restrict your search to SILVA "Ref" TestProbe will only consider hits to full length sequences (>1200 bases for SSU). If you use weighted mismatches the quality and position of the mismatches will be taken into account.
  3. Press "Run TestProbe" to start the calculation.
  4. The progress of the calculation can be monitored in the progress table.
  5. When your job is finished the TestProbe hits are shown in a table. To get additional information about a column you have to hover your mouse over the column header. Clicking on the header will sort the fields according to the values in the clicked column.
  6. If mismatches or weighted mismatches have been allowed, the pie chart on the bottom provides a graphical representation of the hits containing mismatches. Hover you mouse over the chart shows the absolute numbers and percentages of the hits relative to the total amount of hits.
  7. The buttons on the bottom of the result table provides enhanced options:
    • Export All To CSV: download the results in CSV (Comma Separated Values) format for direct processing with Excel or other spreadsheet programs.
    • Download Sequences: create an export job with the result sequences.
    • Add Sequences To Cart: add the result sequences to your cart.
    • Show Results in Browser: show the results in the taxonomic browser.
  8. With "Show Results in Browser" you can visually inspect the coverage of probes or primers in the taxonomic browser and add a specific subtree of your matched sequences to your cart. The percentages shown in the upper part of the browser window provide an overview of how much sequences in the respective group are matched by the probe or primer. In the example shown 56% of the sequences of the genus Nevskia are matched by the probe (shown in bold blue).
  9. A summary of all submitted TestProbe and Export jobs is available on the Download -> Opens internal link in current windowMy data. With "show" former TestProbe jobs can be reviewed.

Probe Evaluation (Cart contains sequences for evaluation)

  1. Select the sequences or group of sequences for probe evaluation and add them to the Cart. You can use the Browser or the Search functions on the SILVA page to do this. Please be aware that when using the SILVA taxonomy in the browser only full length sequences will be selected. In the example all Alphaproteobacteria have been selected and added to the Cart.
  2. Type in or paste your probe or primer into the probe sequence window. Restrict the search to SSURef and allow weighted mismatched if you want. This will relax the search and show also sequences that have mismatches to the probe. For mismatch calculation the quality and position of the mismatch are taken into account. Run "TestProbe" to start the calculation.
  3. The progress table provides additional information about hits, rRNA regions and accession numbers for the TestProbe results and the Cart. Quantity and Progress shows the number of probe/primer hits, Accessions the corresponding number of accession numbers and Regions the number of rRNA regions (sequences). Cart size reflects the number of accession numbers in the Cart and Cart Regions the number of rRNA regions (sequences) in the Cart.
  4. The "Top 30 Sequence Matches" table shows the matches sorted by their match and mismatch status. The "Mismatch Overview" shows the target sequence. In this example you can see that 46 sequences (third row) would match 100% if the probe would end with an Guanin instead of an Thymin.
  5. Sensitivity (hits and non-hits in the Cart) and specificity (hits and non-hits outside the Cart) of the probe/primer with respect to the sequences in the Cart are shown as pie charts. Hover your mouse over the pie chart shows the absolute and relative amount of sequences.

Probe Evaluation including partial sequences (Cart contains sequences for evaluation)

  1. To evaluate a probe or primer against a mixed set of full length and partial sequences you first have to select the sequences using the Browse and Search functions of SILVA. Because currently only full length sequences are classified according to the SILVA taxonomy, taxonomic groups have to be selected based on the RDP or EMBL taxonomies. In the example below the Nevskia group has been selected using the RDP taxonomy.
  2. Adjust parameters and run "TestProbe" to start the calculation.
  3. The results of TestProbe are shown in the table. Sensitivity and specificity of the probe/primer with respect to the sequences in the Cart are shown as pie charts. Additionally, for the sequences that are not targeted by the probe, information is given if the sequences have mismatches or if sequence information is missing (partial sequences). Mouse-over in the pie chart shows the absolute and relative amount of sequences.

ProbeBase Search

  1. Paste a probe sequence or a probe name in the "ProbeBase Search" field and press the search button.
  2. You'll be redirected to the probeBase search page with your results.

Getting subgroups of results with the taxonomic browser

  1. Run your TestProbe job and wait for the results.
  2. Press the "Show Results In Browser" button.
  3. Next you'll find yourself in the taxonomic browser showing the results of your TestProbe job which is indicated by the "Show: Testprobe 8" in the upper left corner. The "8" stands for the job number which is assigned to every job you create. In the example we add all Alphaproteobacteria that where matched in the TestProbe job. Additionally you have the possibility to remove subgroubs when they are already in your cart.