No autocalling even though clusters are observed

Failure of autocalling of SNP results is usually due to problems associated with (select one):

Outlier wells

If an assay shows clustering around the no template control (NTC), search for an outlier sample. In some cases, the software automatically scales the plot to include the outlier giving the other samples the appearance of clustering around the NTC. If the outlier is removed from the analysis, the program rescales the plot and the data can be autocalled.

Allelic discrimination outliers

Remove the outlier(s) and reanalyze. The program will adjust the scaling and distinct clusters can be seen.

7900 SDS Software

Note: The instructions below are for Sequence Detection Software v.2.3. For other versions, see the user guide.

  1. Click the Results tab to view the data.
  2. In the allelic discrimination plot, click and drag the mouse to select the outlier.
  3. Click the Setup tab.
  4. Check Omit Well(s).
  5. If there are additional outliers, repeat Steps 2 through 4.
  6. Click Re-analyze the Plate.


7500 Fast System Software

Note: The instructions below are for Sequence Detection Software v.1.4. For other versions, see the user guide.

  1. Click the Results tab.
  2. In the allelic discrimination plot, click and drag the mouse to select the outlier.
  3. The selected well(s) will appear colored in the plate map below. Click the well(s) by holding down the control button, and press   to bring up the well inspector.
  4. Omit the highlighted wells by clicking the Omit box in the well inspector. Click OK.
  5. Click Re-analyze the Plate.

StepOne™/StepOnePlus™ System Software

  1. In the allelic discrimination plot, click and drag the mouse to select the outlier.
  2. The selected well(s) will appear highlighted in the plate map.
  3. Right click the well(s) and click omit.
  4. Reanalyze the plate.

Software

You can experience problems with the software if (select one):

The autocaller algorithm is disabled

To achieve automatic allele calling of cluster plots generated by an end point read, the automatic allele calling feature must be enabled.
       
Solution:  How to enable the autocaller

7900 SDS Software

Note: The instructions below are for Sequence Detection Software v.2.3. For other versions, see the user guide.

  1. Click the Results tab.
  2. Click the Analysis Settings button.
  3. Click the Autocaller Enabled button for the marker of choice.
  4. Analyze.

7500 Fast System software

Note: The instructions below are for Sequence Detection Software v1.4. For other versions, see the user guide.

  1. Click the Results tab.
  2. Click the Analysis Settings button.
  3. Click the Automatic Allele Calling button for the marker of choice. If the plate has been previously analyzed, deselect the Keep manual calls from previous analysis button.
  4. Analyze.

StepOne™ / StepOnePlus™ System Software 2.0

  1. Autocalling is set to default.
  2. Click the Analysis Settings  button, to switch from Manual Call to Auto Call.
  3. In the Call Settings tab, either:
    • Select Use Default Settings, or
    • Click the Autocaller Enabled box. 
  4. Click Apply Analysis Settings.

You have a low number of clusters

If only two different genotypes are present on the plate, the software will not automatically call them unless Two Cluster Calling under Function is activated.

Solution:

7900 SDS Software

Note: The instructions below are for Sequence Detection Software v.2.3. For other versions, see the user guide.

  1. Click the Analysis Settings button .
  2. Click the 2-cluster calling enabled button for your marker of choice.
  3. Analyze.

7500 Fast System Software

Note: The instructions below are for Sequence Detection Software v1.4. For other versions, see the user guide.

  1. Click the Analysis Settings button
  2. Click the 2-cluster calling on button for your marker of choice. If the plate has been previously analyzed, make sure to deselect the Keep manual calls from previous analysis button.
  3. Analyze.

StepOne™ / StepOnePlus™ System Software 2.0

  1. Click the Analysis Settings button .
  2. In the Call Settings tab, deselect Use default settings and click the 2-cluster calling enabled box.
  3. Click Apply Analysis Settings.