What does it mean when ICE reports that the "quality score is too low"?
The ICE tool requires that the input sequence files are of a high enough quality that they can be properly interpreted and analyzed. If the tool reports that the quality score is too low, it means that either the experiment file or control file does not meet the tool's criteria for trace quality.
What is causing the error?
There are two reasons why the tool may recognize this quality error:
1) The guide RNA target sequence(s) occur too close to either end of the amplicon.
2) The trace peaks in the Sanger sequence file are too noisy for the sequencing provider to make reliable base calls.
What can I do to fix these issues?
1) Check that your guide is not in the first or last 50 bp of the sequencing trace or < 100bp from the end of the sequencing primer. In general, a 400-800 bp PCR amplicon length is best amenable to ICE analysis. It is best to design the forward and reverse primers at least 150 bp from the closest sgRNA cut-site to allow for optimal sequencing across the edit.
2) Check that your sequencing trace for the edited sample is not too noisy before the guide sequence - the noise will typically look like significant secondary trace peaks. If the trace is noisy, we would recommend using an internal nested primer to do your sequencing to get less noise in the trace. If the noise problem persists, you may want to investigate using another Sanger sequencing provider.
3) Be sure to check that your .ab1 file contains the quality score field. If it does not contain this field, contact your sequencing provider to request this information.
If the problem persists, please contact firstname.lastname@example.org with the following information:
- Url link to the analysis results if available
- Control and edited sample files with a description of which control file correspond to which edited file
- Guide sequence(s) and which control/edited file pair it corresponds to
- Screenshots of any error messages or relevant details.