What are Best Focus and No Usable Signal errors?
“Best focus not found,” “Best focus is too near the edge of range,” or “No usable signal found, it is possible clustering has failed,” errors that occur after the first cycle of imaging on the MiSeq indicate there may be insufficient cluster intensity for the instrument to find the best plane of focus.
Possible instrumentation causes include:
Possible library and reagent causes are:
Troubleshooting Best Focus and No Usable Signal errors
To make sure that the instrument’s fluidics, temperature and motion systems are performing as expected, first perform a System Check on the instrument, as described in the MiSeq System Guide:
Once the tests are complete, click Show Details in the lower left corner of the screen to get a full listing of the results. If the volume test fails any position other than the PR2 position, or if any of the other tests fail, email Illumina Technical Support at firstname.lastname@example.org for further assistance.
If the system check passes, the failure may be due to an issue with the library or reagents. To investigate the most common issues:
If there are no apparent issues with the library or reagents, repeat the run with a 20% PhiX control spike in. This will act as a positive control for clustering for the next run to determine if the previous error was caused by an underlying library issue.
If an additional No Usable Signal or Best Focus error occurs at cycle 1 of the next run, contact Illumina Technical Support at email@example.com for further assistance.