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.

Potential error messages:

• Best focus not found
• Best focus is too near the edge of range
• No usable signal found, it is possible clustering has failed
• Through-focus peak did not exceed SNR threshold
• Z Motor attempt to move outside soft limits

Possible instrumentation causes include:

• Poor delivery of reagents involved in cluster generation or first base chemistry
• Flow cell temperature control issues
• Optical system issues (rarely)

Possible library and reagent causes are:

• Use of expired or improperly stored reagents
• Library design and/or quality/quantification issues
• Poor Read 1 primer hybridization due to library design issues
• Use of incompatible primers
• Under- or overclustering

Troubleshooting cycle 1 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:

1. Perform the post run wash as prompted by the instrument
2. Power cycle the instrument as described in the System Guide
3. Go to Manage Instrument, then select System Check
4. Select all motion tests, prime reagent lines, and both thermal ramping, and volume tests

Once the tests are complete, select 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 techsupport@illumina.com 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:

• Check reagent kits for expiration dates and proper storage
• Make sure the library design is compatible to run on Illumina platforms
• Check the quality and quantification of the library using Illumina-recommended methods
• Make sure custom primers are compatible with the 65°C annealing temperature for the MiSeq
• Make sure custom primers are added to the correct cartridge wells (spiked into existing primer wells with no change to the sample sheet, or added to custom primer wells with the sample sheet indicating that custom primers are being used)

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 error occurs at cycle 1 of the next run, contact Illumina Technical Support at techsupport@illumina.com for further assistance.

Additional Resources:

MiSeq System Custom Primers Guide
Spiking custom primers into the Illumina sequencing primers
MiSeq System Denature and Dilute Libraries Guide