Troubleshooting best focus errors occurring mid run on the MiSeq

The following errors may occur after the first cycle of imaging on the MiSeq and indicate there may be insufficient cluster intensity for the instrument to find the best plane of focus.

• 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

• ThroughFocusScan CoarseFocus failed to find focus, and there is no fallback position

  

Either library, instrument, or run set-up issues can lead to a mid-run (after cycle 1) best focus error.

Possible library and reagent causes:

• Use of expired or improperly stored reagents.

• Library design and/or quality/quantification issues.

• Use of incompatible primers if custom primers are used for later reads.

• Under- or overclustering.

Possible instrumentation causes:

• Poor delivery of reagents involved in cluster generation or first base chemistry.

• Flow cell temperature control issues.

• Optical system issues (rare).

Possible run setup causes:

• Setting up run with the total number of cycles greater than what is supported by the kit.

• Selecting a Custom Primer in the run setup when not intended.

Troubleshooting steps:

1. Review run data to confirm run performance. If a library issue is not suspected, troubleshoot the instrument.

   1. Verify total cycles called for in run length is compatible with the reagent kit used.

   2. Check reagent kits for expiration dates and proper storage.

   3. Make sure the library design is compatible to run on Illumina platforms.

   4. Check the quality and quantification of the library using Illumina-recommended methods.

   5. Make sure custom primers are compatible with the MiSeq.

   6. If applicable, 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).

2. To ensure that the instrument fluidics, temperature, and motion systems are performing as expected, first perform a System Check on the instrument as described in the MiSeq System Guide.

3. If the system check passes, set up the next run.

4. If there is a failure in any of the system checks or the following run stops due to another Best Focus error, email Illumina Technical Support at [e-mail] for further assistance.

Additional Notes: If the run stopped after completing the index reads, FASTQ files can be generated. For further information, search for Knowledge Base articles:* Generate FASTQ from an incomplete run in Local Run Manager

• How To Generate FASTQ files from an incomplete run in MiSeq Reporter

Additional Resources:* MiSeq System Denature and Dilute Libraries Guide

• MiSeq System Custom Primers Guide

For further information, search for Knowledge Base articles:* Spiking custom primers into the Illumina sequencing primers

• How to achieve more consistent cluster density on Illumina sequencing platforms

• System checks for Illumina sequencing platforms

• How To Power Cycle the MiSeq

• How To Power Cycle the MiSeq Video

• Troubleshooting best focus errors occurring mid-run on the MiSeq

• Troubleshooting Z motor errors occurring mid-run on the MiSeq

• Troubleshooting No Intensity for Index Read on MiSeq

• Best Focus and No Usable Signal errors on the MiSeq

For any feedback or questions regarding this article (Illumina Knowledge Article #1849), contact Illumina Technical Support [e-mail].