Troubleshooting cycle 1 errors on the MiSeq

The following errors may occur after the first cycle of imaging on the MiSeq and indicate insufficient cluster intensity for the instrument to find the best plane of focus. This can be due to too high of cluster density, too low of cluster density, or a complete failure to cluster.

Potential error texts include:

• 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

• New in MiSeq Control Software (MCS) v4.0: ThroughFocusScan CoarseFocus failed to find focus, and there is no fallback position.

  

Cycle 1 errors can result from library, run setup, instrument, and reagent issues.

Possible instrumentation causes include:

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

• Flow cell temperature control issues.

• Optical system issues (rare).

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.

• Underclustered or overclustered libraries.

• NaOH pH issue.

• Contamination of wash tray.

Cycle 1 errors indicate the instrument was unable to identify sufficient signal at cycle 1 and could not calculate the appropriate focal point on the flow cell. 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.

If a focus error occurs later in the run (such as in the first cycle of the index read or in the middle of Read 1 or Read 4) for further information, search for Knowledge Base article(s): Troubleshooting best focus errors occurring mid-run on the MiSeq, Troubleshooting Z motor errors occurring mid-run, and Troubleshooting No Intensity for Index Read on MiSeq.

Troubleshooting Steps:

To verify 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:

1. Perform the post run wash as prompted by the instrument.

2. Power cycle the instrument as described in the System Guide.

3. Navigate 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 [e-mail] 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:

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

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

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

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

5. 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).

6. Confirm that a fresh dilution of NaOH was used and the pH is above 12.5.

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 the following run stops again with an error at cycle 1, email Illumina Technical Support at [e-mail] for further assistance.

Additional Resources: MiSeq System Denature and Dilute Libraries Guide MiSeq System Custom Primers Guide

For further information, search for Knowledge Base article(s): 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 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 #1430), contact Illumina Technical Support [e-mail].