Run Setup Problems


Possible Cause


Flow cell lever is orange.

The flow cell did not seat properly.

The vacuum did not seal.

Manifolds did not raise.

Remove the flow cell and repeat the cleaning steps.

Make sure that the gaskets are present and well-seated.

Reload the flow cell.

The software did not initialize.

The software was unable to initialize internal hardware devices.

Close the error message and then re-launch the instrument software.

If the problem persists, restart the instrument computer. Make sure that the "Do Not Eject" drive is populated upon restarting the computer.

If the problem persists after restarting the instrument computer, shut down the instrument, wait a minimum of 60 seconds, and then restart the instrument.

Poor fluid delivery.

Potential bubbles in the system.

Reposition the flow cell and confirm that the holes are facing down.

Look for white precipitate around the gaskets. If precipitate is present, replace the gaskets. Always replace gaskets before loading a new flow cell.

Confirm that the sipper assemblies are fully lowered and each sipper is in contact with the reagents.

Performing a Fluidics Check

The Check button on the Start screen allows you to perform a fluidics check when you are not setting up a run. Use this option during instrument installation and fluidics troubleshooting.

  1. Load a used flow cell onto the instrument.
  2. Load eight 250 ml bottles with wash solution or laboratory-grade water, and load the bottles onto the corresponding reagent rack. Load the rack onto the instrument.
  3. Select Check on the start screen.
  4. Select solution 5 (SB2) from the drop-down list. If you are performing a fluidics check with a used flow cell installed, you can select solution 2, which is water.
  5. Enter the following default values: Volume: 250; Aspirate Rate: 250; Dispense Rate: 2000.
  6. Select Pump. If you need to pause the fluidics check, select Pause.
  7. Visually inspect the flow cell for bubbles passing through the lanes and leaks near the manifolds.
  8. If you see excessive bubbles, check the gaskets for obstructions, reduce the aspirate rate to 100, and pump another 250 µl of water to the flow cell.

Performing a Read 2 Rehybridization on a HiSeq Flow Cell

If you see that intensities have dropped significantly in the first cycle of Read 2 and you are using HCS v1.4 or later, you can perform a Read 2 primer rehybridization to rescue the run. Before performing this procedure, email Illumina Technical Support for further evaluation. See Performing a Read 2 Rehybridization on a HiSeq Flow Cell.