How to perform a Maintenance Wash on the NovaSeq X Series Instruments

Background

The NovaSeq X Series instruments perform an automated wash after every successful sequencing run. As part of normal instrument maintenance, an additional maintenance wash is required every 14 days or whenever an automatic post-run wash fails to complete.

The maintenance wash requires:

• Wash Reagent cartridge

• Used Buffer cartridge

• Either Wash flow cell or a used 8-lane sequencing flow cell that has not been removed from the instrument

• Empty used Small and Large Spent Reagents Bottle

Important Notes

For the NovaSeq X Control Software v1.3 and above:

• The wash setup is only available from the Start Menu in the NovaSeq X Control Software, the Wash option has been removed from Settings.

• The Wash Flow Cell usage count is now enforced and will prevent using a Wash Flow Cell with over 20 uses, and present a warning when use count reaches 15.

  • For assistance with replacing the Wash Flow Cell, contact Illumina Technical Support.

  • Illumina recommends performing Maintenance Washes with the used 8-Lane flow cell that has not been removed from the instrument whenever possible.

For the NovaSeq X Control Software v1.2.2 and below:

• The wash setup is available from either the Control Software Settings Menu or the Start menu.

• The Wash Flow Cell usage count is NOT enforced, though the Software will add to the use counter for that Wash Flow Cell.

  • Illumina recommends performing Maintenance Washes with the used 8-Lane flow cell that has not been removed from the instrument whenever possible.

Preparing the Wash Solution Buffer

1. Add 400 mL laboratory-grade water to a 500 ml centrifuge bottle.

2. Add 0.2 mL 100% Tween-20 to the laboratory-grade water to prepare the 0.05% Tween-20 wash solution.

• Using a freshly prepared dilution of Tween-20 limits the introduction of contaminants into the fluidics system.

1. Invert solution several times to mix thoroughly.

2. Add 380 mL of 0.05% Tween-20 wash solution to the large wash solution well in the wash reagent cartridge.

3. Combine the following volumes in a 50 mL centrifuge tube to prepare 49 mL of 0.12% reagent grade NaOCl:

   1. 1.2 mL 5% reagent grade NaOCl.

   2. 48 mL Deionized (DI) water.

4. Invert solution several times to mix thoroughly.

5. Add 49 mL 0.12% NaOCl solution to the bleach well on the wash reagent cartridge.

Setting up the Maintenance Wash (Run Time: ~3 hours)

For NovaSeq X Control Software v1.3 and above:

1. From the Start Menu in the NovaSeq X Series Control Software home screen, select Wash.

For the NovaSeq X Control Software v1.2.2 and below:

1. From the NovaSeq X Series Control Software home screen, select the instrument icon to open the global navigation menu.

2. Select Settings, then select Wash.

3. Select the checkboxes for the instrument side(s) to perform the Maintenance Wash on.

4. If a used 8-Lane sequencing flow cell is still in place from the last sequencing run, the instrument will automatically load the used flow cells and the user can skip to Step 5.

   1. Users can use an 8-lane used sequencing flow cell to perform a maintenance wash only if it remains loaded on the flow cell stage after the previous run. Once the sequencing flow cell is unloaded from the flow cell stage it can no longer be used for a maintenance wash.

   2. If the used 8-lane flow cell has been removed, proceed to step 3.

5. Select the checkbox Load wash flow cells to replace the used sequencing flow cell with aWash flow celland then select the Load wash flow cells button.

• After selecting, the display monitor raises and the flow cell door opens. The flow cell light indicates the instrument side which the wash is being performed.

1. Once the flow cell door has opened, place the wash flow cells in the flow cell stage and select Close flow cell door.

• Wash Flow Cells are currently limited to 20 uses or its expiration date, whichever comes first.

1. On the Wash screen, select Load wash reagent cartridges.

• The instrument doors unlock automatically, and the system displays information on loading the wash reagent and buffer cartridges.

1. Remove the used sequencing reagent cartridge, then load the Wash Reagent Cartridge into the instrument in the right bay of the reagent drawer.

• A previously-used buffer cartridge is required to start a maintenance wash and should be left in place.

1. Empty the used reagent bottles (small and large) then returned the used reagent bottles into the used reagent drawers.

• Dispose of spent reagents according to local EH&S guidelines.

1. Close the reagent drawers, then close the instrument doors.

2. Select Verify wash cartridges.

3. After all wash consumables are verified, select Start wash.

4. After starting the wash, the instrument doors automatically lock. The doors unlock after the wash completes and the instrument returns to the Start screen.

For most sequencing run issues, the instrument will attempt to perform the automatic post-run wash so a maintenance wash will not be required for many run-stopping issues.

For detailed instructions, see the Perform a Maintenance Wash section of the NovaSeq X Plus Product Documentation.

For any feedback or questions regarding this article (Illumina Knowledge Article #7479), contact Illumina Technical Support [e-mail].