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

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.

Wash Buffer Preparation

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 reservoir 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)

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 7.

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

5. To load a Wash Flow Cell, select the checkbox next to Load wash flow cells 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, load the wash flow cells onto the flow cell holder and select Close flow cell door.

• Wash Flow Cells are currently limited to 20 uses or 15 months from manufacture, whichever comes first.

1. On the Wash screen, select Load wash 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 then replace the used reagent bottles into the used reagent drawers.

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

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

3. Select Verify wash cartridges.

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

5. 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].