Proper preparation and inspection of NovaSeq 6000 flow cells before sequencing can reduce the occurrence of vacuum-related issues and potentially prevent instrument leaks. This bulletin explains how to prepare and inspect NovaSeq 6000 flow cells for successful sequencing. 

Best Practices for NovaSeq flow cell preparation and inspection

1. Remove new flow cell package from 2°C to 8°C storage.
2. Set the sealed flow cell package aside for 10–15 minutes to allow the flow cell to reach room temperature. Use the flow cell within 12 hours of removing it from the package.
3. Inspect the top and bottom surface of the flow cell for any damage such as cracks or chips.
   1. Contact Illumina Technical Support to evaluate the flow cell if chips are found on the bottom surface or cracks are found on either surface.
4. Inspect the flow cell for any particulate material within or nearby flow cell gaskets (see Fig. 1).
   1. If particulate is observed, gently wipe away debris or turn the flow cell over. Then, gently tap the flow cell on counter to remove material.
   2. If particulate obstructing the gasket cannot be removed, contact Illumina Technical Support to discuss whether to proceed with use of the flow cell.
 



Figure 1. Example of material blocking the flow cell gasket.

5. Inspect the flow cell for cosmetic defects. Cosmetic defects can include variable iridescence across the flow cell, circular spots within the flow cell, scratches on the surface or within the flow cell, or chips on the top surface of the flow cell (see Fig. 2). Flow cells go through rigorous visual Quality Control (QC) before customer use. Cosmetic defects such as these rarely affect output or quality, and performance is guaranteed.
 



Figure 2. Variable iridescence across flow cell surface (left). Circular spots within the flow cell (middle). Flow cell scratches (right).

6. Before loading the flow cell on the NovaSeq 6000, inspect the flow cell and NovaSeq 6000 flow cell stage for glass shards or any debris.
   1. Glass shards can impact the vacuum seal needed for successful sequencing. See the Troubleshooting NovaSeq 6000 flow cell loading video for additional details.
   2. Inspect the underside of the flow cell for any glass shards. If glass shards are present, use an alcohol wipe to clean the underside of the flow cell. Then dry the flow cell with a clean, dry lint-free tissue.
   3. Inspect the NovaSeq 6000 flow cell stage including the grooves, vacuum holes, and the manifolds.
      1. If the flow cell stage appears free of debris or glass shards, use an alcohol wipe to clean the flow cell loading position, manifolds, and the flow cell clamp. Remove residual alcohol by wiping the flow cell stage with a clean, dry lint-free tissue.
      2. If debris or glass shards are observed (see Fig. 3), remove the debris by blotting the flow cell stage with an alcohol wipe. After blotting, visually inspect the flow cell stage to confirm that the debris has been removed. Remove any residual alcohol by wiping with a clean, dry lint-free tissue.
 



Figure 3. Example of glass shards observed on the NovaSeq 6000 flow cell stage.

7. Contact Illumina Technical Support with any questions or concerns pertaining to the information outlined in this bulletin. Technical Support can assess whether to proceed with flow cell use. Providing a picture of the flow cell and lot information can help Illumina provide appropriate guidance.

Resources:

• NovaSeq 6000 Sequencing System Guide
• Troubleshooting NovaSeq 6000 flow cell loading