How to troubleshoot unable to find patterned fiducials errors on the HiSeq 3000/4000 platforms

Patterned flow cell fiducials are populated with clusters during cluster generation, and these fiducials are then used by the optical system to properly align the flow cell prior to the first cycle of imaging. When the instrument is unable to find these patterned fiducials at the start of the run, it gives the error “Unable to find patterned fiducials or alternate patterned fiducials,” and the run will stop.

The most frequent causes of these errors are:* The flow cell is not sitting flush against the guide pins

• Poor delivery of first base of chemistry

• Poor Read 1 primer hybridization

• Poor ExAmp clustering of the library on the cBot

The most common cause of these errors is a flow cell seating issue. To correct a potential flow cell seating issue, the flow cell must be loaded as a new run.

1. From the Welcome screen in HiSeq Control Software (HCS) select Sequence > New Run.

2. If reloading the flow cell on the same side, HCS will require a water wash before a new run is set up. Alternatively, the flow cell may be moved to the other side of a HiSeq 4000 instrument, or to a different HiSeq 3000/4000 instrument.

3. When prompted by HCS, select the option to prime the SBS reagents over a wash flow cell. This step must be performed before loading the sequencing flow cell.

4. Clean the sequencing flow cell and the HiSeq stage with a moist lab tissue.

5. Reseat the flow cell securely against the guide pins on the top and right sides of the flow cell. Do not keep your finger in contact with the flow cell when engaging the vacuum as it can cause alignment issues.

6. If a solid green light is displayed on the vacuum lever when moved to position 2, proceed with run setup.

7. Verify flow across the sequencing flow cell by pumping SBS reagent from positions 5 or 6 using the default settings.

8. If proper flow is confirmed, start the run.

If another “Unable to find patterned fiducials or alternate patterned fiducials” error occurs after starting a new run, it could indicate an issue with clustering, primer hybridization, or an instrument issue. Contact Illumina Technical Support for further troubleshooting assistance.

For any feedback or questions regarding this article (Illumina Knowledge Article #1346), contact Illumina Technical Support [e-mail].