# Failure to find fiducial errors on the NextSeq 1000/2000

**Background**: If the system is unable to detect clusters in the first cycle of imaging, the run may exit with the following error:

"A failure occurred during sequencing, and consumables cannot be reused. Before setting up a new run, power cycle the instrument per system guide instructions. If the error persists, contact Illumina Technical Support."

![](https://761066130-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FGM9W2DuBTgEXv1ClCm8H%2Fuploads%2Fgit-blob-66775b0c5ae6c64c90bc067d6cd9cb672d9e68dc%2Fimage1.jpg?alt=media)

When seen at cycle 1, approximately 4 hours after run start, the most common root cause is a "Failure to find fiducial" error.

The NextSeq 1000/2000 completes an autocenter process in the blue channel at cycle 1 to find initial focus. With Patterned Flow Cell Technology, the NextSeq 1000/2000 uses clustered fiducials for Autocenter. Successful Autocenter images require signal present in blue channel at cycle 1.

![](https://761066130-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FGM9W2DuBTgEXv1ClCm8H%2Fuploads%2Fgit-blob-355b5d4238f9dfd90ff181bb8aa06a16b4dcdded%2Fimage2.jpg?alt=media\&token=a26124ad-aec0-4a26-94ec-979efbdb81c5)

**Troubleshooting**

Library Considerations

* Use provided RSB+Tween 20 to dilute library to 2nM and then to loading concentration
* Confirm library doesn't have a T overcall at cycle 1
  * If running Illumina mRNA stranded library with T over call at cycle 1, a custom recipe is required\
    \- NextSeq 1000/2000 control software version 1.2 and earlier, use the custom recipe found at [NextSeq 1000/2000 Sequencing System Product Files](https://support.illumina.com/sequencing/sequencing_instruments/nextseq-1000-2000/product-files.html)\
    \- NextSeq 1000/2000 control software version 1.3 and later, custom recipe is integrated into instrument cloud run setup

Run Setup

* Confirm protocol for loading library matches what is chosen in runparameters.xml
  * loaded double stranded DNA and false is listed
  * manual denature and dilute and true is listed
* If using custom primers, confirm they are indicated in runparameters.xml

Reagent Preparation

* Reagent thawed and prepared in accordance with recommendations in [NextSeq 1000/2000 Sequencing System Guide](https://support-docs.illumina.com/IN/NextSeq10002000/Content/IN/NextSeq2000_1000/ThawBaggedCartridge_fNS.htm)

After reviewing library, run setup, and reagent preparation, contact Illumina Technical Support for further troubleshooting and provide the following:

1. Collect run cycle logs located at /usr/local/Illumina/runs/\[runfolder]
2. Collect the AutoCenter Images at /usr/local/Illumina/runs/\[runfolder]/Autocenter
3. Contact [Illumina Technical Support](https://www.illumina.com/company/contact-us.html#/united-states/technical-support) for a file transfer link and further troubleshooting

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| *For any feedback or questions regarding this article (Illumina Knowledge Article #3549), contact Illumina Technical Support* [*techsupport@illumina.com*](mailto:techsupport@illumina.com?subject=Question%2FFeedback%20Regarding%20Illumina%20Knowledge%20Article%20#000003549%20-%20Instrumentation%20\&body=Dear%20Illumina%20Technical%20Support,%0D%0A%0D%0A)*.* |