# Clustering failures on the iSeq 100

**Overview**

A clustering failure on the iSeq 100 can be caused by several potential root causes including the instrument, consumables, or library pool. As such, all three aspects must be assessed during review to ensure the best chance for successful subsequent runs.

**When a Clustering Failure Occurs:**

Confirm Clustering Failure: Review the thumbnail images located at D:\Illumina\iSeq Runs\[RunFolder]\Thumbnail\_Images\L001. Confirm whether vertical black and white striping is present, as this is indicative of an authentic clustering failure (Figure 1).

![](/files/snkeHLtE49rtRknta76M)

**Figure 1.** Thumbnail\_Images showing good clustering and a clustering failure.

**Investigate Library, Reagent Handling and Instrument Root Causes**

* **Library:** Confirm double-stranded DNA was loaded. Review library quality control (QC) and quantification, dilution calculations and ensure the library was loaded at expected Loading Concentration. For further information, search for Knowledge Base article [Library loading concentration considerations for the iSeq 100](/instrumentation/iseq-100/instrumentation-iseq-100-reference_material-list/000002121.md).
* **Reagent Handling:** Confirm cartridge was thawed and prepared properly, see Knowledge Base Article [How to thaw and store sequencing reagents for optimal performance](/instrumentation/general/instrumentation-general-reference_material-list/000001533.md).
* **Instrument:** Confirm instrument performance by running the system checks with the iSeq 100 Reusable Test Kit. For further information, search for Knowledge Base article [Running a System Check on an iSeq 100](/instrumentation/iseq-100/instrumentation-iseq-100-reference_material-list/000002298.md).
  * If the System Check passes and there are no issues identified with the library pool, re-sequence the library pool with a new flow cell and reagent cartridge.\
    \- If successful, the consumables from the first run may be suspected. Contact Illumina Technical Support (<techsupport@illumina.com>) for warranted replacements. Provide the RunParameters.xml files found at D:\Illumina\iSeq Runs\[RunFolder] from both the affected run as well as the successful re-run.\
    \- If unsuccessful, collect the Thumbnail\_Images folder, RunParameters.xml file from both runs (D:\Illumina\iSeq Runs\[RunFolder]), and the System Check results (D:\Illumina\iSeq System Checks\SystemCheckReport.\[date/time]), contact [Illumina Technical Support](https://www.illumina.com/company/contact-us.html#/united-states/technical-support) to request a file share link to upload the requested files. Also provide the library type (and library preparation kit name, if appropriate), library final loading concentration, and the percentage of PhiX added to the pool.

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| *For any feedback or questions regarding this article (Illumina Knowledge Article #1374), contact Illumina Technical Support* [*techsupport@illumina.com*](mailto:techsupport@illumina.com?subject=Question%2FFeedback%20Regarding%20Illumina%20Knowledge%20Article%20#000002298%20-%20Instrumentation%20\&body=Dear%20Illumina%20Technical%20Support,%0D%0A%0D%0A)*.* |


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