# Troubleshooting MiSeq Read 2 low intensity and quality scores

On the MiSeq platform, paired-end turnaround is performed after the i5 index read and before the start of Read 2. Low intensities and quality scores from the start of Read 2 are typically caused by issues with paired-end turnaround or poor priming Read 2.

Issues with paired end turnaround can be caused by the following root causes:

* Instrument fluidics.
* Reagent cartridge consumable quality.

Issues with Read 2 priming can be caused by the following root causes:

* Instrument fluidics.
* Reagent cartridge consumable quality.
* Library design or custom primers.

**Diagnosing**

The Intensity and % ≥ Q30 Data by Cycle charts will show show drops at the start of Read 2.

![](https://761066130-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FGM9W2DuBTgEXv1ClCm8H%2Fuploads%2Fgit-blob-7ba8de5cdb8d6f4a1f9dc7af2f53290eda8c9701%2Fimage1.jpg?alt=media\&token=ab670d49-e927-4bdc-b881-2a55b2bc5f63)

Figure 1: Low Intensities in the Data by Cycle plot at the start of Read 2.

![](https://761066130-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FGM9W2DuBTgEXv1ClCm8H%2Fuploads%2Fgit-blob-9cf7c5705fa85eafa6f8fd58015309241944696b%2Fimage2.jpg?alt=media\&token=8774586a-ccdc-4972-8f91-8bb43103bdf2)

Figure 2: Low quality scores in the Data by Cycle plot at the start of Read 2.

**Troubleshooting steps:**

1. Once the run completes, complete the instrument post-run wash.
2. Perform the 'Fluidics' section of the MiSeq System Checks using the flow cell from the affected sequencing run.

* Instructions for the MiSeq System Checks are found in the **How to perform a System Check on the MiSeq** Knowledge article.

3. If the 'Fluidics' System Checks are all pass, the MiSeq fluidics system is operating within expectation.

* The root cause of the issue was potentially a consumable related issue, proceed with repeat sequencing run if confident in Library Preparation design, quality, and primers.

4. If the 'Fluidics' System Check fails, perform the following actions:
   1. Power cycle the instrument.
   2. Switch to a trusted flow cell from a previously successful run.
   3. Perform a Maintenance wash.
   4. Repeat the 'Fluidics' system checks.

For further information, search for Knowledge articles:

* **How to shut down or power cycle the MiSeq**
* **Wash best practices for the MiSeq**

If the Fluidics system checks are unable to pass after performing these troubleshooting actions, contact [Illumina Technical Support](https://www.illumina.com/company/contact-us.html#/united-states/technical-support) for further assistance.

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| *For any feedback or questions regarding this article (Illumina Knowledge Article #6633), contact Illumina Technical Support* [*techsupport@illumina.com*](mailto:techsupport@illumina.com?subject=Question%2FFeedback%20Regarding%20Illumina%20Knowledge%20Article%20#000006633%20-%20Instrumentation%20\&body=Dear%20Illumina%20Technical%20Support,%0D%0A%0D%0A)*.* |