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.

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

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.

1. 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.

1. 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 for further assistance.

For any feedback or questions regarding this article (Illumina Knowledge Article #6633), contact Illumina Technical Support [e-mail].