Troubleshooting no intensity for index read on MiSeq

If an index read has no intensity for the entire read, or if MiSeq Control Software ends the run with an error that it cannot find clusters or focus at the first cycle of the index read, this is typically caused by incompatibility between the library type and the index sequencing primers. If the index sequencing primer cannot bind to the library, no intensity will be generated during Sequencing-by-Synthesis (SBS) chemistry. Review the troubleshooting steps below to determine the root cause of the issue.

Troubleshooting steps:

1. Sample not indexed

• Some sequencing libraries do not contain an index in the standard position(s). If there is no index in the adapters used to construct a sequencing library, there will be no intensity for the index read. Libraries built with the older Genomic DNA Sample Preparation Kit, Paired-End DNA Sample Preparation Kit, or mRNA-Seq Sample Preparation Kit will have no index. Illumina PhiX libraries have no index. All current Illumina library preparation kits build libraries with on or two indexes.

• If there is no intensity for the index read, this may result in software errors, loss of focus, or poor quality base calls that do not reflect the library. In most cases, the run will continue through the index read, and the results of the index read can be ignored.

1. Custom Library

• It is possible for libraries constructed with nonstandard preparation protocols to fail to provide sufficient intensity for high-quality sequencing. Nonstandard library preparation protocols often require optimization to be compatible with Illumina sequencers. The use of custom sequencing primers may also require optimization on different instruments.

1. Custom sequencing primers

• Some custom libraries do require custom sequencing primers. If a custom sample preparation typically works on the MiSeq but does not work for a specific run, this might be because custom sequencing primers were not used or were not specified in the sample sheet.

• Indicating "Custom Index Sequencing Primers" during run setup will cause the instrument to deliver the Index primers from a different location in the reagent cartridge than normal. If the run does not require Custom Primers, or if primers were loaded into the incorrect location, this may also result in intensity issues during index reads.

1. Delivery issues during index chemistry

• The instrument may have failed to properly deliver the index sequencing primer. Run a conduct volume test to confirm proper fluidic functionality.

For further information, search and review the following Knowledge Base articles:

• How to Perform a System Check on the MiSeq

• Considerations when migrating - non-Illumina libraries between sequencing platforms

• Sequencing primer compatibility of Illumina libraries and recommended library kit for the MiSeq

For any feedback or questions regarding this article (Illumina Knowledge Article #1461), contact Illumina Technical Support [e-mail].