Troubleshooting high cluster density on MiSeq

Overclustering leads to poor run performance, lower Q30 scores, the possible introduction of sequencing artifacts, and, counterintuitively, lower total data output. For a MiSeq v2 reagent chemistry, supported cluster density for well-balanced libraries is 1000-1200K/mm2. For MiSeq v3 reagent chemistry, the supported cluster density for well-balanced libraries is 1200-1400K/mm2. For low diversity libraries, Illumina recommends targeting a cluster density 30-40% beneath the optimal range for the chemistry version and platform used. The optimal amount of reduction required must be empirically determined.

Elevated cluster density does not result from instrument issues or sequencing consumable issues.

Best practices for quantification:

• Accurate quantification and proper quality check of next-generation sequencing libraries are key to a successful sequencing run.

• Different methods for quantification and quality control are recommended depending on the sequencing library kit being used.

• Consider the average size of the library. Because the clustering process preferentially amplifies shorter libraries in a mixture of fragments, large libraries tend to cluster less efficiently than smaller libraries.

Best practices for sequencing low diversity libraries:

• Nucleotide diversity is required for effective template generation (cluster mapping) on Illumina sequencing platforms and is important for generating high-quality data.

• Diversity is especially important during the first 4-7 cycles of the first sequencing read for MiSeq systems. The sequencing software uses images from these early cycles to identify the location of each cluster.

• A minimum of 5% PhiX is required for sequencing low base diversity libraries on MiSeq with v3 reagents.

  • The PhiX Control v3 Library has a diverse base composition (45% GC and 55% AT) that provides the balanced fluorescent signals that low diversity sample libraries lack during each sequencing cycle. This, in turn, assists with template registration and improves overall run quality.

• Reduce the library loading concentration to target a cluster density 30-40% beneath the optimal range for the chemistry version and platform used. The optimal amount of reduction required must be empirically determined.

Additional Resources:

Cluster density guidelines for Illumina sequencing platforms using non-patterned flow cells Cluster Optimization Overview Guide

For further information, search for Knowledge Base articles:

• How to achieve more consistent cluster density on Illumina sequencing platform

• How much PhiX spike-in is recommended when sequencing low diversity libraries on Illumina platforms?

• Library quantification and quality control quick reference guide

• Best practices for library quantification

• Library quantification considerations when using qPCR

• Best practices for manually normalizing library concentrations

For any feedback or questions regarding this article (Illumina Knowledge Article #6801), contact Illumina Technical Support [e-mail].