Recommendations for optimal cluster density and PhiX loading concentrations on the MiSeq System

Cluster density is an important factor in optimizing sequencing data quality and yield. The list below includes the recommended raw cluster densities for balanced libraries (such as the PhiX control library, or other balanced/base diversity libraries like whole genome libraries).

MiSeq Reagent Kit v2: Standard; Micro and Nano kits

• Optimal Cluster density: 1000 - 1200 K/mm2 (for a well-balanced library).

• PhiX loading concentration: 12.5 pM.

MiSeq Reagent Kit v3: Standard kit

• Optimal Cluster density: 1200 - 1400 K/mm2 (for a well-balanced library).

• PhiX loading concentration: 20.0 pM.

Best practices for sequencing low diversity libraries:

• When running low diversity or unbalanced libraries, Illumina recommends reducing the library loading concentration to target a cluster density 30-40% below the optimal range for the chemistry version and platform used. The optimal amount of reduction required must be empirically determined.

• To compensate for low base diversity in libraries, Illumina recommends spiking in a minimum of 5% PhiX Control v3 Library for sequencing when using MiSeq v3 chemistry or 20% when using MiSeq v2 chemistry. PhiX has a diverse base composition (45% GC and 55% AT) that provides the balanced fluorescent signals that low diversity libraries lack during each sequencing cycle.

For further information regarding PhiX on other Illumina sequencers, search for Knowledge Base article How much PhiX spike-in is recommended when sequencing low diversity libraries on Illumina platforms?

For any feedback or questions regarding this article (Illumina Knowledge Article #1676), contact Illumina Technical Support [e-mail].