Loading concentration considerations when migrating Illumina libraries between sequencing platforms

Due to differences in chemistry and hardware, libraries exhibit different clustering efficiencies on different Illumina sequencing platforms. Migrating libraries between platforms requires instrument-specific optimization of cluster density.

Listed are guidelines for determining the loading concentration of a library that is being migrated to a different Illumina sequencing platform:

• Between MiniSeq and NextSeq 500/550 platforms

  • The MiniSeq and NextSeq 500/550 platforms use similar chemistries for cluster generation. As a result, the same library can be expected to cluster at a similar density.

• Between MiSeq and MiniSeq/NextSeq 500/550 platforms

  • The MiniSeq and NextSeq 500/550 platforms require significantly lower cluster densities than the MiSeq platform. Therefore, it is important to redo cluster density optimization when migrating a library from MiSeq to MiniSeq or NextSeq 500/550 platform and vice versa.

• From iSeq 100 to NovaSeq 6000

  • Prior to performing in-depth sequencing of the library on the NovaSeq 6000 system, shallow sequencing on the iSeq 100 can provide a rapid and cost-saving quality check; refer to the Sequencing Library QC with the iSeq System application note and Step-by-step instructions for sequencing library QC with the iSeq 100 System support bulletin. When quantifying the library before sequencing on NovaSeq 6000, rebalancing the library pool based on the actual iSeq 100 reads is as consistent as using the qPCR data.

  • Optimize loading concentration based on the run’s percent occupancy and the percent pass filter; refer to the Plotting %Occupied by %PF to optimize loading for the NovaSeq 6000 and X, MiSeq i100, and iSeq 100 Illumina Knowledge Article. The percentage of duplicate metrics from the secondary analysis also help optimize the loading concentration; refer to the Cluster Optimization Overview.

• From NovaSeq 6000 to NovaSeq X Series

  • For transitioning projects from the NovaSeq 6000 System to the NovaSeq X Series, center titrations at ~30% of the NovaSeq 6000 System loading concentration for the standard onboard clustering workflow. See Maximizing performance on the NovaSeq X Series for more information.

• From MiSeq to MiSeq i100

  • For transitioning projects from the original MiSeq System using the MiSeq Reagent Kit v3 to the MiSeq i100 Series, center titrations at ~6.5× the MiSeq Reagent Kit v3 loading concentration.

  • Recommended center point concentrations vary for different library preparation kits for use with the MiSeq i100 Series, see Maximizing performance on the MiSeq i100 Series (Table 1). For all other cases, it is recommended to use 100 pM for the center point concentration.

  • For additional information, see Knowledge Article Loading Concentration Optimization Recommendations for the MiSeq i100 Series for additional detail.

For further guidance on library loading concentrations across Illumina systems, refer to the PhiX loading concentrations for validation runs on Illumina sequencing platforms article.

For any feedback or questions regarding this article (Illumina Knowledge Article #1509), contact Illumina Technical Support [email protected].