Cluster density considerations when migrating Illumina libraries between sequencing platforms

06/05/20


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 HiSeq™ and MiSeq™ platforms
    Cluster generation on the MiSeq platform and HiSeq platform in rapid run mode typically results in approximately 15%–20% higher cluster density when compared to the same library at the same concentration clustered on a HiSeq High Output flow cell.
     
  • 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 HiSeq/MiSeq and MiniSeq/NextSeq 500/550 platforms
    The MiniSeq and NextSeq 500/550 platforms require significantly lower cluster densities than the HiSeq and MiSeq platforms. Therefore, it is important to redo cluster density optimization when migrating a library from MiSeq or HiSeq to MiniSeq or NextSeq 500/550 platform and vice versa.
     
  • To or from HiSeq 3000/4000 and NovaSeq 6000
    The cluster generation process of the patterned flow cells on the HiSeq 3000/4000 and NovaSeq 6000 systems differs from non-patterned flow cell systems. Therefore, cluster density optimization must be performed when migrating a library from any other system to the HiSeq 3000/4000 or NovaSeq 6000 platforms, and vice versa.
     
    When using NovaSeq Control Software version 1.2 and higher, libraries previously optimized on the HiSeq™ 3000/4000 or HiSeq™ X platforms are loaded at the same concentration for the XP workflow and are loaded at 1.5x that loading concentration for the Standard workflow on the NovaSeq 6000 platform. Optimize subsequent loading concentrations based on the pass filter percentage from primary metrics and percent duplicate metrics from the secondary analysis. Refer to the Cluster Optimization Overview Guide and the Plotting % Occupied by % Pass Filter to optimize loading concentration for NovaSeq 6000 and iSeq 100 platforms support bulletin.
     
  • 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 reads is as consistent as using the qPCR data.
     
    In addition, for the NovaSeq Xp workflow, the library loading concentration is expected to be 2.2x–2.5x the concentration of the iSeq 100 system, and for the standard NovaSeq 6000 workflow, 3.3x–4x the concentration of the iSeq 100. Optimize subsequent loading concentration based on the run’s percentage occupancy and the percentage pass filter; refer to the Plotting % Occupied by % Pass Filter to optimize loading concentration for NovaSeq 6000 and iSeq 100 platforms support bulletin. The percentage duplicate metrics from the secondary analysis also helps optimize the loading concentration; refer to the Cluster Optimization Overview Guide.

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