Cluster density considerations when migrating Illumina libraries between sequencing platforms

11/28/17


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 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
    The cluster generation process of the patterned flow cells on the HiSeq 3000/4000 and NovaSeq systems differ 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 platforms, and vice versa.
  • From HiSeq 3000/4000 to NovaSeq platforms
    When using NovaSeq Control Software version 1.1 and lower, library types previously optimized on the HiSeq 3000/4000 platform should be loaded on the NovaSeq platform at 1.5x the HiSeq 3000/4000 loading concentration. When using NovaSeq Control Software version 1.2 and higher, libraries previously optimized on the HiSeq 3000/4000 platform should be loaded at the same concentration on the NovaSeq platform. Optimize subsequent loading concentrations based on the run’s pass filter percentage and percent duplicate metrics from the secondary analysis.

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