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