Considerations when migrating non-Illumina libraries between sequencing platforms

Sequencing libraries prepared with non-Illumina library preparation methods or kits may require additional optimization on different sequencing platforms. Consider the following differences:

The iSeq 100, MiniSeq, MiSeq, MiSeq i100 Series, NextSeq 500/550, NextSeq 1000/2000, NovaSeq 6000, and NovaSeq X Series flow cells are paired-end flow cells.

• Note: single read sequencing can be performed on these platforms, but paired end type flow cells are a specific flow cell design, distinct from single read flow cell types.

Libraries with paired-end adapter designs can be run as single-read or paired-end sequencing runs, but some older single-read libraries may not sequence successfully on modern paired-end flow cells. As a result, it may not be possible to successfully sequence some libraries developed for single-read applications on the HiSeq 1000/1500/2000/2500 instruments on current sequencing platforms.

For guidance on if a library has a single-read or paired-end adapter design, contact Illumina Technical Support.

Primer Binding and SBS chemistry temperatures

Temperatures used for primer binding, deblocking, and nucleotide incorporation steps can vary between platforms and therefore need to be considered when using custom sequencing primers or primer binding sites.

Platform	Primer Binding	Deblocking	Incorporation
iSeq 100	65° C	63° C	65° C
MiniSeq	60° C	60° C	60° C
MiSeq	65° C	60° C	65° C
MiSeq i100 Series	65° C	56° C	65° C
NextSeq 500/550	60° C	60° C	60° C
NextSeq 1000/2000	60° C	60° C	60° C
NovaSeq 6000	60° C	55° C	60° C
NovaSeq X Series	60° C	60° C	60° C

Clustering efficiency

Libraries can exhibit different clustering efficiencies when migrating from one platform to another. See Loading concentration considerations when migrating Illumina libraries between sequencing platforms for additional information on these variations.

Additional considerations

• Insert sizes more than 550 bp are generally not supported on the MiniSeq or NextSeq 500/550,and may require additional optimization steps.

• Some applications with 550 bp or greater insert sizes are compatible with the NovaSeq 6000 platform, but additional optimization steps may be required.

• Sequencing low diversity libraries can require additional optimization across different instruments.

For any feedback or questions regarding this article (Illumina Knowledge Article #1478), contact Illumina Technical Support [e-mail].