Considerations when migrating non-Illumina libraries between sequencing platforms


All libraries prepared with the current Illumina library preparation kits not requiring a custom sequencing primer are compatible with all Illumina sequencing platforms.

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

  • iSeq 100, MiniSeq, MiSeq, NextSeq 500/550, NextSeq 1000/2000, and NovaSeq flow cells are paired-end — Both single-read and paired-end flow cells are available for the HiSeq platforms.
    Note: Paired-end libraries can be run on single-read flow cells, but single-read libraries may not sequence successfully on paired-end flow cells. As a result, it is not possible to successfully sequence single-read libraries on the iSeq100, MiniSeq, MiSeq, NextSeq 500/550, NextSeq 1000/2000, or NovaSeq.
  • Libraries designed for HiSeq 1000/2000/1500/2500 single-read flow cells may not be compatible with HiSeq 3000/4000 single-read flow cells — Single-read flow cells for the HiSeq 1000/2000/1500/2500 use a different adapter system than flow cells for the HiSeq 3000/4000 platforms (HiSeq X systems only use paired-end flow cells). For this reason, libraries prepared with custom or pre-TruSeq adapters may not be compatible with HiSeq 3000/4000 single-read flow cells. Libraries made with pre-TruSeq adapters remain compatible with HiSeq 1000/2000/1500/2500 single-read flow cells. HiSeq 3000/4000 single-read flow cells are compatible with libraries made from all current Illumina library preparation kits. 
  • 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 primer or primer binding sites.
      Primer Binding Deblocking Nucleotide
    iSeq 100 65°C 63°C 65°C
    MiniSeq 60°C 60°C 60°C
    MiSeq 65°C 60°C 65°C
    NextSeq 500/550 60°C 60°C 60°C
    NextSeq 1000/2000 60°C 60°C 60°C
    HiSeq Rapid Run 65°C 60°C 65°C
    HiSeq High Output v3 60°C 55°C 55°C
    HiSeq High Output v4 60°C 55°C 65°C
    HiSeq 3000/4000* 60°C (R1), 65°C 55°C 60°C
    NovaSeq 60°C 55°C 60°C

    *HiSeq 3000/4000: cBot Read 1 (R1) primer binding at 60°C. Index and Read 2 primers bind at 65°C

  • Clustering efficiency — Libraries can exhibit different clustering efficiencies when migrating from one platform to another. Consult the bulletin, Cluster density 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, NextSeq 500/550, or HiSeq 3000/4000 platforms, and may require additional optimization steps.
    • Some applications with 550 bp or greater insert sizes are compatible with the NovaSeq platform, but additional optimization steps may be required.
    • Current versions of the MiSeq and HiSeq 2500 control software are optimized to run low diversity libraries. MiniSeq, NextSeq 500/550, HiSeq 3000/4000, and NovaSeq software do not. For this reason, running low diversity libraries on these systems requires additional optimization.