Considerations when migrating non-Illumina libraries between sequencing platforms

08/23/18


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:

  • iSeq100, MiniSeq, MiSeq, NextSeq, and NovaSeq flow cells are paired-end — Both single-read and paired-end flow cells are available for the HiSeq platforms. All iSeq100, MiniSeq, MiSeq, NextSeq, and NovaSeq flow cells are paired-end only.
    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, 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. 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
    Incorporation
    iSeq 100 65°C 63°C 65°C
    MiniSeq 60°C 60°C 60°C
    MiSeq 65°C 60°C 65°C
    NextSeq 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 on HiSeq 3000/4000

  • 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, 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, HiSeq 3000/4000, and NovaSeq software do not. For this reason, running low diversity libraries on these systems requires additional optimization.