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
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.
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
- 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.
applications with 550 bp or greater insert sizes are compatible
with the NovaSeq platform, but additional optimization steps may
- 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