⬅️ NGS Handbook
Generating up to 1.5 Tb and 5B reads per run, the HiSeq 3000 / HiSeq 4000 deliver some of the
highest throughputs of any Illumina instrument. Build on the back of HiSeq 2500, HiSeq 3000 and
HiSeq 4000 use patterned flow cell technology originally developed for HiSeq X platforms.
HiSeq 3000 / 4000 run 3 times faster and yield 65% more reads per lane than it's earlier
predecessor. Patterned flow cells contain billions of nanowells at fixed, known positions on the
flow cell. The structured organization enables clustering at higher densities compared to
non-pattern HiSeq designs. See an Illumina video to learn more about
patterned flow cells and kinetic exclusion amplification.
As users on Genohub began ordering services for the HiSeq 3000 / HiSeq 4000 we learned this
innovative patterned technology comes with a cost and definitely introduces limitations that were
otherwise not experienced with the HiSeq 2500.
Search for HiSeq 3000 / 4000 Sequencing Services
HiSeq 3000 / HiSeq 4000 Limitations
- Not recommended for low complexity sequencing. Applications such as non-unique
amplicons, 16S, are currently not recommended. Libraries with low complexity within the first 25
bases of a read are not expected to produce high quality data.
- Library size restrictions. Libraries that are too long can result in polyclonal
clusters that span more than 1 well, these will not pass filter. Smaller libraries will
preferentially amplify with Illumina's new kinetic exclusion amplification so tight library
distributions ranging from 300-500 bp are recommended.
- Very low tolerance for adapter dimers. Even as little as 1% adapter dimer can
take up ~6% of sequencing reads, 10% contamination will take up 84% of reads. Illumina
recommends you keep adapter contamination below 0.5% of your entire library.
- Higher duplication rates as compared to HiSeq 2500.
- Lane bias against lanes 1 and 8, these will have 2-3% fewer reads and or bases
- Low quality read 2 - entire HiSeq 3000 install base is affected - as of January
HiSeq 3000 / HiSeq 4000 Questions & Answers: (updated January 2016)
What types of library preparation are currently supported on the HiSeq 3000 / 4000?
DNA-Seq, RNA-seq , ChIP-Seq, Mate-pair, small RNA and Exome library preparations are currently
compatible applications. Any library preparation where there is enough sequence diversity is
currently supported. Amplicon, 16S and applications with low sequencing diversity are currently not
supported on the HiSeq 3000 / 4000.
What types of multiplexing or barcoding are compatible?
Single and dual indexing options (up to 18 cycles for indexing) are compatible.
What type of read quality is expected from the HiSeq 3000 / 4000 ?
- 2x50: ≥85% bases > Q30
- 2x75: ≥80% bases > Q30
- 2x150: ≥75% of bases >Q30
What are the costs for sequencing on a HiSeq 3000 / 4000 ?