HiSeq 3000 / 4000 Services

⬅️ 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


  1. 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.
  2. 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.
  3. 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.
  4. Higher duplication rates as compared to HiSeq 2500.
  5. Lane bias against lanes 1 and 8, these will have 2-3% fewer reads and or bases at Q30.
  6. Low quality read 2 - entire HiSeq 3000 install base is affected - as of January 2016.

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 ?

  1. 2x50: ≥85% bases > Q30
  2. 2x75: ≥80% bases > Q30
  3. 2x150: ≥75% of bases >Q30


What are the costs for sequencing on a HiSeq 3000 / 4000 ?