Questions & Answers

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  • What is the throughput of the HiSeq 4000 system?

    Each system can generate up to 1.5 Tb in 3.5 days with greater than 75% of bases above Q30 from a 2 x 150 bp run. This throughput enables up to 12 genomes at 30x per run per system. For more information, see HiSeq 3000/4000 System Specifications.

  • Can I upgrade my HiSeq 2500 or earlier model to a HiSeq 4000?

    No. An upgrade package (catalog # SY-401-4002) is available for HiSeq 3000 only.


  • Can custom primers be used on the HiSeq 4000?

    Custom primers have not been tested for use on the HiSeq 4000.


  • What type of library prep is supported on the HiSeq 4000?

    Like the HiSeq 2500, the HiSeq 4000 is designed as an open platform intended to support a broad array of applications. Internally, Illumina has run numerous library prep kits without issue. For sample data demonstrating system performance, see the HiSeq 3000/4000 Sample Datasheets.

    The HiSeq 4000 system is compatible with current paired-end P5 and P7 primers. Single-read and paired-end libraries are compatible with the HiSeq 4000, but must use current adapters. Legacy libraries that are based on the original Illumina single-read adapter do not cluster properly.

  • Can I process a low-diversity sample on a HiSeq 4000?

    True low diversity samples, such as single amplicon and 16S samples, are not expected to produce quality results. However, spiking in a well-balanced genome such as PhiX improves performance.

Reagents and Flow Cells

  • What is a patterned flow cell?

    A patterned flow cell is a flow cell with billions of ordered nano-wells that are manufactured into the flow cell glass. The ordered wells allow for the generation of sequencing clusters in an ordered arrangement. Clusters are aligned more closely together increasing the number of output reads and amount of sequence data generated. The HiSeq 3000/4000 patterned flow cell contains 8 lanes and has the same general dimensions as a HiSeq high-output flow cell.

  • What flow cells and reagents are supported on the HiSeq 4000?

    Reagents and flow cells provided in the HiSeq 3000/4000 SBS Kit and the HiSeq 3000/4000 Cluster Kits are designed and optimized for the HiSeq 4000 and HiSeq 3000 systems. Reagent kits designed for other HiSeq systems are not compatible with the HiSeq 4000 and HiSeq 3000 systems.


  • What sequencing read length is supported on the HiSeq 4000?

    Supported read lengths for the HiSeq 4000 are up to 2 x 150 bp. Three kit sizes (50-cycle, 150-cycle, and 300-cycle) are available to support a range of read lengths.

  • Do I need a cBot to cluster HiSeq 3000/4000 flow cells for sequencing on my HiSeq 4000?

    Yes, a cBot is needed for cluster generation on any 8-lane high output flow cell, including the HiSeq 3000/4000 patterned flow cell. On-instrument clustering is a feature of Rapid Run mode, which is available only on the HiSeq 2500 and HiSeq 1500. You can use any cBot running cBot software v3.0 or later to cluster a HiSeq 3000/4000.

  • What indexing options are available on the HiSeq 4000?

    The HiSeq 3000/4000 reagent kits support single and dual indexing options. SBS kits includes sufficient reagent for 25 additional cycles for indexed sequencing. The indexing primer provides primers for both single and dual indexing.

  • Is the indexing primer for the paired-end flow cell compatible with the single-read flow cell?

    No, indexing primers are specific to the SR or PE cluster kit because the indexing workflow is different for each flow cell type. For details on indexing workflows, see the NextSeq, MiSeq, and HiSeq Systems Indexed Sequencing Guide (part # 15057455).

  • Does paired-end resynthesis happen before the Index 2 Read for dual-indexing?

    Yes for a paired-end flow cell; no for a single-read flow cell. The dual-indexed workflow differs depending on flow cell type. For details on each workflow, see the NextSeq, MiSeq, and HiSeq Systems Indexed Sequencing Guide (part # 15057455).

  • How long does a maintenance wash take?

    Actual wash time for a maintenance wash is approximately 1.5 hours, not including flow check time.

  • How often do wash bottles and tubes need to be renewed?

    Monthly replacement of wash bottles and tubes containing maintenance wash solution is typically sufficient. Wash bottles and tubes containing water are typically replaced every 6 months, although the water is replaced about every week.


  • What control software version is compatible with current kits and library prep methods?

    Use HiSeq Control Software v3.3 and Real-Time Analysis v2.3, or later. Previous versions of HiSeq Control Software and Real-Time Analysis are not compatible.

  • Can I use Sequencing Analysis Viewer (SAV) to view data from a HiSeq 4000 run?

    Yes. SAV 1.8.46 or later is required to view data from a HiSeq 4000 run. You can download SAV for use on a computer or use BaseSpace.

    For the version of SAV compatible with your version of HCS, see the HCS release notes.

  • What is the typical run folder size?

    The run folder size for the maximum read length of 2 x 150 is about 0.6 TB.


  • What data quality can I expect from a run on the HiSeq 4000?

    You can expect greater than 75% of all bases above Q30 with a 2 x 150 bp run. For more information, see HiSeq 3000/HiSeq 4000 System Specifications.

  • What are my options for analyzing HiSeq 4000 data?

    BaseSpace is the preferred analysis solution for the HiSeq 4000. For third-party analysis packages, you can use the bcl2fastq converter. CASAVA and HAS are not supported.

  • If a network outage to the central file server storing our runs occurs, can the HiSeq 4000 cache an entire 2 x 150 run?

    Yes. The server storage size can store 1 run if there is network storage. Real-Time Analysis continues processing and resumes data transfer when the network is restored.

  • How many tiles are imaged on a HiSeq 3000/4000 flow cell?

    Scanning and analysis of a HiSeq 3000/4000 flow cell is performed in 2 swaths per surface on 2 surfaces per lane. Each swath is divided into 28 tiles. Therefore, each flow cell contains 896 tiles.