HiSeq 3000 FAQs

Each system can generate up to 750 Gb in 3.5 days with greater than 75% of bases above Q30 from a 2 x 150 bp run. This throughput enables up to 6 genomes at 30x per run per system. For more information, see HiSeq 3000/4000 System Specifications.

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

Custom primers have not been tested for use on the HiSeq 3000 System.

The HiSeq 3000 is designed as an open platform intended to support a broad array of applications. To date, validated applications are whole genome sequencing, RNA sequencing, and exome sequencing. For more information, see HiSeq 3000/HiSeq 4000 Applications.

Like the HiSeq 2500 System, the HiSeq 3000 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 3000 system is compatible with current paired-end P5 and P7 primers. Single-read and paired-end libraries are compatible with the HiSeq 3000, but must use current adapters. Legacy libraries that are based on the original Illumina single-read adapter do not cluster properly.

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.

A patterned flow cell has billions of ordered nanowells 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 has eight lanes and the same general dimensions as a HiSeq high-output flow cell.

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.

Supported read lengths for the HiSeq 3000 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.

Yes, a cBot is needed for cluster generation on any 8-lane high output flow cell, including the HiSeq 3000/HiSeq 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 systems. You can use any cBot running cBot software v3.0 or later to cluster a HiSeq 3000/4000.

The HiSeq 3000/4000 reagent kits support single and dual indexing options. SBS kits include sufficient reagent for 18 additional cycles of indexed sequencing. The indexing primer provides primers for both single and dual indexing.

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 Indexed Sequencing Overview Guide (document # 15057455).

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 Indexed Sequencing Overview Guide (document # 15057455).

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

Yes. SAV 1.8.46 or later is required to view data from a HiSeq 3000 run. You can download SAV for use on a computer or use BaseSpace Sequence Hub.

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

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.

BaseSpace is the preferred analysis solution for the HiSeq 3000. For third-party analysis packages, you can use the bcl2fastq converter. CASAVA and HAS are not supported.

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

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