Illumina HiSeq 3000/4000

HiSeq 3000/4000 is a sequencer released by Illumina in January 2015. It is based on Sequencing by Synthesis (SBS).

Illumina HiSeq 3000/4000
Photo of HiSeq 4000: by Illumina


Manufacturer Illumina
Technology Sequencing by Synthesis (SBS)
Release date January 2015
Price [2] $900,000
MSRP of HiSeq 4000 as of release date
Type Industrial
Weight 225.9 Kg
Supports real-time analysis No
Demultiplexing support Yes
File format FASTQ
Read length Illumina HiSeq 3000/4000: 1x50bp to 2x150bp
Run time Illumina HiSeq 3000/4000: 24 to 84 hours
Reagent cost per Gb [1] Illumina HiSeq 3000/4000: $8.98
Data quality [3] Illumina HiSeq 3000/4000: ≥ 85% bases above Q30
Maximum output per run (Gb)[4] Illumina HiSeq 3000/4000: 1500.00
Primary error type [5] Substitutions
Notes about the specifications

  1. Cost per Gb: The cost per Gb is calculated by dividing the costs of sequencing reagents by the manufacturer’s maximum output per run. For these runs, the manufacturer may have included their own controls and used specific library preparation kits. Actual performance may vary based on sample and library type and quality, loading concentration, and other experimental factors. The sequencing reagent costs used for calculations do not include any other costs associated with preparing the samples or running the instrument, including reagents used for extraction, quality control or library preparation, labor, electricity, lab consumables, storage, etc. Performance metrics and prices are subject to change.
  2. Price: The final price may be different depending on the supplier, the region and any discounts provided through bulk orders or negotiation. Other factors that may influence the final price are warranty options, maintenance contracts, possible trade-ins of older instruments, and any financing arrangements chosen.
  3. Data quality: This is the maximum data quality that the instrument can offer based on manufacturer’s specifications and optimizations. The quality of a specific run will depend on multiple factors, including sample quality, sample quantity, library prep protocol, run conditions, etc.
  4. Maximum output: This is the maximum output of a full instrument run. This number is based on manufacturer’s specifications and optimizations.
  5. Primary error type: This is the most common type of error that may occur due to sequencing. Other types of error may occur as well.

More about the HiSeq 3000/4000

The Illumina HiSeq 3000/4000 sequencing systems represented a significant step in high-throughput next-generation sequencing (NGS) capabilities. They were designed for mid- to large-scale sequencing projects where substantial data output is needed, but the highest output platforms like the Illumina NovaSeq 6000 are not necessary. These instruments succeeded the popular HiSeq 2500, offering improved speed, data quality, and cost-effectiveness.

It’s important to note that the Illumina HiSeq 3000/4000 has been discontinued, superseded by more powerful instruments like the NovaSeq 6000. However, these systems may still be found in operation in some laboratories.

Key Innovations

The HiSeq 3000/4000 platforms incorporate several advancements over their predecessor:

  • Patterned Flow Cells: These flow cells have billions of nanowells positioned at fixed locations, unlike the random clustering on previous HiSeq systems. This structured design increases cluster density, allowing more sequencing data to be generated per run.
  • Faster Imaging and Computing: Enhanced camera systems and powerful onboard computers significantly reduce run times compared to the HiSeq 2500, translating into faster time-to-results.

Features and Technologies

  • Sequencing Chemistry: The HiSeq 3000/4000 uses Illumina's proven Sequencing by Synthesis (SBS) chemistry. This involves fluorescently labeled nucleotides, ensuring high accuracy and reliable base calling.
  • Read Lengths: Supports standard 150 bp paired-end reads, sufficient for many common NGS applications.
  • Data Output: Capable of generating up to 1.5 terabases (Tb) of data per run, enabling sequencing of multiple human genomes in a single run.
  • Run Times: Runs are completed in approximately 3-4 days, significantly faster than the HiSeq 2500 high-output mode.

Intended Uses and Specific Applications

The HiSeq 3000/4000 is a versatile platform suited for various NGS applications:

  • Whole Genome Sequencing (WGS): Ideal for deep-coverage sequencing of human or other complex genomes. Suitable library prep kits include Illumina TruSeq DNA PCR-Free and TruSeq DNA Nano.
  • Exome Sequencing: Cost-effective targeted sequencing of protein-coding regions. Library prep options include Illumina Nextera DNA Flex and various capture-based kits.
  • RNA Sequencing (RNA-Seq): Reveals gene expression patterns and transcriptome analysis. Compatible with kits like the Illumina TruSeq Stranded mRNA.
  • Other Applications: Targeted sequencing, ChIP-Seq, microbial sequencing, and more.

Comparison with Competing Instruments

  • Illumina NovaSeq 6000: The NovaSeq 6000 offers much higher throughput (up to 3 Tb per run) at faster speeds and flexible run configurations. It's better suited for larger, population-scale projects. The HiSeq 3000/4000 may be a more practical choice for labs that don't require that level of output.
  • Complete Genomics DNBSEQ-G400: This platform uses DNA nanoball (DNB) sequencing technology. It provides less data output to the HiSeq 3000/4000, although it can reach longer read lengths and can offer shorter run times, especially if the data output needed isn’t high.

Special Considerations

The HiSeq 3000/4000 is a good choice for laboratories that:

  • Need high throughput but not the highest levels of output offered by platforms like the NovaSeq.
  • Want faster run times and higher data density than was possible with the HiSeq 2500.
  • Favor the Illumina SBS chemistry for its well-established accuracy and reliability.
  • Still have access to the reagents needed, as Illumina discontinued support on March 31, 2024.

Need sequencing done on a Illumina HiSeq 3000/4000 or a specific instrument? Tell us about your project here, and we'll connect you with a qualified provider.

This information on this page is up-to-date as of April 22, 2024 and based on the spec sheets published by the manufacturer.