Illumina HiSeq 2500

HiSeq 2500 is a sequencer released by Illumina in June 2012. It is based on Sequencing by Synthesis (SBS).

Illumina HiSeq 2500
Photo of HiSeq 2500: by Illumina

HiSeq 2500 is available in the following models:
  • HiSeq High-Output v3
  • HiSeq High-Output v4
  • HiSeq Rapid Run


Manufacturer Illumina
Technology Sequencing by Synthesis (SBS)
Release date June 2012
Price [2] Discontinued
Type Benchtop
Weight 221.4 Kg
Supports real-time analysis No
Demultiplexing support Yes
File format FASTQ
Read length Illumina HiSeq High-Output v3: 1x36bp to 2x125bp
Illumina HiSeq High-Output v4: 1x36bp to 2x125bp
Illumina HiSeq Rapid Run: 1x36bp to 2x250bp
Run time Illumina HiSeq High-Output v3: 29 to 120 hours
Illumina HiSeq High-Output v4: 29 to 144 hours
Illumina HiSeq Rapid Run: 7 to 60 hours
Reagent cost per Gb [1] Illumina HiSeq High-Output v3: Unavailable
Illumina HiSeq High-Output v4: Unavailable
Illumina HiSeq Rapid Run: Unavailable
Data quality [3] Illumina HiSeq High-Output v3: ≥ 85% of bases above Q30
Illumina HiSeq High-Output v4: ≥ 85% of bases above Q30
Illumina HiSeq Rapid Run: ≥ 85% of bases above Q30
Maximum output per run (Gb)[4] Illumina HiSeq High-Output v3: 800.00
Illumina HiSeq High-Output v4: 1000.00
Illumina HiSeq Rapid Run: 300.00
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 2500

The Illumina HiSeq 2500 was a pivotal next-generation sequencing (NGS) system known for its high throughput and efficient generation of massive sequencing datasets. Designed for large-scale, production-level projects, the HiSeq 2500 revolutionized applications like whole-genome sequencing and exome sequencing. While Illumina has discontinued the model in favor of newer platforms, the HiSeq 2500 remains a powerful option, often available on the secondary market.

Key Innovations

The HiSeq 2500 improved upon earlier models with several key features:

  • Patterned Flow Cells: Previous Illumina sequencers used random clustering on flow cells, leading to uneven density and reduced sequencing efficiency. The HiSeq 2500 introduced patterned flow cells, guiding DNA clusters into pre-defined positions. This maximized cluster density and data output.
  • Four-Channel SBS Chemistry: Sequencing-by-Synthesis (SBS) involves adding labeled nucleotides and measuring fluorescence to determine DNA sequences. The HiSeq 2500 used four-channel SBS chemistry, where four fluorescent nucleotides competed for binding at a given cycle.

Features and Technologies

  • Flexibility: The HiSeq 2500 offered the choice of 'Rapid Run' and 'High Output' modes. Rapid Run mode delivered results in as little as 27 hours for a human genome at 30x coverage, while High Output mode maximized data generation.
  • SBS Chemistry: Illumina's trusted SBS chemistry underpins HiSeq 2500 function, ensuring high base-calling accuracy and reliable read quality.
  • Data Output: In High Output mode, the HiSeq 2500 could produce up to 1Tb of data per run.

Intended Uses and Specific Applications

The HiSeq 2500 excelled in several large-scale NGS applications:

  • Whole Genome Sequencing (WGS): For human genomes, population-level studies, and more. (Library prep kits: Illumina TruSeq DNA PCR-Free, Illumina TruSeq Nano).
  • Exome Sequencing: To analyze protein-coding regions of the genome. (Library prep kits: Illumina Nextera DNA Flex, Roche KAPA HyperExome)
  • RNA Sequencing (RNA-Seq): For gene expression profiling and transcriptome analysis (Library prep kits: Illumina TruSeq RNA, NEBNext Ultra II RNA)

Comparison with Competing Instruments

Here's how the HiSeq 2500 compares to similar-output systems:

  • Illumina HiSeq X: HiSeq X series instruments exceeded the HiSeq 2500 in raw data output, and had similar average run time to the high-output mode. However, the HiSeq X did not have as much flexibility as the HiSeq 2500 with its different read lengths and run modes.
  • Element Biosciences AVITI: The AVITI offers longer read lengths for similar run times and similar max output to the HiSeq’s rapid run mode. However, the HiSeq still has the high output mode for greater flexibility.
  • DNBSEQ-G400: The DNBSEQ-G400 can offer longer read lengths with slightly faster run times on average, especially when compared to the HiSeq 2500’s high output mode. However, the HiSeq 2500 still offers higher max output.

Special Considerations

When deciding if the HiSeq 2500 is the right NGS solution, consider:

  • Project scale: The HiSeq 2500 is best suited to those with consistent demand for high volumes of sequencing data.
  • Cost: While the HiSeq 2500 may now be available at lower prices on the secondary market, reagent costs remain consistent with Illumina's SBS chemistry.
  • Support: As a discontinued system, support and software updates for the HiSeq 2500 may become less readily available over time.

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This information on this page is up-to-date as of April 18, 2024 and based on the spec sheets published by the manufacturer.