Illumina MiSeq

MiSeq is a sequencer released by Illumina in January 2011. It is based on Sequencing by Synthesis (SBS).

Illumina MiSeq
Photo of MiSeq: by Illumina

MiSeq is available in the following models:
  • MiSeq
  • MiSeq v2
  • MiSeq v2 Micro
  • MiSeq v2 Nano
  • MiSeq v3


Manufacturer Illumina
Technology Sequencing by Synthesis (SBS)
Release date January 2011
Price [2] $99,000
MSRP as of March 2024
Type Benchtop
Weight 57.2 Kg
Supports real-time analysis No
Demultiplexing support Yes
File format FASTQ
Read length Illumina MiSeq: 1x36bp to 2x250bp
Illumina MiSeq v2: 1x36bp to 2x250bp
Illumina MiSeq v2 Micro: 2x150bp
Illumina MiSeq v2 Nano: 2x150bp to 2x250bp
Illumina MiSeq v3: 2x75bp to 2x300bp
Run time Illumina MiSeq: 4 hours to 39 hours
Illumina MiSeq v2: 4 hours to 39 hours
Illumina MiSeq v2 Micro: 19 hours
Illumina MiSeq v2 Nano: 17 to 28 hours
Illumina MiSeq v3: 21 to 56 hours
Reagent cost per Gb [1] Illumina MiSeq: $179.76
Illumina MiSeq v2: $179.76
Illumina MiSeq v2 Micro: $475.83
Illumina MiSeq v2 Nano: $908.00
Illumina MiSeq v3: $132.60
Data quality [3] Illumina MiSeq: > 90% bases higher than Q30
Illumina MiSeq v2: > 90% bases higher than Q30
Illumina MiSeq v2 Micro: > 90% bases higher than Q30
Illumina MiSeq v2 Nano: > 90% bases higher than Q30
Illumina MiSeq v3: > 85% bases higher than Q30
Maximum output per run (Gb)[4] Illumina MiSeq: 8.50
Illumina MiSeq v2: 8.50
Illumina MiSeq v2 Micro: 1.20
Illumina MiSeq v2 Nano: 0.50
Illumina MiSeq v3: 15.00
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 MiSeq

The Illumina MiSeq is a benchtop next-generation sequencing (NGS) instrument known for its speed, flexibility, and relatively affordable price point. The MiSeq belongs to the low to mid-throughput segment of the NGS market, making it ideally suited for smaller-scale sequencing projects that nevertheless require high-quality data.

Key Innovations

The MiSeq introduced several notable innovations that enhanced its versatility and streamlined the sequencing workflow:

  • Simplified Workflow: The MiSeq features a cartridge-based system that integrates all the steps from sample loading to primary data analysis within the instrument. This eliminates the need for complex fluidics handling and manual transfers, minimizing the risk of errors and reducing hands-on time for researchers. This streamlined approach allows researchers to focus on experiment design and data analysis rather than intricate protocols.
  • Speed: The MiSeq delivers significantly faster turnaround times than its predecessors. Runs can be completed in as little as 7 hours for a 50 base pair single-end read, making it ideal for researchers who need results quickly to inform the next steps in their experiments. This speed is achieved through several factors, including the 2-channel SBS chemistry that allows for faster sequencing cycles, and the streamlined workflow that minimizes hands-on time and eliminates inter-instrument transfer steps.
  • Flexibility: The MiSeq's multiple flow cell options and adaptable read lengths make it suitable for a wide range of sequencing applications. The standard flow cell offers a balance of throughput and cost, generating up to 25 million reads with read lengths of up to 300 base pairs. Researchers can also opt for the low throughput flow cell for targeted sequencing projects where a smaller number of high-quality reads are needed. Additionally, the MiSeq can accommodate a wide range of read lengths, from short 36 base pair reads for targeted resequencing up to 2x300 base pair paired-end reads for applications like amplicon sequencing or small genome projects. This flexibility allows researchers to tailor their sequencing run to the specific needs of their experiment, optimizing data output, read length, and overall cost.

Features and Technologies

  • Sequencing by Synthesis (SBS): Like other Illumina systems, the MiSeq uses SBS chemistry. Fluorescently labeled nucleotides are added one at a time, imaged after incorporation, and the fluorescent labels are chemically removed to allow the next cycle.
  • 2-Channel SBS: The MiSeq was the first Illumina instrument to introduce 2-channel SBS, allowing for faster sequencing cycles and run times compared to the 4-channel systems widely in use today.
  • Paired-End Reads: Paired-end sequencing, generating reads from both ends of a DNA fragment, is critical for many applications. The MiSeq supports paired-end reads up to 300 bases long.
  • Data Output: The MiSeq can generate up to 15 gigabases (Gb) of data and 25 million sequencing reads per run. Several flow cell options are available to fine-tune the data output depending on the project's requirements.
  • Data Quality: MiSeq instruments generate high-quality data with accuracy exceeding 99% (Q30).

Intended Uses and Specific Applications

The MiSeq's combination of features makes it well-suited for various sequencing applications, including:

  • Targeted Sequencing: Sequencing of specific genomic regions to identify variants (kits include TruSeq Custom Amplicon)
  • Small Genome Sequencing: Sequencing of microbial, viral, and organellar genomes (kits include Nextera XT)
  • Amplicon Sequencing: Analysis of 16S rRNA genes for microbiome studies or sequencing PCR-amplified targets (kits include Nextera XT and 16S Metagenomic Sequencing Kit)
  • Metagenomics: Characterization of microbial communities in environmental or clinical samples (kits include Nextera XT and Nextera DNA Flex)

Comparison with Competing Instruments

  • Illumina MiniSeq: The MiniSeq offers similar functionality as the MiSeq, but with an even smaller footprint and lower throughput. The MiniSeq also supports a rapid run mode, further reducing turnaround time. Tradeoffs exist between the MiniSeq and MiSeq, with the MiSeq offering higher throughput and flexibility while the MiniSeq provides faster speeds and lower costs.
  • Complete Genomics DNBSEQ-E25: The DNBSEQ-E25, using DNA nanoball (DNB) technology, is a lower-throughput alternative to the MiSeq. In general, the DNBSEQ-E25 has shorter available read lengths and shorter run times, although it depends on which MiSeq reagent kit is used.
  • Thermo Fisher Ion GeneStudio S5: The Ion GeneStudio S5 Series sequencers use semiconductor sequencing technology, offering advantages such as rapid turnaround times and longer read lengths, although they are not paired end. The Ion GeneStudio S5 series also offers higher max output than the MiSeq at a lower upfront cost, although the MiSeq is slightly more accurate and can offer paired end reads.

Special Considerations

The Illumina MiSeq strikes a compelling balance for several scenarios:

  • Established NGS workflow: Researchers already familiar with Illumina's SBS chemistry and bioinformatics ecosystem will experience a seamless transition with the MiSeq, as the underlying technology and data analysis pipelines are very similar to its other systems.
  • Emphasis on Accuracy and Reliability: The MiSeq delivers highly accurate sequencing data that is essential for applications like variant discovery and clinical research, making it a strong contender if these are major priorities.
  • Flexibility: The MiSeq's diverse range of sequencing applications and variable read lengths give it an advantage when research projects are varied and may require adjustments to the experimental design.

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This information on this page is up-to-date as of April 24, 2024 and based on the spec sheets published by the manufacturer.