{ "id": "ep0njy931t", "slug": "solexa-sequencing", "title": "Solexa sequencing", "updated": "2026-10-15", "topic_path": [ { "id": "life", "label": "Life and health", "api_url": "https://www.edgepedia.org/api/v1/topics/life" }, { "id": "life.biology", "label": "Biological foundations", "api_url": "https://www.edgepedia.org/api/v1/topics/life.biology" }, { "id": "life.biology.genetics", "label": "Genetics and genomic reference", "api_url": "https://www.edgepedia.org/api/v1/topics/life.biology.genetics" }, { "id": "life.biology.genetics.genomics_projects_databases", "label": "Genomics, sequencing, and genome resources", "api_url": "https://www.edgepedia.org/api/v1/topics/life.biology.genetics.genomics_projects_databases" }, { "id": "life.biology.genetics.genomics_projects_databases.dna-sequencing-technologies", "label": "DNA sequencing technologies", "api_url": "https://www.edgepedia.org/api/v1/topics/life.biology.genetics.genomics_projects_databases.dna-sequencing-technologies" } ], "geo": [ { "id": "geo.nongeo.t2001.life.biology.genetics.genomics_projects_databases", "label": "Non-geographic · 2001 to 2020: Genomics, sequencing, and genome resources", "api_url": "https://www.edgepedia.org/api/v1/geo/geo.nongeo.t2001.life.biology.genetics.genomics_projects_databases", "path": [ { "id": "geo.nongeo", "label": "Non-geographic", "api_url": "https://www.edgepedia.org/api/v1/geo/geo.nongeo" }, { "id": "geo.nongeo.t2001", "label": "Non-geographic · 2001 to 2020", "api_url": "https://www.edgepedia.org/api/v1/geo/geo.nongeo.t2001" }, { "id": "geo.nongeo.t2001.life", "label": "Life and health", "api_url": "https://www.edgepedia.org/api/v1/geo/geo.nongeo.t2001.life" }, { "id": "geo.nongeo.t2001.life.biology", "label": "Biological foundations", "api_url": "https://www.edgepedia.org/api/v1/geo/geo.nongeo.t2001.life.biology" }, { "id": "geo.nongeo.t2001.life.biology.genetics", "label": "Genetics and genomic reference", "api_url": "https://www.edgepedia.org/api/v1/geo/geo.nongeo.t2001.life.biology.genetics" }, { "id": "geo.nongeo.t2001.life.biology.genetics.genomics_projects_databases", "label": "Genomics, sequencing, and genome resources", "api_url": "https://www.edgepedia.org/api/v1/geo/geo.nongeo.t2001.life.biology.genetics.genomics_projects_databases" } ] } ], "excerpt": "Solexa sequencing is a massively parallel DNA sequencing method that reads clusters base by base by synthesis, and is the core chemistry of Illumina's instruments.", "snippet": "Solexa sequencing is a massively parallel DNA sequencing method that reads clusters base by base by synthesis, and is the core chemistry of Illumina's instruments.", "node": "life.biology.genetics.genomics_projects_databases.dna-sequencing-technologies", "markdown": "# Solexa sequencing\n\nSolexa sequencing is a massively parallel method that amplifies DNA fragments into clusters on a flow cell surface and reads them base by base by synthesis with fluorescently tagged reversible terminators. It is the core chemistry of Illumina's short-read instruments and today delivers up to 16 terabases (Tb) per run on the NovaSeq X Plus.[1](https://pmc.ncbi.nlm.nih.gov/articles/PMC2581791/) • [2](https://emea.illumina.com/science/technology/next-generation-sequencing/illumina-sequencing-history.html) • [3](https://assets.dev-web.illumina.com.cn/content/dam/illumina/gcs/assembled-assets/marketing-literature/novaseq-x-series-spec-sheet-m-us-00197/novaseq-x-series-specification-sheet-m-us-00197.pdf)\n\n| Key fact | Detail |\n|---|---|\n| Chemistry | Four 3'-O-azidomethyl blocked, fluorophore-labeled nucleotides added simultaneously; TCEP removes dye and block each cycle[1](https://pmc.ncbi.nlm.nih.gov/articles/PMC2581791/) |\n| Clustering | Isothermal bridge amplification builds clusters of up to about 1,000 identical copies, at densities up to ten million clusters per square centimeter[4](https://www.enterprise.cam.ac.uk/10th-anniversary-story-solexa/) • [5](https://courses.cs.duke.edu/spring22/compsci260/resources/GenomeSequencingTechnology/Illumina.Solexa.sequencing.pdf) |\n| First instrument | Genome Analyzer, launched 2006, 1 Gb per run[2](https://emea.illumina.com/science/technology/next-generation-sequencing/illumina-sequencing-history.html) |\n| Landmark result | 2008 human genome from >30x depth of paired 35-base reads: four million SNPs and four hundred thousand structural variants[1](https://pmc.ncbi.nlm.nih.gov/articles/PMC2581791/) |\n| Dye schemes | Four-channel on MiSeq/HiSeq; two-channel (T green, C red, A both, G dark) on MiniSeq, NextSeq, and NovaSeq; one-channel on iSeq 100[6](https://emea.support.illumina.com/content/dam/illumina-marketing/documents/products/techspotlights/cmos-tech-note-770-2013-054.pdf) |\n| Current output | NovaSeq X Plus: up to 16 Tb (52 billion single reads) per dual flow cell run; 2 × 150 bp dual 25B run in about 48 hours[3](https://assets.dev-web.illumina.com.cn/content/dam/illumina/gcs/assembled-assets/marketing-literature/novaseq-x-series-spec-sheet-m-us-00197/novaseq-x-series-specification-sheet-m-us-00197.pdf) |\n\n## How it works\n\nThe method reads DNA by sequencing by synthesis with reversible terminators: each cycle extends every template by exactly one base, images the incorporated base, then strips the label so the next cycle can proceed. The terminators are 3'-O-azidomethyl 2'-deoxynucleoside triphosphates (A, C, G, and T), each carrying a different removable fluorophore.[1](https://pmc.ncbi.nlm.nih.gov/articles/PMC2581791/) Because the 3' block prevents a second incorporation, polymerase-directed extension proceeds one base at a time without over-incorporation, and all four nucleotides can be added simultaneously rather than sequentially, which minimizes the risk of mis-incorporation.[1](https://pmc.ncbi.nlm.nih.gov/articles/PMC2581791/) After imaging, tris(2-carboxyethyl)phosphine (TCEP) removes the fluorescent dye and side-arm from the linker on the base and simultaneously regenerates the 3' hydroxyl, readying the chain for the next cycle.[1](https://pmc.ncbi.nlm.nih.gov/articles/PMC2581791/) The whole process is an extension–termination–cleavage–extension cycle repeated at every position.[7](https://pmc.ncbi.nlm.nih.gov/articles/PMC4357665/)\n\n## How it is done\n\nA run follows three stages: library preparation, cluster generation by in situ amplification, and sequencing by synthesis.[8](https://experiments.springernature.com/articles/10.1007/978-1-0716-1099-2_2) Prepared fragments are attached to the flow cell surface and copied by isothermal \"bridging\" amplification, which forms DNA \"clusters\" from each fragment.[1](https://pmc.ncbi.nlm.nih.gov/articles/PMC2581791/) Each cluster holds up to about 1,000 identical copies within a diameter of one micron or less, and densities reach up to ten million single-molecule clusters per square centimeter because no photolithography, spotting, or bead positioning is required.[4](https://www.enterprise.cam.ac.uk/10th-anniversary-story-solexa/) • [5](https://courses.cs.duke.edu/spring22/compsci260/resources/GenomeSequencingTechnology/Illumina.Solexa.sequencing.pdf)\n\nBefore sequencing, the cluster DNA is made single-stranded and a universal primer is added.[1](https://pmc.ncbi.nlm.nih.gov/articles/PMC2581791/) The instrument then cycles through nucleotide addition, imaging, and cleavage. [Base calling](https://www.edgepedia.org/base-calling) quantifies the per-cycle fluorescent signal and assigns phred-scaled Q values (logarithmic per-base sequencing quality scores), which are used to weight alignment and variant detection; mixed clusters are discarded.[1](https://pmc.ncbi.nlm.nih.gov/articles/PMC2581791/)\n\n## Origin\n\nThe concept of clonal arrays and massively parallel solid-phase sequencing of short reads with reversible terminators emerged from a series of discussions in the lab and at a local pub, including an evening at the Panton Arms where Balasubramanian sketched the idea.[2](https://emea.illumina.com/science/technology/next-generation-sequencing/illumina-sequencing-history.html) • [4](https://www.enterprise.cam.ac.uk/10th-anniversary-story-solexa/) With seed funding from Abingworth Management, Solexa was formed in 1998.[2](https://emea.illumina.com/science/technology/next-generation-sequencing/illumina-sequencing-history.html) The company acquired molecular clustering technology, a method that amplified DNA strands into clusters of about 1,000 copies, improving signal-to-noise and reducing phase errors.[2](https://emea.illumina.com/science/technology/next-generation-sequencing/illumina-sequencing-history.html) • [4](https://www.enterprise.cam.ac.uk/10th-anniversary-story-solexa/) In 2005 Solexa re-sequenced bacteriophage ΦX174 with more than 99.9% accuracy, and in 2006 its first machine, the 1G Genetic Analyzer priced at $400,000, shipped.[4](https://www.enterprise.cam.ac.uk/10th-anniversary-story-solexa/)\n\nThe first large-scale demonstration on a human genome was reported by David R. Bentley and colleagues in Nature in 2008, building an accurate consensus from more than 30x average depth of paired 35-base reads.[9](https://doi.org/10.1038/nature07517) The method's contemporary competitor, 454 pyrosequencing, had been reported by Marcel Margulies and colleagues in Nature in 2005, using emulsion amplification in picolitre reactors.[10](https://doi.org/10.1038/nature03959)\n\n## Variants\n\n**Paired-end reads** are obtained by regenerating double-stranded templates from clusters, or by circularizing roughly 2 kb fragments to create junction fragments that sequence both ends.[1](https://pmc.ncbi.nlm.nih.gov/articles/PMC2581791/)\n\n**Dye chemistry** has been reduced from four colors to fewer. Four-channel SBS uses four dyes and four images per cycle on MiSeq and HiSeq systems. Two-channel SBS, used on the MiniSeq, NextSeq, and NovaSeq, labels thymine green, cytosine red, adenine both colors, and leaves guanine dark, cutting reagent consumption and imaging time per base.[6](https://emea.support.illumina.com/content/dam/illumina-marketing/documents/products/techspotlights/cmos-tech-note-770-2013-054.pdf) • [11](https://link.springer.com/article/10.1186/s13059-026-04081-3) The iSeq 100 goes further with one-channel SBS on a CMOS chip, using two chemistry and imaging steps per cycle: adenine labeled in the first image only, cytosine in the second only, thymine in both, and guanine dark.[6](https://emea.support.illumina.com/content/dam/illumina-marketing/documents/products/techspotlights/cmos-tech-note-770-2013-054.pdf)\n\n**Flow cells and instruments** have also changed. Patterned flow cells use ExAmp chemistry so that each nanowell generates a single clonal cluster aligned over a photodiode.[6](https://emea.support.illumina.com/content/dam/illumina-marketing/documents/products/techspotlights/cmos-tech-note-770-2013-054.pdf) Major platform updates were the HiSeq 2000 in 2010, HiSeq X Ten in 2014, NovaSeq 6000 in 2017, and NovaSeq X in 2022.[11](https://link.springer.com/article/10.1186/s13059-026-04081-3)\n\n## Applications\n\nShort reads are viable for re-sequencing because reference sequences exist for the human and many other genomes, allowing reads to be aligned to a reference instead of the traditional 400–800 base pair reads of [Sanger sequencing](https://www.edgepedia.org/sanger-sequencing).[1](https://pmc.ncbi.nlm.nih.gov/articles/PMC2581791/) The 2008 human genome demonstration characterized four million single-nucleotide polymorphisms and four hundred thousand structural variants, many previously unknown.[1](https://pmc.ncbi.nlm.nih.gov/articles/PMC2581791/)\n\nThroughput has grown by orders of magnitude. The original Genome Analyzer generated 1–2 Gb of high-quality purity-filtered sequence per flow cell from about 60 million single 35-base reads, or 2–4 Gb in paired-read mode.[1](https://pmc.ncbi.nlm.nih.gov/articles/PMC2581791/) The HiSeq 2000 produced 100–150 bp reads and 600 Gb in 8 days, and the NovaSeq X Plus delivers up to 16 Tb (52 billion single reads) per dual flow cell run, with the X Series reducing cost per gigabase by up to 60% versus the NovaSeq 6000.[7](https://pmc.ncbi.nlm.nih.gov/articles/PMC4357665/) • [3](https://assets.dev-web.illumina.com.cn/content/dam/illumina/gcs/assembled-assets/marketing-literature/novaseq-x-series-spec-sheet-m-us-00197/novaseq-x-series-specification-sheet-m-us-00197.pdf) Run times on the NovaSeq X Plus are about 17–18 hours for 2 × 50 bp and about 48 hours for 2 × 150 bp on dual 25B flow cells, with 85–90% or more of bases exceeding Q30 depending on read length.[3](https://assets.dev-web.illumina.com.cn/content/dam/illumina/gcs/assembled-assets/marketing-literature/novaseq-x-series-spec-sheet-m-us-00197/novaseq-x-series-specification-sheet-m-us-00197.pdf)\n\n## Limitations and alternatives\n\n**Phasing and pre-phasing** are error modes of the chemistry. Phasing arises from incomplete removal of 3' terminators and fluorophores and from cluster sequences missing an incorporation cycle; pre-phasing arises from incorporation of nucleotides without effective 3'-blocking. The affected proportion of each cluster increases with cycle number, hampering correct base identification late in reads.[12](https://link.springer.com/article/10.1186/1471-2164-12-382) Base calling is further complicated by strong correlation of the A and C intensities and of the G and T intensities, because the fluorophores have similar emission spectra and optical filters separate them imperfectly.[12](https://link.springer.com/article/10.1186/1471-2164-12-382) In early ultra-short read data, error rates ranged from 0.3% at the beginning of reads to 3.8% at the end.[13](https://pubmed.ncbi.nlm.nih.gov/18660515/) The two-color chemistry carries a recurrent artifact that affects somatic variant detection.[11](https://link.springer.com/article/10.1186/s13059-026-04081-3) The XLEAP-SBS chemistry on NovaSeq X addresses phasing directly, giving up to 2× faster cycle times and up to 3× greater accuracy than standard SBS.[3](https://assets.dev-web.illumina.com.cn/content/dam/illumina/gcs/assembled-assets/marketing-literature/novaseq-x-series-spec-sheet-m-us-00197/novaseq-x-series-specification-sheet-m-us-00197.pdf)\n\n**Compared with other platforms**, in one published comparison, Roche/454 delivered 500–1000 bp reads and 700 Mb in 23 hours, [Ion Torrent](https://www.edgepedia.org/ion-torrent) 318 delivered 200 bp and 1 Gb in 2 hours, SOLiD5500 delivered 60 bp and 180 Gb in 14 days, and HiSeq2000 delivered 100–150 bp and 600 Gb in 8 days.[7](https://pmc.ncbi.nlm.nih.gov/articles/PMC4357665/) In the ABRF benchmark across HiSeq/NovaSeq, Ion, PacBio CCS, Oxford Nanopore, and BGI instruments, HiSeq 4000 and X Ten gave the most consistent, highest genome coverage among short-read instruments while BGI/MGISEQ showed the lowest sequencing error rates; PacBio CCS and PromethION/MinION mapped best in repeat-rich areas and across homopolymers, and NovaSeq 6000 with 2 × 250 bp chemistry was the most robust instrument for capturing known insertion/deletion events.[14](https://www.nature.com/articles/s41587-021-01049-5)\n\n## References\n\n1. [Accurate Whole Human Genome Sequencing using Reversible Terminator Chemistry (Bentley et al., Nature 2008)](https://pmc.ncbi.nlm.nih.gov/articles/PMC2581791/)\n2. [History of Illumina Sequencing & Solexa Technology (Illumina)](https://emea.illumina.com/science/technology/next-generation-sequencing/illumina-sequencing-history.html)\n3. [NovaSeq X and NovaSeq X Plus Sequencing Systems specification sheet](https://assets.dev-web.illumina.com.cn/content/dam/illumina/gcs/assembled-assets/marketing-literature/novaseq-x-series-spec-sheet-m-us-00197/novaseq-x-series-specification-sheet-m-us-00197.pdf)\n4. [10th anniversary story: Solexa – Cambridge Enterprise](https://www.enterprise.cam.ac.uk/10th-anniversary-story-solexa/)\n5. [DNA Sequencing with Solexa (technology overview)](https://courses.cs.duke.edu/spring22/compsci260/resources/GenomeSequencingTechnology/Illumina.Solexa.sequencing.pdf)\n6. [Illumina CMOS Chip and One-Channel SBS Chemistry (tech note)](https://emea.support.illumina.com/content/dam/illumina-marketing/documents/products/techspotlights/cmos-tech-note-770-2013-054.pdf)\n7. [The History and Advances of Reversible Terminators Used in New Generations of Sequencing Technology](https://pmc.ncbi.nlm.nih.gov/articles/PMC4357665/)\n8. [The Illumina Sequencing Protocol and the NovaSeq 6000 System (Springer protocol chapter)](https://experiments.springernature.com/articles/10.1007/978-1-0716-1099-2_2)\n9. [David R. Bentley and colleagues (2008). Accurate whole human genome sequencing using reversible terminator chemistry. Nature.](https://doi.org/10.1038/nature07517)\n10. [Marcel Margulies and colleagues (2005). Genome sequencing in microfabricated high-density picolitre reactors. Nature.](https://doi.org/10.1038/nature03959)\n11. [A recurrent sequencing artifact on Illumina sequencers with two-color fluorescent dye chemistry and its impact on somatic variant detection (Genome Biology)](https://link.springer.com/article/10.1186/s13059-026-04081-3)\n12. [Addressing challenges in the production and analysis of Illumina sequencing data (BMC Genomics)](https://link.springer.com/article/10.1186/1471-2164-12-382)\n13. [Substantial biases in ultra-short read data sets from high-throughput DNA sequencing](https://pubmed.ncbi.nlm.nih.gov/18660515/)\n14. [Performance assessment of DNA sequencing platforms in the ABRF Next-Generation Sequencing Study](https://www.nature.com/articles/s41587-021-01049-5)\n\n---\n*Topic: Encyclopedia › Life and health › Biological foundations › Genetics and genomic reference › Genomics, sequencing, and genome resources › DNA sequencing technologies*\n\n*Initially written Sep 29, 2026 · Edited: Oct 15, 2026*\n\n*Copyright 2026 EdgeChat AI, a subsidiary of Biostate AI.*\n\nLicense: Edgepedia Community License 1.0, https://www.edgepedia.org/edgepedia/license\n", "same_as": [], "url": "https://www.edgepedia.org/solexa-sequencing", "markdown_url": "https://www.edgepedia.org/solexa-sequencing.md", "license": { "name": "Edgepedia Community License 1.0", "url": "https://www.edgepedia.org/edgepedia/license", "summary": "Free with credit, commercial use included. AI training is open to everyone. For other uses, organizations over USD 100M in revenue or 100M monthly users license separately.", "spdx": "LicenseRef-Edgepedia-Community-1.0" }, "credit": "\"Solexa sequencing\", Edgepedia (EdgeChat), https://www.edgepedia.org/solexa-sequencing. Edgepedia Community License 1.0.", "credit_md": "\"[Solexa sequencing](https://www.edgepedia.org/solexa-sequencing)\", Edgepedia (EdgeChat), [https://www.edgepedia.org/solexa-sequencing](https://www.edgepedia.org/solexa-sequencing). [Edgepedia Community License 1.0](https://www.edgepedia.org/edgepedia/license).", "credit_html": "\"Solexa sequencing\", Edgepedia (EdgeChat), https://www.edgepedia.org/solexa-sequencing. Edgepedia Community License 1.0.", "speakable": "Solexa sequencing is a massively parallel DNA sequencing method that reads clusters base by base by synthesis, and is the core chemistry of Illumina's instruments." }