Illumina/Solexa Sequencing: How Sequencing by Synthesis Works

Short answer

Illumina/Solexa sequencing is sequencing by synthesis (SBS): DNA fragments are copied one base at a time using fluorescently labeled reversible terminator nucleotides, and a camera records which colour each fragment gives off in every cycle. “Solexa” is the legacy name for this chemistry; today it is sold under the Illumina name. Ion sequencing is a different method that detects hydrogen ions instead of light.

Definition and scope: Solexa versus Illumina SBS

Solexa sequencing and Illumina sequencing by synthesis refer to the same family of chemistry. Solexa, Inc. was founded in 1998 by Shankar Balasubramanian and David Klenerman, and Illumina completed its acquisition of the company on 26 January 2007. The name Solexa survives mainly in older papers and in the legacy Solexa quality encoding found in some FASTQ files (see Phred quality scores and FASTQ format explained). This page covers short-read SBS and contrasts it with ion semiconductor sequencing; for the wider field see the DNA sequencing overview and, for the older chain-termination method, the Sanger sequencing guide.

How sequencing by synthesis works

Fluorescent reversible terminators

Illumina describes SBS as a method in which a fluorescently labeled, reversible terminator is imaged as each dNTP is added to the growing strand. After imaging, the terminator is cleaved so the next base can be incorporated. All four terminator-bound nucleotides are present in every cycle, and Illumina states that this competition between the four bases minimizes incorporation bias. The company also says the approach virtually eliminates errors and missed calls in homopolymer runs (a vendor claim).

Clusters on a flat surface

The 2008 paper by Bentley et al. in Nature describes the original arrangement: DNA is attached to a flat surface and amplified in place by isothermal bridge amplification, forming clusters about 1 micron in diameter, which are then sequenced by synthesis using 3′-O-azidomethyl reversible terminator nucleotides. Illumina’s current SBS page adds that patterned flow cells dramatically increase throughput and that the method supports both single-read and paired-end libraries.

How the first benchmark was reported

Bentley et al. sequenced a male Yoruba individual (NA18507) with 35-base reads, usually paired, to an average depth above 30x. They reported about 4 million SNPs and about 400,000 further variants (the abstract calls them structural variants; the results attribute most of them to short insertions and deletions). In a test against bacterial artificial chromosome (BAC) sequence, roughly 90% of 35-base reads matched the reference perfectly, consensus accuracy exceeded 99.999%, SNP false-positive and false-negative rates were each below 1%, and the false discovery rate for novel SNPs tested was 2.4%. These figures describe 2008 chemistry and read lengths, not current instruments.

Related concepts: ion sequencing versus SBS

Ion Torrent sequencing, from Thermo Fisher, is the method most often meant by “ion sequencing”. It is not optical. The table compares what the two vendors’ own technology pages state.

Feature	Illumina SBS	Ion Torrent
What is detected	Fluorescent label on a reversible terminator, imaged as each base is added	Hydrogen ion (H+) released when polymerase incorporates a nucleotide, read as a pH change
Optics	Imaging of the label each cycle	None: no optics, scanning, cameras or light; a solid-state ion sensor reads the chip
Nucleotide delivery	All four terminator-bound bases present in each cycle	Nucleotides flow over the chip one at a time; no match means no voltage change
Repeated identical bases	Terminator design; vendor says homopolymer errors are virtually eliminated	Two identical bases give double the voltage, so signal scales with run length

The practical consequence is that the two platforms measure different physical signals, so a pipeline tuned for one is not automatically valid for the other. Platform-level comparison with nanopore sequencing is covered in Illumina vs Nanopore.

Where it matters for labs and cores

• Reading older data: papers and files from the Solexa era may use Solexa-specific conventions; check the quality encoding before analysis.
• Library design: SBS supports single-read and paired-end libraries, so the read layout is a decision made at library preparation, not afterwards.
• Run quality: see PhiX control and index hopping detection for two checks that accompany Illumina runs.
• Reading vendor claims: accuracy statements such as the XLEAP-SBS figures (up to 2x faster incorporation and up to 3x greater accuracy than standard SBS, according to Illumina) are vendor comparisons against the vendor’s own earlier chemistry.

Common misunderstandings

• “Solexa and Illumina are different technologies.” Solexa is the earlier name for the same reversible-terminator SBS approach.
• “Ion sequencing is SBS with a different dye.” Ion Torrent uses no light at all; it detects a pH change from released hydrogen ions.
• “The 2008 accuracy figures apply to current runs.” They describe a 35-base-read experiment on one genome and should not be quoted as specifications for present instruments.

FAQ

What is Solexa sequencing?

It is the original name for fluorescent reversible-terminator sequencing by synthesis on clusters of amplified DNA fixed to a flat surface, now marketed by Illumina.

Is Solexa the same as Illumina?

For sequencing chemistry, yes: Illumina SBS is the continuation of the Solexa approach. The name Solexa persists mostly in legacy literature and the Solexa FASTQ quality encoding.

What is sequencing by synthesis?

A method that reads DNA by watching each nucleotide as it is incorporated by a polymerase. In Illumina’s version a fluorescent reversible terminator is imaged, then cleaved so the next cycle can proceed.

How is ion sequencing different?

Ion Torrent sequencing detects the hydrogen ion released on each incorporation as a pH change on a semiconductor chip, with no optics, whereas SBS images a fluorescent label.

Sources

• Illumina, Sequencing by synthesis technology, checked 2026-10-29.
• Bentley DR et al., Accurate whole human genome sequencing using reversible terminator chemistry, Nature 2008;456(7218):53-59, doi:10.1038/nature07517, full text at PMC2581791, checked 2026-10-29.
• Thermo Fisher Scientific, Ion Torrent next-generation sequencing technology, checked 2026-10-29.
• Wikipedia, Illumina, Inc. (Solexa founding and acquisition), checked 2026-10-29.