When you hear Next Generation Sequencing (NGS), you think Illumina Inc. Illumina is widely regarded as the giant of NGS, with an estimated 80% share of the global gene sequencing market. At the heart of Illumina’s nucleic acid sequencing technologies is sequencing-by-synthesis (SBS), a chemistry technology with a humble and fascinating backstory.
The concept of SBS chemistry can be traced to a summer’s evening in 1997, when four chemists of the University of Cambridge frequented the pub for a regular laboratory team discussion, a “beer summit”. The quaint city of Cambridge has been at the centre of some of the greatest developments in nucleic acid science, and no telling of this tale is complete without consideration of them. In 1953, James Watson and Francis Crick of Cambridge’s Cavendish Lab burst through The Eagle’s pub doors and proclaimed to have discovered “the secret of life”. Of course, they were referring to their resolution of the three-dimensional structure of DNA. A very British eureka, we hear you say. Another Cambridge alumnus, Frederick Sanger, is accredited with the major breakthrough in DNA sequencing, thus addressing the pressing question of how to “read” the sequence of nucleotide bases. Fred’s ‘Sanger sequencing method’ of 1977 would become the principle DNA sequencing method for over 20 years, until the emergence of SBS chemistry.
In 1997, Shankar Balasubramanian and David Klenerman were using fluorescently labelled nucleotides to observe the motion of a polymerase at the single molecule level as it synthesized DNA immobilized to a surface. Their laboratory work was supplemented with discussions at the Panton Arms, a pub located a stone’s throw away from the department, on account of the “lack of conference rooms” within the Chemistry building. The beer summit of 26 August 1997 would see Balasubramanian and Klenerman, with their postdocs, Mark Osborne and Colin Barnes formulate the “Solexa Idea”. Balasubramanian and Klenerman & Co. realised that if they were watching the polymerase copying a sequence they were also inadvertently reading the sequence. Ideas sparked and the beginnings of the modern SBS chemistry were formulated. Calculations of the possible “reading speed” of such a sequencing method implied a revolutionary technology that could increase the rate and lower the cost of gene sequencing by [a factor of] 104 or 105. The next day came “the acid test”, as described by Balasubramanian: the transfer of the ideas from beer garden to lab benchside.
The central principle of SBS is sequencing single-strands of nucleic acids as they are synthesised by a polymerase using reversibly-terminated fluorescently-labelled nucleotides. The reversible nature of the terminated nucleotides creates a brief pause in synthesis for the fluorescence of a nucleotide to be captured, and a ‘base call’ determined. Iterations of this workflow, occurring in massively parallel array presented over a 100-fold increase in sequencing power relative to the Sanger sequencing method that dominated the market.
Following preliminary works, Balasubramanian and Klenerman met with venture capitalists Abingworth Management in November 1997. Equipped with four acetates and a concept to improve DNA sequencing by 100,000 fold, Balasubramanian and Klenerman pitched the idea under the start-up “Solexa Ltd.”, so named in reference to light and single molecules of DNA.
From 1998 to 2001, Solexa Ltd. operated from Balasubramanian’s laboratory at the University of Cambridge’s Department of Cambridge using seed and Series A funding fronted by Abingworth. Barnes and Osborne, the post-docs from the fateful beer summit, contributed to the early development and research of the Solexa idea. Following Series A funding, Solexa moved to Chesterford Research Park, and built their management team; hiring Nick McCooke as CEO and Dr. Tony Smith as CSO. In these first critical years, the SBS chemistry dreamt of in the pub was translated and re-designed into the technology that we recognise today; sample prep, surface chemistry, sequencing chemistry and the bioinformatics were all revamped. Like any research team, Solexa had their highs and lows: detecting fluorescent tags on single DNA molecules was never going to be an easy feat. Indeed, stories of Klenerman desperately using a loudspeaker blasting high-frequency sound waves to make the DNA stand on end have been told.
In 2004, Solexa acquired molecular clustering technology from Manteia. The amplification of single DNA molecules into clusters enhanced the fidelity and accuracy of gene calling, while reducing the cost of the system optics through generation of a stronger signal. One weekend in February 2005, Solexa attempted to sequence a full genome, that of the ΦX174 virus that Fred Sanger first sequenced 25 years earlier. On a Sunday afternoon, an email was sent with the unmistakable subject, “WE’VE DONE IT !!!!”. The viral genome had been sequenced with more than 99.9 percent accuracy.
Solexa went from strength to strength, in 2005 Solexa Ltd. acquired instrumentation company Lynx Therapeutics in a reverse merger, becoming the international public company, Solexa Inc. (NASDAQ). Solexa’s new state-side engineering and software production team got to work on turning the successful Solexa prototype into a commercial sequencing instrument. In 2006, the first Solexa sequencer, the Genome Analyzer, was launched: offering the power to sequence 1 gigabases (Gb) (i.e. 1 × 109 bases, or 1,000,000,000 bases) of data in a single run.
In 2007, Solexa was acquired by Illumina in a stock-for-stock merger deal valued at $650 million. SBS technologies continue to play an integral role in Illumina’s various sequencing platforms, including the iSeq 100, MiniSeq, MiSeq series and NextSeq series, as well as their flagship NovaSeq series. Today, it is possible to sequence 16 terabytes (Tb) (i.e. 16 × 1012 bases, or 16,000,000,000,000 bases!) of data in a single run, and within a timeframe of half a day to two days. Innovation still continues at Illumina and promises to deliver increased sequencing power and even faster turnaround times. A far cry from the beer summit of 25 years ago!