In 1998, Solexa sequencing emerged at the University of Cambridge from pioneering work of Sir Shankar Balasubramanian and Sir David Klenerman, Dr Colin Barnes and Dr Mark Osborne.
The breakthrough tech revolutionized genomics by paving the way for next-generation sequencing and the reading of the genetic code.
Solexa's innovative approach, later acquired by Illumina, marked a pivotal moment in genomic research and set the path toward targeted medicine and personalised healthcare.
From small seeds...
The genesis of Solexa sequencing was shaped by separate but connected studies on the DNA-polymerase interaction and methods for single molecule DNA immobilisation and detection from the molecular biology labs of Shankar and physical chemistry labs of David.
Conducted by PhD, Scott Furey and post-doc, Mark ('96-'97), these classically academic and curiosity driven studies provided the foundation for novel experiments designed to follow DNA synthesis in real-time, at the single molecule level, to elucidate structural changes in the double-helix at the single strand interface during nucleotide (base) incorporation.
At this time single molecule studies were limited to point detection on a bespoke confocal fluorescence microscope, so immobilisation of DNA on a bead (200 nm) was the only solution providing a visible handle to locate and position the DNA in the diffraction-limited laser volume; a truly primitive platform but near state-of-the-art for '90s!
Moreover, the random nature of DNA to bead attachment produces a Poission distribution of molecules per bead. The stats simply mean that, for an average of one DNA per bead, just over a third of beads have a single molecule DNA attached, the same number have no DNA and a near-quarter have more than one molecule.
Not best for reliable single molecule hits, more so given the microscope back then was entirely manual, requiring the patience of a saint to position the bead, focus the laser, detect fluorescence and repeat!
The case was made to move to widefield, total internal reflection fluorescence (TIRF) imaging, a mechanical stage and arrays of single molecules on glass, to capture more molecules per detection area (0.1 x 0.1 mm) and acquire image sets of single DNA dispersions in a semi-automated way.
Postdoc, Colin Barnes was appointed lead on DNA immobilisation chemistry to switch away from bio-couplings to more robust silane chemistries, while Mark continued redesign and build of the microscope.
Best in class scientific camera in '97 was the ICCD (Pentamax, Princeton Inst.) with ample gain to detect the few hundred photons that make it to the CCD chip from a single fluorescently tagged DNA molecule.
Prism-type TIRF and a powerful microscope lens combined to allow the detection of many 100s of individual molecules across an area with a diameter no bigger than that of the proverbial human hair! Advanced imaging for the 90s, although remarkably VHS remained the only medium with capacity for recording single molecule movies!
A bit of coding in Scion Image (NIH precursor to ImageJ) allowed a decent workflow of image acquisition and data processing, but mechanical drift on the microscope required "best guess" refocusing between image capture and image based "autofocusing" algorithms failed due to SM signal loss only a few steps (10s of nm) from focus.
The neat solution to a steady focus would have to wait and for now a launch and leave for coffee during data acquisition was out of the question.
As the first flickers of fluorescence from the DNA single molecule arrays (SMAs) rolled off the scope, ideas of how the arrays could be put to use evolved, recognising the shear numbers of oligos observed and the inherent "purity" of each DNA molecule displayed, alongside comparisons with emerging tech such as the hybridisation chips of Affymetrix, assays of Evotec and the sequencing by degradation concepts of SEQ.
Scaling the 100s of DNA per "hair" to a typical sample 100X larger (1x1 cm), placed numbers in the 10s of millions and the typical 30 base oligonucleotide used to the demonstration of the DNA SMAs showed that total base numbers per "chip" would approach the 3Bn bases of the human genome.
If the nucleic acids derived from "chopped-up" genome and their sequences read through cycles of fluorescent base incorporation and detection, then that's it, the basis for what evolved to be Solexa sequencing.
The single molecule advantage lay in the "purity" of each nucleic acid sequence, avoiding the contamination issues from DNA amplification and phasing problems associated with "bulk" sequencing methods.
Talks over coffee, chats over beer followed as ideas developed, but the "beer-summit" in the Summer of '97 is seen as a pivotal moment in the formulation of Solexa sequencing in many writings of the story across the years (2010 bio-itworld.com, 2016 enterprise.cam.ac.uk, 2017 illumina.com, 2021 cancerresearchuk.org, 2023 marks-clerk.com).
End of the millennium party begins
As the VHS recordings and SMA preps continued in the lab and numbers were crunched on SMA uniformity and reproducibility, in search of best SMA immobilisation chemistries, Shankar and David presented the sequencing concept to the life sciences VC, Abingworth Bioventures, and Solexa (name but no logo) was seeded at £0.6M in the Autumn of '98 following first patent filings with a first intro and reporting on progress at the London offices in early '99.
By then, estimates of sequencing rates could be made based on hardware limits assuming realistic reductions in image exposure time from 1 s, at that time, to video rate at 30 ms. For a typical SMA of 1000 molecules per image the read rate would approach 50 000 bases/s or 17 hrs per "genome".
Perhaps a naive calculation at the time, with synthesis and wash steps between scans not accounted for, along with the excess DNA required to compensate for failed incorporations and false negative readouts.
However, the numbers set a target for a whole genome read speeds that descendants of Solexa's first commercial sequencer, the Genome Analyzer (2006), would reach routinely over a decade of development later (Illumina Inc., 2007-2017).
Discussions turned to applications and markets, from the obvious in healthcare and personalised medicine, to surprising angles on the sequencing of pets and even thoroughbreds for cloning and pedigree!
Work accelerated through '99, with all hands on the deck (Colin, Scott, Mark, David, Shankar) to resolve challenges in DNA immobilisation chemistries.
By the Summer a flow cell design was realised for the first attempts at on-scope DNA hybridisation experiments and the first attempts at dNTP incorporation on a single molecule DNA array. Initial positives were countered by controls that set the next challenge of reducing non-specific absorption of fluorescent nucleotides at the DNA chip surface.
But ahead of developments in superresolution microscopy, we were localising and colocalising molecules on SMAs according to Bobroff's formulation (Rev. Sci. Instrum. 57, 1152–1157, 1986) to demonstrate functional DNA arrays. Results would be published later after securing IP.
A switch of microscope (Nikon Diaphot to TE2000) with the addition of filter wheels and laser shutters, synchronised through commercial imaging software, Metamorph (Universal Imaging Corp, discontinued) and, a straight to disk PCI frame grabber took the scope a step closer to a "hands-free" instrument (just the autofocus to crack).
At the end of '99 board meeting the milestone of demonstrating two sequencing cycles on a DNA SMA was set for the start of the new millennium.
After many early starts and late nights in the lab, a bucket load of chips had been SMAed, scanned and analysed with numerous coupling chemistries, surface passivations, oligo sequences and concoctions for incorporations.
By early '00, evidence for sequencing success on a SMA of model DNA hairpins, had been generated from stats of two colour colocalisations (dUTP-Cy3, dCTP-Cy5) from large image sets.
Following presentation of the latest data at the first quarterly SAB of '00, Alan Williamson (appointed chairman of Solexa) emailed the news we worked hard to hear, "The key milestone that was set after the last SAB, ...was met to the satisfaction of all the SAB members'.
The milestone triggered a second raise of £1.6M to ultimately spinout Solexa (now with logo) from Cambridge University. The search for new labs and new recruits was on in the Summer of '00 and included the Science Park and even plans drawn to convert space at the Babraham Institute, but by the fall, the team opted for space at Chesterford Research Park (nr Hixton, Sanger Institute, edge of Essex), an old agrochem research facility (Aventis).
A large packing room became the laser lab, with annex for surface chemistry, and separate formulation and application areas were turned to labs for nucleotide chemistry and enzymology.
Colin and Mark became the first employees of Solexa Ltd and along with new premises came new management, CEO Nick McCooke and CTO Harold Swerdlow. Around the same time came new members of the "bench science" band, Jason Bryant on instruments, Xiaohai Liu on nucleotides, David Earnshaw on enzymology and Anil Kumar on surfaces.
The ship sets sail to the future of sequencing
By February '01 end, transfer to Chesterford was complete, new laser tables installed and components ready for two clones of the TIRF microscope to be built. In the same month the first draft human genome made the cover of Nature, 15 years of work from 20 groups across the globe at cost of $300M.
Nick left a copy in the coffee room as reminder of the grand plan, the $1000 genome, in 24 hours! The promo brochure followed. Further recruitment (Niall Gormley, Bojan Obradovic, Vicky Harris, Darren Ellis) grew the team to a rugby squad (with ops manager Konrad, Helen in resources and John Rogers, the go to engineer) and Tony Smith (CSO) was later appointed to build out the team to include bioinformatics headed by Clive Brown.
New ambitious milestones were set through '01 and, after training new arrivals on the objectives and technicals, some hurdles hopped.
On the scope, the focus drift was nailed with an autofocus IP filing (Nikon PFS beat us on publication) and laser drift seen over large area scans resolved with a new TIR-coupling prism design.
Finally, an instrument that allowed a "set and go" (for coffee) operation for automated acquisitions of large, well-focused SMA image sets. A flow-cell redesign (colab with Scientific Generic) further streamlined chip assembly and accuracy of sample rescans.
By late Spring 2001, three incorporation cycles on model DNA had been achieved revealing stats on full extensions, stalled syntheses and missed reads.
But it was increasingly clear that a number of factors (fluorescence bleaching, incorporation inefficiencies, residual NTP adsorption) would couple to reduce the overall read efficiency below that required to make single molecule genome sequencing viable.
Signal amplification and a DNA "excess" was required to account for fluorescence instabilities and deficiencies in polymerase activity.
Nonetheless progress was sufficient for the board to woo a consortium of investors (inc. Schroder Ventures, Amadeus, Oxford Biosciences) that would raise £12M by the Autumn of '01.
On the horizon was a technology from Manteia that allowed local amplification of the DNA at each single molecule site on the SMA to produce 1000s of copies in a cluster.
Rights to the technology would later be purchased by Solexa and the cluster tech, coupled with some clever chemistry to realise reversible terminators (Colin, Shankar, Xiaohai, Harold, John Milton) and bioinformatics (Clive Brown, '02) would accelerated the R&D in the years ahead to the first sequencer, the 1G Genome Analyzer and first resequence, the PhiX174 virus genome in 2005, and ultimately the first human genome in 2008 using the NGS tech.
By the Summer of '01 the Royal Society Research Fellowship I (Mark) had been awarded a year earlier but had deferred to ride the first wave and spinout of Solexa, was finally calling me back to research! After 6 years on DNA and Solexa, it was time to move on to set up a spectroscopy and nanomaterials lab at Sussex University on the south coast.
The Solexa band laid on a memorable farewell (with original merch) and I passed on a bottle of fizz for the team to celebrate their first completed genome.
Consultancy until '07 allowed me the occasional visit to Chesterford Research Park, to pass on any single molecule wisdom from the Sussex lab, see old friends and follow progress as Solexa transitioned to Illumina following a $600m deal in '06.
Some 10 years after publication of the first draft genome and spinout of Solexa, the true potential of Solexa sequencing was perfectly pitched in the 2010 BBC Horizon special "Miracle Cure? A Decade of the Human Genome".
To watch banks of Genome Analyzers (GA)in operation at the Sanger Institute, all fired up and flashing the same fluorescence images of DNA arrays, unchanged from those first video exposures recorded in the basement lab at Cambridge was truly stunning and stirring.
Fast forward to '22 and by now the GA and it's decendants had made impact on a number of crucial sequencing projects, from the 100000 genomes of Genomics England, to the tracking of Covid 19 mutations and genetic susceptibility to the virus.
Then, following prestigious awards of the Millenium Technology Prize and Breakthrough Prize in Life Science to Shanker and David in '21, an invite to a "Celebration of Solexa" saw me back in Cambridge, catching up with original band members and meeting others (John West, Kevin Hall, David Bentley) that were instrumental in making Illumina and Solexa sequencing the powerhouse in NGS that it is today.
And to end the story, at a pre-dinner tour of the UK Illumina HQ, Granta Park, Kevin Hall revealed the champagne bottle I had left Solexa all those years before, had become a "lucky" baton that would be passed on through management as Solexa went from strength to strength.
Perhaps it was the sentiments on the label that remained forever the motivator to make Solexa the future of DNA sequencing.
* Links accessed Dec 2023