Illuminating the code of life: the colourful revolution of next-generation sequencing

This story delves into the quest to understand the essence of life, the uniqueness of each individual and the reasons behind people’s susceptibility to both rare and very common diseases. It highlights how this knowledge empowers us to discover, diagnose and ultimately cure these conditions. The narrative unfolds around two chemists and their two postdoctoral fellows who, while conversing over a pint of beer at a local pub, stumbled on a groundbreaking idea.

“We were merely experimenting, to be honest. However, it is worth mentioning that we were in Cambridge, a place with a rich history of interest in DNA, thanks to predecessors like Watson and Crick and, of course, Fred Sanger. Their stories are well known and frequently discussed in Cambridge, making it impossible to ignore, regardless of one’s interests,” explains Shankar Balasubramanian, Herchel Smith Professor of Medicinal Chemistry, Yusuf Hamied Department of Chemistry at the University of Cambridge.

The Human Genome Project, conducted not far away at the Wellcome Sanger Institute, also played a significant role. 

“In many ways, our journey is a synthesis of all this history. The Human Genome Project aimed to sequence a single genome over 13 years. However, since people are inherently diverse, understanding the genetic basis for our differences requires sequencing numerous human genomes. During our discussions, we realised that our method could be adapted to read the DNA code much faster,” says David Klenerman, Royal Society GSK Research Professor, Yusuf Hamied Department of Chemistry at the University of Cambridge. 

Ability to see single molecules

In 1994, when David Klenerman and Shankar Balasubramanian joined the University of Cambridge, neither was contemplating DNA sequencing, nor did they know each other. 

“We were different types of scientists: David was a physical chemist, and I was an organic chemist-biochemist. We joined the department around the same time,” recalls Shankar Balasubramanian. 

Shankar Balasubramanian, with his background in organic chemistry, completed his PhD and then worked at Pennsylvania State University in the United States before joining Cambridge’s Yusuf Hamied Department of Chemistry. David Klenerman, after being doing postdoctoral studies at Stanford University returned to the United Kingdom to work at BP Research for seven years. 

“We were both young academics in search of interesting problems to tackle. In my field, there was a burgeoning revolution in the ability to see and analyse single molecules, which enabled us to observe biology in action,” clarifies David Klenerman. 

Met over a cup of tea 

Shankar Balasubramanian was also exploring the dynamics of DNA polymerase – a cellular copy machine for DNA, reading the original genetic blueprint to ensure that every new cell receives an exact copy of the genetic material. 

“We had submitted a manuscript, and a reviewer requested an experiment that required time-resolved laser fluorescence detection, which was not available in my lab. I was trying to find someone who could assist with that experiment. Then my former supervisor, Chris Abell, who knew both of us, suggested that I talk to David Klenerman. He was known for his expertise in lasers, making him the perfect person for the task,” explains Shankar Balasubramanian. 

This is how Shankar Balasubramanian and David Klenerman met – over a cup of tea. They addressed the reviewer’s question, and their article was subsequently accepted, marking their first co-authored publication. 

“That experience helped us get to know each other better and understand our interests and areas of specialisation. During these conversations, we developed the idea to combine David’s expertise in single-molecule fluorescence spectroscopy, a groundbreaking approach at the time, with my research interests,” says Shankar Balasubramanian. 

Professor Shankar Balasubramanian. Photo: Nathan Pitt, University of Cambridge.

Very specific and fundamental 

The two new partners decided to embark on a joint project, although it presented challenges. 

“Because we were newcomers, our research groups were small and our fields were not well established. We were seeking something intriguing to pursue. Thus, we embarked on a basic project,” explains David Klenerman. 

Their idea involved building a single-molecule fluorescence microscope to observe the dynamics of DNA polymerase. By linking fluorophores – chemical compounds that emit light upon light excitation – they aimed to monitor the enzyme as it bound to its DNA substrate, extended the DNA and synthesised new DNA strands. 

“Our first joint grant application was funded, providing the necessary resources. We still keep that grant application as a memento. It is fascinating to look back at it, especially since sequencing was not mentioned. Our goal was to observe the process and learn from it, a very specific and fundamental objective,” he adds. 

From years to days 

However, the experiment did not proceed as smoothly as they had hoped. To commiserate and brainstorm solutions, they visited the Panton Arms pub with their two postdoctoral fellows, Mark Osborne and Colin Barnes. The Wellcome Sanger Institute, playing a pivotal role in the Human Genome Project nearby, was determining the 3 billion bases of the human genome for the first time. 

“The Human Genome Project underscored the need for a more rapid and cost-effective sequencing method. There was significant interest in this field, making it the right place and time for us, working on fundamental science that happened to be highly applicable to an important problem,” says Shankar Balasubramanian. 

During their discussions at the Panton Arms, the researchers realised that their work could be adapted to meet this need. 

“The Human Genome Project sequenced one genome in 13 years, but our new idea could potentially reduce the time required to sequence a human genome by a factor of 100,000 – from years to days. I joked with the landlord that I would make him very famous one day. ‘If you do, free beers for life,’ he responded.” 

David Klenerman and Shankar Balasubramanian at Pantom Arms. Photo: Nathan Pitt, University of Cambridge.

Series of colours changing 

Reading genomic DNA is akin to reading ordinary books but is in some ways much simpler, since it comprises only four letters: adenine, cytosine, guanine and thymine – A, C, G and T. In addition, the two strands of DNA helices that form our genomes are chemically linked, pairing A with T and C with G. 

“To read the sequence, we anchored one end of a DNA strand and began building its complementary strand. Our idea, based on our fluorescence colour-coding experiments, was to colour code each of the four building blocks in green, blue, red and magenta. This would enable us to read the code as the strand extended by observing the emitted colours,” explains David Klenerman. 

The researchers spaced single DNA molecules far enough apart on a surface to differentiate them, enabling them to observe the colours emitted as the corresponding DNA strands were synthesised. 

“By recording these colour changes, we could determine the sequence of the DNA molecule on the surface. We planned to remove the bases at all spots and then remove the fluorophore and repeat the sequencing cycle, recording the colour of the dye incorporated at each spot. This process would be repeated multiple times.”

Tearing the book apart 

In hindsight, brilliant ideas often seem simple and obvious. The true genius and simplicity of the new sequencing method lay in its parallelisation. Instead of sequencing a single molecule at a time, researchers would sequence millions simultaneously. This approach was crucial for navigating through the 3 billion letters of the human genome. This task was roughly equivalent to reading the entire Lord of the Rings series, including The Hobbit prequel, 1000 times. 

“While traditional sequencing approaches would typically only read small sections of a book, our approach was to read very short fragments from millions of different positions within the book, and then piece together the content afterwards,” explains Shankar Balasubramanian. 

Balasubramanian and Klenerman would fragment the DNA into millions of tiny pieces and then attempt to reassemble it. Each piece contained a number of “words”. By identifying the overlapping words between pieces, they could determine how the pieces connected, gradually reassembling the entire genomic “book”. This process was akin to solving a giant puzzle, with each piece representing a small part of the story. By matching these parts, they could recreate the complete genome. 

“We would extract genomic DNA, fragment it and attach the millions of fragments to a surface, sequencing the millions of pieces simultaneously. The Human Genome Project provided what we now call a reference sequence, which offers insight into the structure of a genome. By sequencing DNA fragments, we could use a computer to determine where each sequence originated from and realign it to the reference human genome, thereby constructing a new genome,” elaborates David Klenerman. 

We can do this 

In November 1997, Shankar Balasubramanian and David Klenerman met with venture capitalists from the bioscience investment firm Abingworth, armed with just a few slides detailing their ambitious ideas and calculations. They proposed that their concept, if realised, could increase the rate and reduce the cost of gene sequencing by 10,000 to 100,000 times. This proposal was sufficient to convince the investors to support the Cambridge chemists. 

“In 1998, we founded the start-up company Solexa. We chose the name Solexa because sol signifies light, and it was a contraction of solo, as in single molecules of DNA,” explains Shankar Balasubramanian. 

Like any technology, the Solexa sequencing technique required extensive review and innovation. With an investment of several hundred thousand British pounds, they established Solexa’s first physical laboratory and began recruiting experts. The key challenges of synthesising the blocked nucleotides, mutating the polymerase, arraying the DNA on a surface and optimising the surface chemistry were all overcome by a talented team of researchers at Solexa. 

However, to solve the remaining challenge of simplifying the imaging method and obtaining larger signals, Solexa licensed a patent for surface amplification developed by Pascal Mayer and colleagues at Manteia Medical Technologies: the last component needed to get the sequencing technology fully functional.

“Instead of studying a single molecule of DNA, we finally ended up amplifying every DNA molecule into thousands of copies of DNA sequences at the same location. This amplification resulted in spots of blue, green, red and yellow that were easier to detect, simplifying the method. Consequently, we obtained millions of coloured spots on a chip the size of a human hair, each corresponding to a fragment of DNA,” says David Klenerman. 

A genome per hour 

The Solexa team designed chips, surfaces, enzymes, nucleotides, fluorescent labels, optics and engineering components to construct the first next-generation sequencing machine – dubbed 1G because it could sequence a billion bases of DNA in a single experimental run, matching the global sequencing capacity around the time when Shankar Balasubramanian and David Klenerman initiated their project.

 People often ask whether there was a single moment of inspiration. However, it was not as straightforward as two guys walking into a pub and emerging with a sequencing system. It was more about establishing a series of connections, since several conceptual challenges needed to be addressed. We tackled each area separately and then considered how to integrate them. It was through this iterative thinking that we reached a point where we felt confident: ‘We can do this,’” recalls Shankar Balasubramanian. 

In 2006, the team showcased the Solexa sequencing of the PhiX174 bacteriophage, a virus that infects bacteria. In November 2008, the sequencing of the first African genome was published in Nature, alongside the first Asian genome and the first cancer genome, demonstrating Solexa’s capability for rapid and accurate genomic sequencing. 

“The most recent sequencing systems can now sequence several trillion bases per run – a further 1,000 to 10,000-fold improvement on the first Solexa instrument. Thus, these instruments can sequence the equivalent of a genome in roughly about an hour. The Human Genome Project took about 13 years and cost several billion US dollars. Today, sequencing a genome costs less than 1,000 US dollars.” 

We are all different 

Even though it was still considered fantasy or at least theoretical at the time, Solexa sequencing – today known as nextgeneration sequencing – attracted significant investment interest because of its potential to drastically reduce the cost and time required for DNA sequencing. 

“We are, of course, all different. Understanding the genetic basis for what makes us all unique, including disease-related aspects, would require sequencing many human genomes. This required a method that was much faster and more affordable than the methods available at that time,” explains David Klenerman. 

Potential applications included personalised medicine, in which genetic information could guide individualised treatment plans. In November 2006, United States–based Illumina made a USD 650 million offer to buy Solexa, making the project one of the greatest commercialisation success stories to emerge from the University of Cambridge. 

“We had meetings discussing the possibilities if one could sequence the human genome. But the applications and the impact have gone well beyond what we speculated about at those meetings, including early diagnosis of cancer and prenatal screening. If you improve anything by a factor of a million, predicting how the technology will be used can be difficult.” 

Decoding cancer 

If you consider human DNA as a complex instruction manual, errors can lead to cancer. Next-generation sequencing acts as a sophisticated tool, detecting these errors with precision. Thus, this technology has already transformed how cancer is understood, offering insight into its genetic basis and paving the way for targeted treatments. 

“Initiatives like the International Cancer Genome Consortium and Genomics England represent monumental efforts in mapping the cancer genome, aiding in identifying key mutations responsible for various cancers.” 

For instance, the discovery of a mutation in the BRAF gene in about half of melanoma cases has revolutionised treatment options. 

“Today, doctors prescribe a drug that targets the protein encoded by BRAF. This results in a very good response for tumours, especially melanomas that have this mutation. This is an example of using information from your genome and the tumour’s genome to prescribe a drug that will be more effective for that specific case.” 

This knowledge significantly improves patient outcomes. This level of precision in treatment underscores the value of understanding the genetic landscape of tumours. Today, whole-genome sequencing of many types of cancer and certain complex patterns of mutations are helping clinicians to make treatment decisions. 

“I predict that we will see significant advancements over the next two decades. But clinicians are already using data in this way. It turns out that cancer cells release some of their DNA content into the bloodstream. So, by drawing blood and isolating this DNA, which is floating around without associated cells, early detection of cancer becomes one of the most promising ways to ensure better survival outcomes and help patients.” 

Unlocking answers for rare genetic diseases 

Diagnosing rare genetic diseases is akin to finding a needle in a haystack. Next-generation sequencing has offered a new, powerful solution, dramatically improving the ability to diagnose these conditions and treat the people who have them swiftly and accurately. 

“Unfortunately, young children are sometimes born with developmental issues. When they visit the doctor, the doctor may struggle to diagnose the problem or devise a treatment plan. The new sequencing techniques have facilitated the rapid diagnosis of conditions such as Menkes disease among young children. By comparing the DNA of the child with that of their parents, clinicians were able to pinpoint the mutation within days,” says Shankar Balasubramanian. 

This rapid diagnostic process exemplifies how next-generation sequencing is changing the landscape for treating rare diseases. By significantly reducing the time to diagnosis, next-generation sequencing enables more rapid and targeted therapeutic interventions, making a profound difference in patient outcomes.

 “This rapid process is particularly important in neonatal intensive care. Thus, next-generation sequencing is already revolutionising the approach to rare genetic diseases, offering hope for more effective diagnosis and treatment strategies. Its integration into healthcare systems marks a significant advancement in personalised medicine.” 

Sir David Klenerman and Sir Shankar Balasubramanian. Photo: Millenium Technology Prize.

Navigating the pandemic 

The COVID-19 pandemic underscored the universal threat posed by infectious diseases. Again, next-generation sequencing was instrumental in deciphering the genetic code of the virus, enabling the tracking of its spread and the evolution of new variants worldwide. 

“An international effort led to the sequencing of more than 1 million SARS-CoV-2 genomes. This global initiative was crucial for understanding the dynamics of the pandemic and guiding public health strategies worldwide,” explains David Klenerman. 

And the application of next-generation sequencing goes beyond tracking; it is about understanding why certain individuals are more severely affected by SARS-CoV-2 and informing the development of future vaccines. This wealth of genetic information is key to combatting not just COVID-19 but future infectious threats as well. 

“The extensive sequencing of SARS-CoV-2 showcases the critical role of next-generation sequencing in pandemic response and preparedness. It highlights the importance of global collaboration in addressing public health crises and the potential of genomics in shaping future healthcare strategies.” 

Afterlife 

The impact of next-generation sequencing, envisioned by David Klenerman and Shankar Balasubramanian, now extends beyond their initial dreams. It has transformed forensic science, enhancing the accuracy of criminal investigations. Moreover, it promises to advance agricultural biotechnology by facilitating the development of crops more resilient to climate change and pests and aids in environmental monitoring and biodiversity identification. 

“Obviously, as the company has matured, you can start to play different roles. I actually stayed involved as a scientific adviser and consultant for another 10 years after Illumina acquired Solexa. Have we been struggling to find anything worthwhile to work on since then? So, on a personal level, people always talk to me about sequencing, but I want to talk about all the other stuff we have been excited about over the past 10–15 years,” explains Shankar Balasubramanian. 

Throughout his career, Shankar Balasubramanian has focused on DNA research: first, understanding the primary sequence of DNA, which led to the development of sequencing technology. 

“Second, I have delved into the world of epigenetics, inventing methods for sequencing chemical modifications on DNA, such as methylation and hydroxymethylation. This work saw the birth in 2012 of the forerunner of Biomodal Limited, advancing our ability to read these epigenetic marks with precision. Lastly, I have explored the secondary structure of DNA, specifically the four-stranded G quadruplex structures.” 

Key pathological features 

These complex DNA structures have been controversial because, for years, their existence and significance in living cells have been debated. Critics questioned their biological relevance and role. 

“Recent breakthroughs over the past decade confirm their real presence in human cells and their crucial role in regulating gene expression and affecting cell identity, particularly in cancer. These structures reveal the complex and fascinating nature of DNA. I have been wrestling with this problem for 25 years and will take it with me to the afterlife if I have not cracked it.” 

Beyond sequencing, David Klenerman’s interests have broadened to include the study of protein misfolding diseases, such as Alzheimer’s and Parkinson’s, through innovative single-molecule fluorescence techniques. These efforts aim to better understand the molecular mechanisms of these diseases and to develop diagnostic and therapeutic strategies. 

“Transitioning to Alzheimer’s and Parkinson’s research, we applied similar principles of high-resolution molecular analysis, but this time focusing on proteins rather than nucleic acids. We used single-molecule fluorescence techniques to study the misfolding and aggregation of proteins, which are key pathological features in both diseases. In this way, we hope to uncover the mechanisms leading to these neurodegenerative diseases,” says David Klenerman. 

Professor David Klenerman. Foto: Nathan Pitt, University of Cambridge.

Were not trying to do anything useful 

What literally started with an idea drawn on a piece of paper in the Panton Arms in 1997 has today brought about several revolutions. For Balasubramanian and Klenerman, this is a great testament to how empowering basic science is and how, if implemented well, it can have a major impact. 

“Scientists working together can solve major problems in society. I think this should still be very evident right now, with the COVID-19 pandemic only a few years behind us. We all need to think about where science can go, but it is equally important to realise that the idea came from basic science. So, the message is: if you want the next breakthrough technology, please fund basic science.” 

How to optimally foster innovation is a big topic of debate around the world. According to Shankar Balasubramanian and David Klenerman, there is no single correct answer to this. However, they are concerned about the popular trend of encouraging people in universities to engage more and more in applied or translational research. 

“My view is that this is not the first thing I would do with resources. Our story did start with basic science. The grants that funded the basic work had no application in mind. We were not even trying to do anything useful. It was just curiosity that drove this. It is often about observations but also the context in which the observation was made, steering things in a certain direction. And these can lead to unexpected and sometimes momentous outcomes,” explains Shankar Balasubramanian. 

Do take risks 

Shankar Balasubramanian is concerned that if organisations force university researchers to pursue only “useful” research, projects have to be so well defined that they will be short term and incremental. “ Transitioning to Alzheimer’s and Parkinson’s research, we applied similar principles of highresolution molecular analysis, but this time focusing on proteins rather than nucleic acids.” David Klenerman 

“This is unlikely to be transformative. So, I emphasise highlighting the absolute importance of funding basic science very strongly if you want true innovations to emerge. Without strong support for basic science, your innovation pipeline will quickly dry up.” 

Equally important is who you decide to fund. The Solexa journey started when Shankar Balasubramanian and David Klenerman were relatively junior and more prepared to take risks than they are now. 

“I would say, especially to the young researchers out there: do take risks, and try to identify and tackle big questions that you feel could make a significant impact if you can. Opportunities like that are rare, but if you see them in what you are doing, I strongly encourage you to go for it and give it a try. The worst thing that can happen is that you may fail. And then you try again,” concludes David Klenerman.