The Sequencing Revolution

Dr. Nicholas Navin, Department of Genetics
G&D 2012 Newsletter

In the 1970s, Fred Sanger developed a revolutionary method using reversible-terminator chemistry that enabled him to sequence nucleotides of DNA. He used this method to delineate all 5386 nucleotides of the Phi174 bacteriophage genome, and was awarded the Nobel prize for his groundbreaking work. Further improvements to the first-generation or “Sanger” sequencing method enabled 700 bases to be sequenced at a time, and the technique became widely adopted by the research community. In 1990 the Human Genome Project was proposed, an audacious project to sequence all 3.18 billion bases in the human genome. It was a monumental effort that took over 10 years to complete, cost about $2.7 billion, and required thousands of sequencing machines and scientists from around the world to collaborate.

About five years later, technical advances in parallelizing sequencing reactions resulted in second or next-generation sequencing (NGS) technologies that were pioneered by Illumina and 454. These methods had the capacity to sequence millions of bases in a single run. In 2005, these technologies were used to sequence the entire genome of James Watson over a period of a just a few months for a cost of around $100,000. Thanks to fierce industrial competition and rapid innovations, the cost of sequencing a human genome has since plummeted to just $4000 per genome in 2012. The timeframe has also decreased substantially – we can now sequence an entire human genome in under one week. These technologies have since been applied to sequence a myriad of genomes, including the black plague bacterium, the orangutan, the platypus, the banana, the shark, a Neanderthal genome, the wine grape, and let us not forget the all-important Ozzy Osbourne genome.

So what have we learned from all of the massive genome sequencing? One of the most important applications has been to improve our understanding of human cancers. NIH and NCI funded a large-scale genome sequencing project called The Cancer Genome Atlas (TCGA) in which they are sequencing hundreds of tumor genomes from different cancer types. Surprisingly, these studies have identified very few mutations (TP53, KRAS, BRAF) that are common in a specific cancer type. Instead, they have found thousands of unique mutations that reflect the tumor’s own evolutionary history. Finding the driver mutations in each cancer is a formidable challenge due to the presence of thousands of passenger mutations, which are unlikely to be contributing to the disease. However, in principle, if we can identify the driver mutations in each tumor, we can treat the patient with a cocktail of specific drugs that are custom-tailored towards their tumor. This personalized medicine approach would be a major advancement over current clinical practice in which chemotherapies are used to target all of the rapidly dividing cells in a human body, often resulting in severe side effects. MD Anderson is ambitiously pursuing personalized medicine with its Institute for Personalized Cancer Therapy where patients can have their tumors sequenced and clinicians will prescribe drugs that target specific mutations. In the next few years, we may see profound changes in the way cancer patients are diagnosed and treated.

In research labs, NGS has become a powerful tool to study the genomics of DNA, RNA and epigenetics. In the last decade, NGS was performed almost exclusively at sequencing centers. However, with the precipitous drop in the cost of sequencing, NGS has been democratized and is now accessible to most university research labs. At MD Anderson we have a DNA sequencing core facility in the Mitchell Basic Sciences Research Building where samples can be processed for NGS on the Illumina HiSeq2000 platform. While the generation of sequencing data has become easier, the analysis remains a formidable challenge. Millions of sequence reads must be aligned to a human genome and the resulting data must be mined to identify mutations or measure expression levels. The problem is that these tasks are computationally intensive and most tools were developed by academics to run on the Unix operating system, presenting a challenge to most biologists. Fortunately, MD Anderson has a Bioinformatics Department with many faculty who are experienced in NGS analysis and eager to collaborate. Thus, MD Anderson is a great place to dive into NGS experiments, which can often provide powerful, unbiased data for generating hypotheses in our research projects.

Peering into the crystal ball, we can see several major innovations that are likely to be forthcoming in the next few years. One of which my own laboratory is deeply involved in: developing new technologies for sequencing the genomes of single cells. These methods will allow us to delineate intratumor heterogeneity and trace the evolution of single cells as they form malignant masses and disseminate into the circulatory system to seed metastatic tumors. The $1000 genome will likely be achieved in the near future by several companies. We will also see several third-generation sequencing technologies emerge, extending the length of sequence reads to thousands of base pairs and providing direct measurements for epigenetic modifications on nucleotides. Another invention that I am particularly excited about was announced at the AGBT conference earlier this year. Oxford Nanopore gave a presentation that literally caused the audience to fall out of their seats – the development of a new plug-and-play USB thumb-drive sequencer, shown at right. With this device, it will be possible to directly apply a DNA sample, plug the device into a laptop and retrieve the sequencing data in a few hours, all for a cost of under $1000. This device would have been considered science fiction only a few years ago, but is now scheduled for commercial release in about a year. These ground-breaking innovations will continue to fuel the next-generation sequencing revolution, and provide powerful new tools that will not only benefit, but transform basic research and clinical practice in the coming years.

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